US20150315243A1 - Methods for the synthesis of dicarba bridges in organic compounds - Google Patents
Methods for the synthesis of dicarba bridges in organic compounds Download PDFInfo
- Publication number
- US20150315243A1 US20150315243A1 US14/804,693 US201514804693A US2015315243A1 US 20150315243 A1 US20150315243 A1 US 20150315243A1 US 201514804693 A US201514804693 A US 201514804693A US 2015315243 A1 US2015315243 A1 US 2015315243A1
- Authority
- US
- United States
- Prior art keywords
- metathesis
- peptide
- cross
- pair
- dicarba
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 288
- 150000002894 organic compounds Chemical class 0.000 title claims abstract description 81
- 230000015572 biosynthetic process Effects 0.000 title claims description 208
- 238000003786 synthesis reaction Methods 0.000 title claims description 106
- 238000005686 cross metathesis reaction Methods 0.000 claims abstract description 167
- 230000000295 complement effect Effects 0.000 claims abstract description 85
- 150000001875 compounds Chemical class 0.000 claims abstract description 66
- 230000001939 inductive effect Effects 0.000 claims abstract description 20
- 230000005855 radiation Effects 0.000 claims abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 326
- 238000006243 chemical reaction Methods 0.000 claims description 268
- 235000001014 amino acid Nutrition 0.000 claims description 168
- 150000001413 amino acids Chemical class 0.000 claims description 166
- 150000001336 alkenes Chemical class 0.000 claims description 124
- 238000005984 hydrogenation reaction Methods 0.000 claims description 116
- 239000003054 catalyst Substances 0.000 claims description 95
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 claims description 92
- WNNNWFKQCKFSDK-UHFFFAOYSA-N allylglycine Chemical compound OC(=O)C(N)CC=C WNNNWFKQCKFSDK-UHFFFAOYSA-N 0.000 claims description 84
- 239000000203 mixture Substances 0.000 claims description 83
- 239000002904 solvent Substances 0.000 claims description 64
- 239000007787 solid Substances 0.000 claims description 63
- QSNZCLMYJQOHAD-UHFFFAOYSA-N 2-(3-methylbut-2-enylamino)acetic acid Chemical compound CC(C)=CCNCC(O)=O QSNZCLMYJQOHAD-UHFFFAOYSA-N 0.000 claims description 55
- 229920006395 saturated elastomer Polymers 0.000 claims description 47
- 238000009876 asymmetric hydrogenation reaction Methods 0.000 claims description 29
- 238000011068 loading method Methods 0.000 claims description 24
- 230000000903 blocking effect Effects 0.000 claims description 18
- 125000000217 alkyl group Chemical group 0.000 claims description 17
- 239000002253 acid Substances 0.000 claims description 16
- 230000000694 effects Effects 0.000 claims description 16
- 229910052739 hydrogen Inorganic materials 0.000 claims description 13
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 10
- 235000018417 cysteine Nutrition 0.000 claims description 10
- 150000002148 esters Chemical class 0.000 claims description 10
- 125000000524 functional group Chemical group 0.000 claims description 10
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 9
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 9
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 9
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 9
- 125000001424 substituent group Chemical group 0.000 claims description 8
- 150000001993 dienes Chemical class 0.000 claims description 7
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 4
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- 150000002825 nitriles Chemical class 0.000 claims description 3
- 239000000816 peptidomimetic Substances 0.000 claims description 3
- 235000018102 proteins Nutrition 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 125000003158 alcohol group Chemical group 0.000 claims 2
- 125000001475 halogen functional group Chemical group 0.000 claims 1
- 125000004356 hydroxy functional group Chemical group O* 0.000 claims 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 367
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 245
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 205
- 239000011347 resin Substances 0.000 description 171
- 229920005989 resin Polymers 0.000 description 171
- 229940024606 amino acid Drugs 0.000 description 165
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 114
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 91
- 239000000243 solution Substances 0.000 description 87
- 239000000047 product Substances 0.000 description 79
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 75
- FCDPQMAOJARMTG-UHFFFAOYSA-M benzylidene-[1,3-bis(2,4,6-trimethylphenyl)imidazolidin-2-ylidene]-dichlororuthenium;tricyclohexylphosphanium Chemical compound C1CCCCC1[PH+](C1CCCCC1)C1CCCCC1.CC1=CC(C)=CC(C)=C1N(CCN1C=2C(=CC(C)=CC=2C)C)C1=[Ru](Cl)(Cl)=CC1=CC=CC=C1 FCDPQMAOJARMTG-UHFFFAOYSA-M 0.000 description 68
- 238000006798 ring closing metathesis reaction Methods 0.000 description 68
- 238000001819 mass spectrum Methods 0.000 description 66
- 238000005649 metathesis reaction Methods 0.000 description 66
- 102000004196 processed proteins & peptides Human genes 0.000 description 65
- 230000002829 reductive effect Effects 0.000 description 65
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 64
- 238000005859 coupling reaction Methods 0.000 description 59
- 238000003776 cleavage reaction Methods 0.000 description 56
- 238000004611 spectroscopical analysis Methods 0.000 description 56
- 230000007017 scission Effects 0.000 description 55
- 229910001868 water Inorganic materials 0.000 description 55
- 239000000539 dimer Substances 0.000 description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 54
- 238000010183 spectrum analysis Methods 0.000 description 52
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 51
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 48
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 48
- 239000011734 sodium Substances 0.000 description 45
- PNPBGYBHLCEVMK-UHFFFAOYSA-L benzylidene(dichloro)ruthenium;tricyclohexylphosphane Chemical compound Cl[Ru](Cl)=CC1=CC=CC=C1.C1CCCCC1P(C1CCCCC1)C1CCCCC1.C1CCCCC1P(C1CCCCC1)C1CCCCC1 PNPBGYBHLCEVMK-UHFFFAOYSA-L 0.000 description 43
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 42
- 230000008878 coupling Effects 0.000 description 42
- 238000010168 coupling process Methods 0.000 description 42
- 239000003153 chemical reaction reagent Substances 0.000 description 41
- -1 homo-amino acids Chemical class 0.000 description 39
- 230000001404 mediated effect Effects 0.000 description 38
- IAQRGUVFOMOMEM-ARJAWSKDSA-N cis-but-2-ene Chemical compound C\C=C/C IAQRGUVFOMOMEM-ARJAWSKDSA-N 0.000 description 35
- 230000004913 activation Effects 0.000 description 33
- 239000000758 substrate Substances 0.000 description 33
- 239000010948 rhodium Substances 0.000 description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 31
- IAQRGUVFOMOMEM-UHFFFAOYSA-N but-2-ene Chemical compound CC=CC IAQRGUVFOMOMEM-UHFFFAOYSA-N 0.000 description 31
- 238000000746 purification Methods 0.000 description 31
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 30
- 230000035484 reaction time Effects 0.000 description 29
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 28
- LKQSFPOXQAQZJB-NSCUHMNNSA-N 2-[[(e)-but-2-enyl]amino]acetic acid Chemical compound C\C=C\CNCC(O)=O LKQSFPOXQAQZJB-NSCUHMNNSA-N 0.000 description 27
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 27
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 27
- 238000003818 flash chromatography Methods 0.000 description 26
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 26
- 239000011541 reaction mixture Substances 0.000 description 26
- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 description 25
- 239000011995 wilkinson's catalyst Substances 0.000 description 25
- IFMXNBRHEQLZMI-VAYQAVKTSA-N (3s)-3-[[(2s)-2-[[(2r)-2-[[(2r)-2-[(2-aminoacetyl)amino]-3-sulfanylpropanoyl]amino]-3-sulfanylpropanoyl]amino]-3-hydroxypropanoyl]amino]-4-[(2s)-2-[[(2s)-1-[[(2r)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2r)-1-amino-1-oxo-3-sulfanylpropan-2-yl]amino]-5-(diaminomet Chemical class N([C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(N)=O)C(=O)[C@@H]1CCCN1C(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)NC(=O)[C@H](CS)NC(=O)CN IFMXNBRHEQLZMI-VAYQAVKTSA-N 0.000 description 24
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 24
- 108050003126 conotoxin Proteins 0.000 description 24
- 235000019439 ethyl acetate Nutrition 0.000 description 24
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 24
- UTODFRQBVUVYOB-UHFFFAOYSA-P wilkinson's catalyst Chemical group [Cl-].C1=CC=CC=C1P(C=1C=CC=CC=1)(C=1C=CC=CC=1)[Rh+](P(C=1C=CC=CC=1)(C=1C=CC=CC=1)C=1C=CC=CC=1)P(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 UTODFRQBVUVYOB-UHFFFAOYSA-P 0.000 description 24
- 239000007821 HATU Substances 0.000 description 23
- TUCNEACPLKLKNU-UHFFFAOYSA-N acetyl Chemical compound C[C]=O TUCNEACPLKLKNU-UHFFFAOYSA-N 0.000 description 23
- 230000003197 catalytic effect Effects 0.000 description 23
- 229910052757 nitrogen Inorganic materials 0.000 description 23
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 22
- 241000786363 Rhampholeon spectrum Species 0.000 description 22
- 150000001793 charged compounds Chemical class 0.000 description 22
- 238000010276 construction Methods 0.000 description 22
- 239000000377 silicon dioxide Substances 0.000 description 22
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 20
- 229910052681 coesite Inorganic materials 0.000 description 20
- 229910052906 cristobalite Inorganic materials 0.000 description 20
- 229910052682 stishovite Inorganic materials 0.000 description 20
- 229910052905 tridymite Inorganic materials 0.000 description 20
- 238000005160 1H NMR spectroscopy Methods 0.000 description 19
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical class C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 19
- 125000004122 cyclic group Chemical group 0.000 description 19
- 239000005977 Ethylene Substances 0.000 description 18
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 18
- 239000012043 crude product Substances 0.000 description 18
- 239000012071 phase Substances 0.000 description 18
- 230000009257 reactivity Effects 0.000 description 18
- 230000009467 reduction Effects 0.000 description 18
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 16
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 16
- 238000010511 deprotection reaction Methods 0.000 description 16
- 238000010647 peptide synthesis reaction Methods 0.000 description 16
- 125000001844 prenyl group Chemical group [H]C([*])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 239000001375 methyl (2E,4E)-hexa-2,4-dienoate Substances 0.000 description 15
- BKOOMYPCSUNDGP-UHFFFAOYSA-N 2-methylbut-2-ene Chemical compound CC=C(C)C BKOOMYPCSUNDGP-UHFFFAOYSA-N 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 229960003067 cystine Drugs 0.000 description 14
- XNMQEEKYCVKGBD-UHFFFAOYSA-N dimethylacetylene Natural products CC#CC XNMQEEKYCVKGBD-UHFFFAOYSA-N 0.000 description 14
- 238000009905 homogeneous catalytic hydrogenation reaction Methods 0.000 description 14
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 14
- 238000004949 mass spectrometry Methods 0.000 description 14
- 229960002429 proline Drugs 0.000 description 14
- 108010069514 Cyclic Peptides Proteins 0.000 description 13
- 102000001189 Cyclic Peptides Human genes 0.000 description 13
- 150000001408 amides Chemical class 0.000 description 13
- 241000894007 species Species 0.000 description 13
- IAQRGUVFOMOMEM-ONEGZZNKSA-N trans-but-2-ene Chemical compound C\C=C\C IAQRGUVFOMOMEM-ONEGZZNKSA-N 0.000 description 13
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 12
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 12
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 12
- 229910052799 carbon Inorganic materials 0.000 description 12
- 235000019253 formic acid Nutrition 0.000 description 12
- 239000000710 homodimer Substances 0.000 description 12
- 239000001257 hydrogen Substances 0.000 description 12
- 239000000543 intermediate Substances 0.000 description 12
- 230000008569 process Effects 0.000 description 12
- 150000003839 salts Chemical class 0.000 description 12
- 125000002837 carbocyclic group Chemical group 0.000 description 11
- 229960002433 cysteine Drugs 0.000 description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 11
- 238000011084 recovery Methods 0.000 description 11
- 239000007858 starting material Substances 0.000 description 11
- 230000009466 transformation Effects 0.000 description 11
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 10
- KYVBNYUBXIEUFW-UHFFFAOYSA-N 1,1,3,3-tetramethylguanidine Chemical compound CN(C)C(=N)N(C)C KYVBNYUBXIEUFW-UHFFFAOYSA-N 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 10
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 10
- VZUAUHWZIKOMFC-ARJAWSKDSA-N [(z)-4-acetyloxybut-2-enyl] acetate Chemical compound CC(=O)OC\C=C/COC(C)=O VZUAUHWZIKOMFC-ARJAWSKDSA-N 0.000 description 10
- 229910052786 argon Inorganic materials 0.000 description 10
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 10
- 239000007789 gas Substances 0.000 description 10
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- 235000013930 proline Nutrition 0.000 description 10
- 229960001153 serine Drugs 0.000 description 10
- 239000007790 solid phase Substances 0.000 description 10
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 9
- 239000004793 Polystyrene Substances 0.000 description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 9
- WNNNWFKQCKFSDK-BYPYZUCNSA-N (2s)-2-aminopent-4-enoic acid Chemical compound OC(=O)[C@@H](N)CC=C WNNNWFKQCKFSDK-BYPYZUCNSA-N 0.000 description 8
- GVIYUKXRXPXMQM-BPXGDYAESA-N 221231-10-3 Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]1C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CSSC1)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C(C)C)=O)C1=CC=C(O)C=C1 GVIYUKXRXPXMQM-BPXGDYAESA-N 0.000 description 8
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 8
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 8
- 239000003875 Wang resin Substances 0.000 description 8
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 8
- 150000001412 amines Chemical class 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 239000012298 atmosphere Substances 0.000 description 8
- 125000005647 linker group Chemical group 0.000 description 8
- 229920002223 polystyrene Polymers 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 125000006239 protecting group Chemical group 0.000 description 8
- 238000010992 reflux Methods 0.000 description 8
- 235000004400 serine Nutrition 0.000 description 8
- 230000003595 spectral effect Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 108010070305 AOD 9604 Proteins 0.000 description 7
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 7
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 125000005610 enamide group Chemical class 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000010348 incorporation Methods 0.000 description 7
- 238000006317 isomerization reaction Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 229910052707 ruthenium Inorganic materials 0.000 description 7
- 108010048412 somatotropin (177-191) Proteins 0.000 description 7
- ADOHASQZJSJZBT-SANMLTNESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C12=CC=CC=C2N(C(=O)OC(C)(C)C)C=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ADOHASQZJSJZBT-SANMLTNESA-N 0.000 description 6
- SCEJQXRUWDSIBB-UHFFFAOYSA-N 2,2-diaminooctanedioic acid Chemical class OC(=O)C(N)(N)CCCCCC(O)=O SCEJQXRUWDSIBB-UHFFFAOYSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 6
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 125000000649 benzylidene group Chemical group [H]C(=[*])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 6
- 239000013522 chelant Substances 0.000 description 6
- 239000007822 coupling agent Substances 0.000 description 6
- YMWUJEATGCHHMB-DICFDUPASA-N dichloromethane-d2 Chemical compound [2H]C([2H])(Cl)Cl YMWUJEATGCHHMB-DICFDUPASA-N 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 238000006772 olefination reaction Methods 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 239000002243 precursor Substances 0.000 description 6
- 230000008961 swelling Effects 0.000 description 6
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 6
- 238000000844 transformation Methods 0.000 description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 6
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 5
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 5
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 5
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 5
- 239000004912 1,5-cyclooctadiene Substances 0.000 description 5
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 5
- 108010016626 Dipeptides Proteins 0.000 description 5
- VQTUBCCKSQIDNK-UHFFFAOYSA-N Isobutene Chemical group CC(C)=C VQTUBCCKSQIDNK-UHFFFAOYSA-N 0.000 description 5
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 5
- 239000012190 activator Substances 0.000 description 5
- 125000002947 alkylene group Chemical group 0.000 description 5
- 125000004429 atom Chemical group 0.000 description 5
- 239000006227 byproduct Substances 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 230000001010 compromised effect Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- 150000004702 methyl esters Chemical class 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 231100000614 poison Toxicity 0.000 description 5
- 239000002574 poison Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 229960002898 threonine Drugs 0.000 description 5
- 239000002435 venom Substances 0.000 description 5
- 231100000611 venom Toxicity 0.000 description 5
- 210000001048 venom Anatomy 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 4
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- KMSVANWURMHBAC-UHFFFAOYSA-N 2-acetamido-2-hydroxyacetic acid Chemical compound CC(=O)NC(O)C(O)=O KMSVANWURMHBAC-UHFFFAOYSA-N 0.000 description 4
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 description 4
- SEPQTYODOKLVSB-UHFFFAOYSA-N 3-methylbut-2-enal Chemical compound CC(C)=CC=O SEPQTYODOKLVSB-UHFFFAOYSA-N 0.000 description 4
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 4
- 241001638933 Cochlicella barbara Species 0.000 description 4
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 4
- 238000003072 Ellman's test Methods 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 238000006546 Horner-Wadsworth-Emmons reaction Methods 0.000 description 4
- 108010000521 Human Growth Hormone Proteins 0.000 description 4
- 102000002265 Human Growth Hormone Human genes 0.000 description 4
- 239000000854 Human Growth Hormone Substances 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 108010033276 Peptide Fragments Chemical group 0.000 description 4
- 102000007079 Peptide Fragments Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 125000001118 alkylidene group Chemical group 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- GCWCVCCEIQXUQU-UHFFFAOYSA-N alpha-hydroxyhippuric acid Chemical compound OC(=O)C(O)NC(=O)C1=CC=CC=C1 GCWCVCCEIQXUQU-UHFFFAOYSA-N 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- IVYACSIMCHEJJO-UHFFFAOYSA-N amino pent-4-enoate Chemical compound NOC(=O)CCC=C IVYACSIMCHEJJO-UHFFFAOYSA-N 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 4
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 4
- HOXDXGRSZJEEKN-UHFFFAOYSA-N cycloocta-1,5-diene;rhodium Chemical compound [Rh].C1CC=CCCC=C1 HOXDXGRSZJEEKN-UHFFFAOYSA-N 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- FJKIXWOMBXYWOQ-UHFFFAOYSA-N ethenoxyethane Chemical compound CCOC=C FJKIXWOMBXYWOQ-UHFFFAOYSA-N 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000036571 hydration Effects 0.000 description 4
- 238000006703 hydration reaction Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- BTPLQRUPGWLEPL-UHFFFAOYSA-N methyl 2-benzamido-2-methoxyacetate Chemical compound COC(=O)C(OC)NC(=O)C1=CC=CC=C1 BTPLQRUPGWLEPL-UHFFFAOYSA-N 0.000 description 4
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 230000006919 peptide aggregation Effects 0.000 description 4
- FAIAAWCVCHQXDN-UHFFFAOYSA-N phosphorus trichloride Chemical compound ClP(Cl)Cl FAIAAWCVCHQXDN-UHFFFAOYSA-N 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- CYTQBVOFDCPGCX-UHFFFAOYSA-N trimethyl phosphite Chemical compound COP(OC)OC CYTQBVOFDCPGCX-UHFFFAOYSA-N 0.000 description 4
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 3
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 3
- 229940117976 5-hydroxylysine Drugs 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 241000237970 Conus <genus> Species 0.000 description 3
- 238000010485 C−C bond formation reaction Methods 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- 229960003767 alanine Drugs 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 229960005261 aspartic acid Drugs 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 125000002619 bicyclic group Chemical group 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 3
- 150000003857 carboxamides Chemical class 0.000 description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000003196 chaotropic effect Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000011097 chromatography purification Methods 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000012044 organic layer Substances 0.000 description 3
- 230000007030 peptide scission Effects 0.000 description 3
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 239000010453 quartz Substances 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000012453 solvate Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- WLPUWLXVBWGYMZ-UHFFFAOYSA-N tricyclohexylphosphine Chemical compound C1CCCCC1P(C1CCCCC1)C1CCCCC1 WLPUWLXVBWGYMZ-UHFFFAOYSA-N 0.000 description 3
- 230000007306 turnover Effects 0.000 description 3
- 108091058550 ω-conotoxin Proteins 0.000 description 3
- KJPRLNWUNMBNBZ-QPJJXVBHSA-N (E)-cinnamaldehyde Chemical compound O=C\C=C\C1=CC=CC=C1 KJPRLNWUNMBNBZ-QPJJXVBHSA-N 0.000 description 2
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 2
- VXNZUUAINFGPBY-UHFFFAOYSA-N 1-Butene Chemical compound CCC=C VXNZUUAINFGPBY-UHFFFAOYSA-N 0.000 description 2
- LKQSFPOXQAQZJB-UHFFFAOYSA-N 2-(but-2-enylamino)acetic acid Chemical compound CC=CCNCC(O)=O LKQSFPOXQAQZJB-UHFFFAOYSA-N 0.000 description 2
- UPMGJEMWPQOACJ-UHFFFAOYSA-N 2-[4-[(2,4-dimethoxyphenyl)-(9h-fluoren-9-ylmethoxycarbonylamino)methyl]phenoxy]acetic acid Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(O)=O)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 UPMGJEMWPQOACJ-UHFFFAOYSA-N 0.000 description 2
- CDUUKBXTEOFITR-BYPYZUCNSA-N 2-methyl-L-serine Chemical compound OC[C@@]([NH3+])(C)C([O-])=O CDUUKBXTEOFITR-BYPYZUCNSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- SKDHHIUENRGTHK-UHFFFAOYSA-N 4-nitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC=C(C(Cl)=O)C=C1 SKDHHIUENRGTHK-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical group NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 2
- 229940127007 Compound 39 Drugs 0.000 description 2
- 241000237942 Conidae Species 0.000 description 2
- 241000237972 Conus geographus Species 0.000 description 2
- 241001495101 Conus imperialis Species 0.000 description 2
- 241001459904 Conus regius Species 0.000 description 2
- 241000237974 Conus textile Species 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 2
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical group COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 2
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 description 2
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- QQONPFPTGQHPMA-UHFFFAOYSA-N Propene Chemical compound CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PJANXHGTPQOBST-VAWYXSNFSA-N Stilbene Natural products C=1C=CC=CC=1/C=C/C1=CC=CC=C1 PJANXHGTPQOBST-VAWYXSNFSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- ACWQBUSCFPJUPN-UHFFFAOYSA-N Tiglaldehyde Natural products CC=C(C)C=O ACWQBUSCFPJUPN-UHFFFAOYSA-N 0.000 description 2
- BAECOWNUKCLBPZ-HIUWNOOHSA-N Triolein Natural products O([C@H](OCC(=O)CCCCCCC/C=C\CCCCCCCC)COC(=O)CCCCCCC/C=C\CCCCCCCC)C(=O)CCCCCCC/C=C\CCCCCCCC BAECOWNUKCLBPZ-HIUWNOOHSA-N 0.000 description 2
- PHYFQTYBJUILEZ-UHFFFAOYSA-N Trioleoylglycerol Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCCCCCCCC)COC(=O)CCCCCCCC=CCCCCCCCC PHYFQTYBJUILEZ-UHFFFAOYSA-N 0.000 description 2
- VZUAUHWZIKOMFC-ONEGZZNKSA-N [(e)-4-acetyloxybut-2-enyl] acetate Chemical compound CC(=O)OC\C=C\COC(C)=O VZUAUHWZIKOMFC-ONEGZZNKSA-N 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- ZHWLPDIRXJCEJY-UHFFFAOYSA-N alpha-hydroxyglycine Chemical class NC(O)C(O)=O ZHWLPDIRXJCEJY-UHFFFAOYSA-N 0.000 description 2
- CDUUKBXTEOFITR-UHFFFAOYSA-N alpha-methylserine Natural products OCC([NH3+])(C)C([O-])=O CDUUKBXTEOFITR-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- CREXVNNSNOKDHW-UHFFFAOYSA-N azaniumylideneazanide Chemical group N[N] CREXVNNSNOKDHW-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- PNPBGYBHLCEVMK-UHFFFAOYSA-N benzylidene(dichloro)ruthenium;tricyclohexylphosphanium Chemical compound Cl[Ru](Cl)=CC1=CC=CC=C1.C1CCCCC1[PH+](C1CCCCC1)C1CCCCC1.C1CCCCC1[PH+](C1CCCCC1)C1CCCCC1 PNPBGYBHLCEVMK-UHFFFAOYSA-N 0.000 description 2
- 150000001576 beta-amino acids Chemical class 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- 238000005356 chiral GC Methods 0.000 description 2
- 238000012505 colouration Methods 0.000 description 2
- KJQOYUHYAZGPIZ-PIJHVLQJSA-N conotoxin vc1.1 Chemical compound C([C@H]1C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(N[C@@H](CSSC[C@@H]2NC(=O)[C@@H](NC(=O)CN)CSSC[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]3CCCN3C(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC2=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H](CC(O)=O)C(=O)N1)C(N)=O)=O)[C@@H](C)CC)C1=CN=CN1 KJQOYUHYAZGPIZ-PIJHVLQJSA-N 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000003292 diminished effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 229960002449 glycine Drugs 0.000 description 2
- 125000005843 halogen group Chemical group 0.000 description 2
- 239000000833 heterodimer Substances 0.000 description 2
- NIDHFQDUBOVBKZ-UHFFFAOYSA-N hex-4-enoic acid Chemical compound CC=CCCC(O)=O NIDHFQDUBOVBKZ-UHFFFAOYSA-N 0.000 description 2
- 239000002815 homogeneous catalyst Substances 0.000 description 2
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 229960003646 lysine Drugs 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical group [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 description 2
- LGYKXKOQUCLBJO-UHFFFAOYSA-N methyl 2-acetamido-2-hydroxyacetate Chemical compound COC(=O)C(O)NC(C)=O LGYKXKOQUCLBJO-UHFFFAOYSA-N 0.000 description 2
- FKNMSDVAGYOANC-UHFFFAOYSA-N methyl 2-acetamido-2-methoxyacetate Chemical compound CC(=O)NC(OC)C(=O)OC FKNMSDVAGYOANC-UHFFFAOYSA-N 0.000 description 2
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- MOOYVEVEDVVKGD-UHFFFAOYSA-N oxaldehydic acid;hydrate Chemical compound O.OC(=O)C=O MOOYVEVEDVVKGD-UHFFFAOYSA-N 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 108010026901 peptide 106 Proteins 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 239000002798 polar solvent Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 239000012508 resin bead Substances 0.000 description 2
- 150000003303 ruthenium Chemical class 0.000 description 2
- YAYGSLOSTXKUBW-UHFFFAOYSA-N ruthenium(2+) Chemical compound [Ru+2] YAYGSLOSTXKUBW-UHFFFAOYSA-N 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 238000010530 solution phase reaction Methods 0.000 description 2
- 238000007614 solvation Methods 0.000 description 2
- 230000000707 stereoselective effect Effects 0.000 description 2
- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical compound C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 description 2
- 235000021286 stilbenes Nutrition 0.000 description 2
- TYFQFVWCELRYAO-UHFFFAOYSA-N suberic acid Chemical compound OC(=O)CCCCCCC(O)=O TYFQFVWCELRYAO-UHFFFAOYSA-N 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 229940034208 thyroxine Drugs 0.000 description 2
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 2
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 2
- 229940117972 triolein Drugs 0.000 description 2
- 229960004441 tyrosine Drugs 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- ASGMFNBUXDJWJJ-JLCFBVMHSA-N (1R,3R)-3-[[3-bromo-1-[4-(5-methyl-1,3,4-thiadiazol-2-yl)phenyl]pyrazolo[3,4-d]pyrimidin-6-yl]amino]-N,1-dimethylcyclopentane-1-carboxamide Chemical compound BrC1=NN(C2=NC(=NC=C21)N[C@H]1C[C@@](CC1)(C(=O)NC)C)C1=CC=C(C=C1)C=1SC(=NN=1)C ASGMFNBUXDJWJJ-JLCFBVMHSA-N 0.000 description 1
- XGPXBCKGQLCHDW-ZCTOJWETSA-M (1z,5z)-cycloocta-1,5-diene;(2s,5s)-1-[2-[(2s,5s)-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane;rhodium;trifluoromethanesulfonate Chemical compound [Rh].C\1C\C=C/CC\C=C/1.[O-]S(=O)(=O)C(F)(F)F.CC[C@H]1CC[C@H](CC)P1C1=CC=CC=C1P1[C@@H](CC)CC[C@@H]1CC XGPXBCKGQLCHDW-ZCTOJWETSA-M 0.000 description 1
- MTKRKAHKLRCDGI-JEDNCBNOSA-N (2S)-2-aminohex-4-enoic acid hydrochloride Chemical compound Cl.CC=CC[C@H](N)C(O)=O MTKRKAHKLRCDGI-JEDNCBNOSA-N 0.000 description 1
- GVVCHDNSTMEUCS-UAFMIMERSA-N (2r,5r)-1-[2-[(2r,5r)-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane Chemical compound CC[C@@H]1CC[C@@H](CC)P1C1=CC=CC=C1P1[C@H](CC)CC[C@H]1CC GVVCHDNSTMEUCS-UAFMIMERSA-N 0.000 description 1
- AJNZWRKTWQLAJK-KLHDSHLOSA-N (2r,5r)-1-[2-[(2r,5r)-2,5-dimethylphospholan-1-yl]phenyl]-2,5-dimethylphospholane Chemical compound C[C@@H]1CC[C@@H](C)P1C1=CC=CC=C1P1[C@H](C)CC[C@H]1C AJNZWRKTWQLAJK-KLHDSHLOSA-N 0.000 description 1
- YVBLQCANYSFEBN-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pent-4-enoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC=C)C(=O)O)C3=CC=CC=C3C2=C1 YVBLQCANYSFEBN-SFHVURJKSA-N 0.000 description 1
- GAUUPDQWKHTCAX-VIFPVBQESA-N (2s)-2-amino-3-(1-benzothiophen-3-yl)propanoic acid Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CSC2=C1 GAUUPDQWKHTCAX-VIFPVBQESA-N 0.000 description 1
- DEMIRSVUSWJCFT-YFKPBYRVSA-N (2s)-5,5-dimethylpyrrolidine-2-carboxylic acid Chemical compound CC1(C)CC[C@@H](C(O)=O)N1 DEMIRSVUSWJCFT-YFKPBYRVSA-N 0.000 description 1
- AJNZWRKTWQLAJK-VGWMRTNUSA-N (2s,5s)-1-[2-[(2s,5s)-2,5-dimethylphospholan-1-yl]phenyl]-2,5-dimethylphospholane Chemical compound C[C@H]1CC[C@H](C)P1C1=CC=CC=C1P1[C@@H](C)CC[C@@H]1C AJNZWRKTWQLAJK-VGWMRTNUSA-N 0.000 description 1
- YQZHANAPVDIEHA-WDSKDSINSA-N (2s,7s)-2,7-bis(azaniumyl)octanedioate Chemical compound [O-]C(=O)[C@@H]([NH3+])CCCC[C@H]([NH3+])C([O-])=O YQZHANAPVDIEHA-WDSKDSINSA-N 0.000 description 1
- UDQTXCHQKHIQMH-KYGLGHNPSA-N (3ar,5s,6s,7r,7ar)-5-(difluoromethyl)-2-(ethylamino)-5,6,7,7a-tetrahydro-3ah-pyrano[3,2-d][1,3]thiazole-6,7-diol Chemical compound S1C(NCC)=N[C@H]2[C@@H]1O[C@H](C(F)F)[C@@H](O)[C@@H]2O UDQTXCHQKHIQMH-KYGLGHNPSA-N 0.000 description 1
- ZRPFJAPZDXQHSM-UHFFFAOYSA-L 1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazole;dichloro-[(2-propan-2-yloxyphenyl)methylidene]ruthenium Chemical compound CC(C)OC1=CC=CC=C1C=[Ru](Cl)(Cl)=C1N(C=2C(=CC(C)=CC=2C)C)CCN1C1=C(C)C=C(C)C=C1C ZRPFJAPZDXQHSM-UHFFFAOYSA-L 0.000 description 1
- RUKVGXGTVPPWDD-UHFFFAOYSA-N 1,3-bis(2,4,6-trimethylphenyl)imidazolidine Chemical group CC1=CC(C)=CC(C)=C1N1CN(C=2C(=CC(C)=CC=2C)C)CC1 RUKVGXGTVPPWDD-UHFFFAOYSA-N 0.000 description 1
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 1
- LJCZNYWLQZZIOS-UHFFFAOYSA-N 2,2,2-trichlorethoxycarbonyl chloride Chemical compound ClC(=O)OCC(Cl)(Cl)Cl LJCZNYWLQZZIOS-UHFFFAOYSA-N 0.000 description 1
- QPLJYAKLSCXZSF-UHFFFAOYSA-N 2,2,2-trichloroethyl carbamate Chemical compound NC(=O)OCC(Cl)(Cl)Cl QPLJYAKLSCXZSF-UHFFFAOYSA-N 0.000 description 1
- WGFNXGPBPIJYLI-UHFFFAOYSA-N 2,6-difluoro-3-[(3-fluorophenyl)sulfonylamino]-n-(3-methoxy-1h-pyrazolo[3,4-b]pyridin-5-yl)benzamide Chemical compound C1=C2C(OC)=NNC2=NC=C1NC(=O)C(C=1F)=C(F)C=CC=1NS(=O)(=O)C1=CC=CC(F)=C1 WGFNXGPBPIJYLI-UHFFFAOYSA-N 0.000 description 1
- YQZHANAPVDIEHA-UHFFFAOYSA-N 2,7-bis(azaniumyl)octanedioate Chemical class OC(=O)C(N)CCCCC(N)C(O)=O YQZHANAPVDIEHA-UHFFFAOYSA-N 0.000 description 1
- VVCMGAUPZIKYTH-VGHSCWAPSA-N 2-acetyloxybenzoic acid;[(2s,3r)-4-(dimethylamino)-3-methyl-1,2-diphenylbutan-2-yl] propanoate;1,3,7-trimethylpurine-2,6-dione Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O.CN1C(=O)N(C)C(=O)C2=C1N=CN2C.C([C@](OC(=O)CC)([C@H](C)CN(C)C)C=1C=CC=CC=1)C1=CC=CC=C1 VVCMGAUPZIKYTH-VGHSCWAPSA-N 0.000 description 1
- MTKRKAHKLRCDGI-UHFFFAOYSA-N 2-aminohex-4-enoic acid;hydrochloride Chemical compound Cl.CC=CCC(N)C(O)=O MTKRKAHKLRCDGI-UHFFFAOYSA-N 0.000 description 1
- XTXGLOBWOMUGQB-UHFFFAOYSA-N 2-azaniumyl-3-(3-methoxyphenyl)propanoate Chemical compound COC1=CC=CC(CC(N)C(O)=O)=C1 XTXGLOBWOMUGQB-UHFFFAOYSA-N 0.000 description 1
- BGAJNPLDJJBRHK-UHFFFAOYSA-N 3-[2-[5-(3-chloro-4-propan-2-yloxyphenyl)-1,3,4-thiadiazol-2-yl]-3-methyl-6,7-dihydro-4h-pyrazolo[4,3-c]pyridin-5-yl]propanoic acid Chemical compound C1=C(Cl)C(OC(C)C)=CC=C1C1=NN=C(N2C(=C3CN(CCC(O)=O)CCC3=N2)C)S1 BGAJNPLDJJBRHK-UHFFFAOYSA-N 0.000 description 1
- XFDUHJPVQKIXHO-UHFFFAOYSA-N 3-aminobenzoic acid Chemical compound NC1=CC=CC(C(O)=O)=C1 XFDUHJPVQKIXHO-UHFFFAOYSA-N 0.000 description 1
- NYPYPOZNGOXYSU-UHFFFAOYSA-N 3-bromopyridine Chemical compound BrC1=CC=CN=C1 NYPYPOZNGOXYSU-UHFFFAOYSA-N 0.000 description 1
- 238000004679 31P NMR spectroscopy Methods 0.000 description 1
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 1
- GONFBOIJNUKKST-UHFFFAOYSA-N 5-ethylsulfanyl-2h-tetrazole Chemical compound CCSC=1N=NNN=1 GONFBOIJNUKKST-UHFFFAOYSA-N 0.000 description 1
- GDUANFXPOZTYKS-UHFFFAOYSA-N 6-bromo-8-[(2,6-difluoro-4-methoxybenzoyl)amino]-4-oxochromene-2-carboxylic acid Chemical compound FC1=CC(OC)=CC(F)=C1C(=O)NC1=CC(Br)=CC2=C1OC(C(O)=O)=CC2=O GDUANFXPOZTYKS-UHFFFAOYSA-N 0.000 description 1
- 229940027041 8-mop Drugs 0.000 description 1
- BAAKAPDBWZEGDD-UHFFFAOYSA-N 9h-fluoren-1-ylmethyl n-(2,5-dioxopyrrolidin-1-yl)carbamate Chemical compound C=1C=CC(C2=CC=CC=C2C2)=C2C=1COC(=O)NN1C(=O)CCC1=O BAAKAPDBWZEGDD-UHFFFAOYSA-N 0.000 description 1
- ZZOKVYOCRSMTSS-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl carbamate Chemical compound C1=CC=C2C(COC(=O)N)C3=CC=CC=C3C2=C1 ZZOKVYOCRSMTSS-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 101710193470 Alpha-conotoxin ImI Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102000016904 Armadillo Domain Proteins Human genes 0.000 description 1
- 108010014223 Armadillo Domain Proteins Proteins 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- JQUCWIWWWKZNCS-LESHARBVSA-N C(C1=CC=CC=C1)(=O)NC=1SC[C@H]2[C@@](N1)(CO[C@H](C2)C)C=2SC=C(N2)NC(=O)C2=NC=C(C=C2)OC(F)F Chemical compound C(C1=CC=CC=C1)(=O)NC=1SC[C@H]2[C@@](N1)(CO[C@H](C2)C)C=2SC=C(N2)NC(=O)C2=NC=C(C=C2)OC(F)F JQUCWIWWWKZNCS-LESHARBVSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 101710115643 Cathelicidin-1 Proteins 0.000 description 1
- 208000000094 Chronic Pain Diseases 0.000 description 1
- 201000003874 Common Variable Immunodeficiency Diseases 0.000 description 1
- 241001516648 Conus amadis Species 0.000 description 1
- 241000855803 Conus lynceus Species 0.000 description 1
- 241000146344 Conus marmoreus Species 0.000 description 1
- 101000922006 Conus textile Epsilon-conotoxin TxVA Proteins 0.000 description 1
- 241000237980 Conus tulipa Species 0.000 description 1
- 241001343482 Conus victoriae Species 0.000 description 1
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 1
- 241000289632 Dasypodidae Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical class Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- FSBIGDSBMBYOPN-VKHMYHEASA-N L-canavanine Chemical compound OC(=O)[C@@H](N)CCONC(N)=N FSBIGDSBMBYOPN-VKHMYHEASA-N 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- 125000003290 L-leucino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 108090000543 Ligand-Gated Ion Channels Proteins 0.000 description 1
- 102000004086 Ligand-Gated Ion Channels Human genes 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- QXKHYNVANLEOEG-UHFFFAOYSA-N Methoxsalen Chemical compound C1=CC(=O)OC2=C1C=C1C=COC1=C2OC QXKHYNVANLEOEG-UHFFFAOYSA-N 0.000 description 1
- 101100328158 Mus musculus Clmp gene Proteins 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 1
- 108010053775 Nisin Proteins 0.000 description 1
- 102000008092 Norepinephrine Plasma Membrane Transport Proteins Human genes 0.000 description 1
- 108010049586 Norepinephrine Plasma Membrane Transport Proteins Proteins 0.000 description 1
- FSBIGDSBMBYOPN-UHFFFAOYSA-N O-guanidino-DL-homoserine Natural products OC(=O)C(N)CCON=C(N)N FSBIGDSBMBYOPN-UHFFFAOYSA-N 0.000 description 1
- 241001465382 Physalis alkekengi Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102000003743 Relaxin Human genes 0.000 description 1
- 108090000103 Relaxin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 102000016913 Voltage-Gated Sodium Channels Human genes 0.000 description 1
- 108010053752 Voltage-Gated Sodium Channels Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 125000005210 alkyl ammonium group Chemical group 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 125000000746 allylic group Chemical group 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 229940124277 aminobutyric acid Drugs 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 1
- 230000002783 anti-lipogenic effect Effects 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 125000003289 ascorbyl group Chemical group [H]O[C@@]([H])(C([H])([H])O*)[C@@]1([H])OC(=O)C(O*)=C1O* 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid group Chemical group C(C1=CC=CC=C1)(=O)O WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- PUJDIJCNWFYVJX-UHFFFAOYSA-N benzyl carbamate Chemical compound NC(=O)OCC1=CC=CC=C1 PUJDIJCNWFYVJX-UHFFFAOYSA-N 0.000 description 1
- KPLXXWHGAQYTID-UHFFFAOYSA-N benzylidene-[1,3-bis(2,4,6-trimethylphenyl)imidazolidin-2-ylidene]ruthenium;tricyclohexylphosphane Chemical compound C1CCCCC1P(C1CCCCC1)C1CCCCC1.CC1=CC(C)=CC(C)=C1N(CCN1C=2C(=CC(C)=CC=2C)C)C1=[Ru]=CC1=CC=CC=C1 KPLXXWHGAQYTID-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- 125000004369 butenyl group Chemical group C(=CCC)* 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- NQZFAUXPNWSLBI-UHFFFAOYSA-N carbon monoxide;ruthenium Chemical group [Ru].[Ru].[Ru].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-] NQZFAUXPNWSLBI-UHFFFAOYSA-N 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000002579 carboxylato group Chemical group [O-]C(*)=O 0.000 description 1
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003694 chemoselective catalyst Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 229940125936 compound 42 Drugs 0.000 description 1
- 239000000039 congener Substances 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013058 crude material Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- HVJJUDAMOHYMRL-UHFFFAOYSA-L dichloro-[(2-propan-2-yloxyphenyl)methylidene]ruthenium Chemical compound CC(C)OC1=CC=CC=C1C=[Ru](Cl)Cl HVJJUDAMOHYMRL-UHFFFAOYSA-L 0.000 description 1
- WBKFWQBXFREOFH-UHFFFAOYSA-N dichloromethane;ethyl acetate Chemical compound ClCCl.CCOC(C)=O WBKFWQBXFREOFH-UHFFFAOYSA-N 0.000 description 1
- LEFPWWWXFFNJAA-UHFFFAOYSA-N dicyclohexylphosphorylcyclohexane Chemical compound C1CCCCC1P(C1CCCCC1)(=O)C1CCCCC1 LEFPWWWXFFNJAA-UHFFFAOYSA-N 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- PIJUCRAUUJLFEQ-UHFFFAOYSA-N dimethyl 2-acetamido-7-benzamidooct-4-enedioate Chemical compound COC(=O)C(NC(C)=O)CC=CCC(C(=O)OC)NC(=O)C1=CC=CC=C1 PIJUCRAUUJLFEQ-UHFFFAOYSA-N 0.000 description 1
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000011982 enantioselective catalyst Substances 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- PYGSKMBEVAICCR-UHFFFAOYSA-N hexa-1,5-diene Chemical group C=CCCC=C PYGSKMBEVAICCR-UHFFFAOYSA-N 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 238000007172 homogeneous catalysis Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- UWYVPFMHMJIBHE-OWOJBTEDSA-N hydroxymaleic acid group Chemical group O/C(/C(=O)O)=C/C(=O)O UWYVPFMHMJIBHE-OWOJBTEDSA-N 0.000 description 1
- WVDDGKGOMKODPV-UHFFFAOYSA-N hydroxymethyl benzene Natural products OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- HXEACLLIILLPRG-RXMQYKEDSA-N l-pipecolic acid Natural products OC(=O)[C@H]1CCCCN1 HXEACLLIILLPRG-RXMQYKEDSA-N 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 230000002366 lipolytic effect Effects 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 1
- TZOPFVPQJVFNCE-AWEZNQCLSA-N methyl (2s)-6-acetyloxy-2-benzamidohex-4-enoate Chemical compound CC(=O)OCC=CC[C@@H](C(=O)OC)NC(=O)C1=CC=CC=C1 TZOPFVPQJVFNCE-AWEZNQCLSA-N 0.000 description 1
- MIBMMKTXTRJVOP-UHFFFAOYSA-N methyl 2-acetamidopent-4-enoate Chemical compound COC(=O)C(CC=C)NC(C)=O MIBMMKTXTRJVOP-UHFFFAOYSA-N 0.000 description 1
- WHHNAMFLUXPVNV-UHFFFAOYSA-N methyl 2-aminopent-4-enoate;hydrochloride Chemical compound Cl.COC(=O)C(N)CC=C WHHNAMFLUXPVNV-UHFFFAOYSA-N 0.000 description 1
- TZOPFVPQJVFNCE-UHFFFAOYSA-N methyl 6-acetyloxy-2-benzamidohex-4-enoate Chemical compound CC(=O)OCC=CCC(C(=O)OC)NC(=O)C1=CC=CC=C1 TZOPFVPQJVFNCE-UHFFFAOYSA-N 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 230000007604 neuronal communication Effects 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 239000004309 nisin Substances 0.000 description 1
- 235000010297 nisin Nutrition 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 239000012454 non-polar solvent Substances 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid group Chemical group C(CCCCCCC\C=C/CCCCCCCC)(=O)O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 150000002902 organometallic compounds Chemical class 0.000 description 1
- 125000003232 p-nitrobenzoyl group Chemical group [N+](=O)([O-])C1=CC=C(C(=O)*)C=C1 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- QUCQEUCGKKTEBI-UHFFFAOYSA-N palmatine Chemical compound COC1=CC=C2C=C(C3=C(C=C(C(=C3)OC)OC)CC3)[N+]3=CC2=C1OC QUCQEUCGKKTEBI-UHFFFAOYSA-N 0.000 description 1
- IPCSVZSSVZVIGE-UHFFFAOYSA-N palmitic acid group Chemical group C(CCCCCCCCCCCCCCC)(=O)O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
- 230000001769 paralizing effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N pentanoic acid group Chemical class C(CCCC)(=O)O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 238000005897 peptide coupling reaction Methods 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000009522 phase III clinical trial Methods 0.000 description 1
- WLJVXDMOQOGPHL-UHFFFAOYSA-N phenylacetic acid Chemical compound OC(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-N 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- GWLJTAJEHRYMCA-UHFFFAOYSA-N phospholane Chemical compound C1CCPC1 GWLJTAJEHRYMCA-UHFFFAOYSA-N 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- OCAAZRFBJBEVPS-UHFFFAOYSA-N prop-2-enyl carbamate Chemical compound NC(=O)OCC=C OCAAZRFBJBEVPS-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000012217 radiopharmaceutical Substances 0.000 description 1
- 229940121896 radiopharmaceutical Drugs 0.000 description 1
- 230000002799 radiopharmaceutical effect Effects 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- QBERHIJABFXGRZ-UHFFFAOYSA-M rhodium;triphenylphosphane;chloride Chemical compound [Cl-].[Rh].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 QBERHIJABFXGRZ-UHFFFAOYSA-M 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000011986 second-generation catalyst Substances 0.000 description 1
- 230000009834 selective interaction Effects 0.000 description 1
- 238000005872 self-metathesis reaction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003354 serine derivatives Chemical class 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000003711 snail venom Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical class C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229940075620 somatostatin analogue Drugs 0.000 description 1
- 239000003203 stereoselective catalyst Substances 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 150000003441 suberic acid derivatives Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XBXCNNQPRYLIDE-UHFFFAOYSA-N tert-butylcarbamic acid Chemical compound CC(C)(C)NC(O)=O XBXCNNQPRYLIDE-UHFFFAOYSA-N 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229940124598 therapeutic candidate Drugs 0.000 description 1
- 238000005979 thermal decomposition reaction Methods 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- FQDIANVAWVHZIR-OWOJBTEDSA-N trans-1,4-Dichlorobutene Chemical compound ClC\C=C\CCl FQDIANVAWVHZIR-OWOJBTEDSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 102000038650 voltage-gated calcium channel activity Human genes 0.000 description 1
- 108091023044 voltage-gated calcium channel activity Proteins 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- BPKIMPVREBSLAJ-QTBYCLKRSA-N ziconotide Chemical compound C([C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]2C(=O)N[C@@H]3C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CSSC2)C(N)=O)=O)CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CSSC3)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(N1)=O)CCSC)[C@@H](C)O)C1=CC=C(O)C=C1 BPKIMPVREBSLAJ-QTBYCLKRSA-N 0.000 description 1
- 229960002811 ziconotide Drugs 0.000 description 1
- 108091058551 α-conotoxin Proteins 0.000 description 1
- XJKFZICVAPPHCK-NZPQQUJLSA-N ω cgtx Chemical compound C([C@@H](C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H]1N(C[C@H](O)C1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CS)NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H]1N(C[C@H](O)C1)C(=O)[C@H](CO)NC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H]1N(C[C@H](O)C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CS)[C@@H](C)O)C1=CC=C(O)C=C1 XJKFZICVAPPHCK-NZPQQUJLSA-N 0.000 description 1
- KNJNGVKTAFTUFL-OCMUWRIYSA-N ω-conotoxin Chemical compound N([C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H]1C(N[C@@H](CSSC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H]1C(N[C@@H](CCCN=C(N)N)C(=O)N[C@H](CO)C(=O)NCC(=O)N[C@H](CCCCN)C(=O)N[C@H](CSSC1)C(N)=O)=O)=O)C(=O)[C@@H]1CSSC[C@@H](N)C(=O)N[C@H](CCCCN)C(=O)NCC(=O)N[C@H](CCCCN)C(=O)NCC(=O)N[C@H](C)C(=O)N[C@@H](CCCCN)C(=O)N1 KNJNGVKTAFTUFL-OCMUWRIYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B37/00—Reactions without formation or introduction of functional groups containing hetero atoms, involving either the formation of a carbon-to-carbon bond between two carbon atoms not directly linked already or the disconnection of two directly linked carbon atoms
- C07B37/04—Substitution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2/00—Preparation of hydrocarbons from hydrocarbons containing a smaller number of carbon atoms
- C07C2/02—Preparation of hydrocarbons from hydrocarbons containing a smaller number of carbon atoms by addition between unsaturated hydrocarbons
- C07C2/42—Preparation of hydrocarbons from hydrocarbons containing a smaller number of carbon atoms by addition between unsaturated hydrocarbons homo- or co-oligomerisation with ring formation, not being a Diels-Alder conversion
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2/00—Preparation of hydrocarbons from hydrocarbons containing a smaller number of carbon atoms
- C07C2/02—Preparation of hydrocarbons from hydrocarbons containing a smaller number of carbon atoms by addition between unsaturated hydrocarbons
- C07C2/50—Diels-Alder conversion
- C07C2/52—Catalytic processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
- C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
- C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
- C07C2603/18—Fluorenes; Hydrogenated fluorenes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The present invention relates to methods for forming dicarba bridges in organic compounds. This involves the use of a pair of complementary metathesisable groups on the organic compound, and subjecting the compound to cross-metathesis under microwave radiation conditions. In an alternative, the compounds contain a turn-inducing group between the pair of cross-metathesisable groups to facilitate the cross-metathesis.
Description
- The present application broadly relates to methods for forming dicarba bridges in organic compounds, and compounds such as peptides containing dicarba bridges.
- Cystine (—S—S—) bridges are common structural motifs in naturally occurring cyclic peptides. In some cases, these disulfide bridges act as reactive functional groups. In many other cases however, the cystine bridge serves only a skeletal, structural role, maintaining secondary and tertiary structure. Disulfide bonds in peptides and other compounds are highly reactive under broad-ranging conditions, and therefore useful peptides containing disulfide bonds which have a structural role are at risk of denaturation, resulting in loss of properties. There is accordingly some interest in developing methods for creating more robust bridges in such compounds—such as dicarba (—C—C—) containing bridges, which are not as reactive, so as to produce compounds having the activity of, or similar activity to, the disulfide-containing polypeptides, but with better biostability.
- Once a suitably strategy for forming such dicarba bridges is established, it is of additional interest to be able to form multiple dicarba bridges—selectively. By way of explanation, a peptide possessing four cysteine residues, and two cystine bridges, has three topoisomers—the [1,3],[2,4]-isomer (globule), the [1,4],[2,3]-isomer (ribbon) and the [1,2],[3,4]-isomer (bead). It would be useful to be able to selectively form one of these isomers, without any of the other two topoisomers. It is also of interest to be able to form one or more dicarba bridges using chemistry that does not destroy any disulfide bridges that are present, so that dicarba-disulfide containing compounds can additionally be formed. It is of further interest to have a dicarba bridge forming method that can take place despite the presence of disulfide, which could otherwise interfere with dicarba bridge-forming reactions.
- Once this is achievable, it is of interest to be able to form dicarba-containing analogues of a range of disulfide-containing peptides, such as conotoxins. It is also of interest to form peptide and non-peptide compounds containing one or more intramolecular dicarba bridge, and an olefin-handle enabling reaction to other moieties.
- According to the present invention, there is provided a range of methods for forming dicarba bridges, as well as new compounds containing dicarba bridges and a range of new compounds that facilitate the construction of these bridges.
- According to a first aspect, there is provided a method for the synthesis of an organic compound with a dicarba bridge, comprising:
-
- providing a reactable organic compound having a pair of unblocked complementary metathesisable groups, or two or more reactable organic compounds having between them a pair of unblocked complementary, metathesisable groups, and
- subjecting the reactable organic compound or compounds to cross-metathesis under microwave radiation conditions to form an organic compound with an unsaturated dicarba bridge.
- As explained in further detail below, cross-metathesis involves the formation of an unsaturated dicarba bridge (inter- or intramolecular, depending on whether there are one or two reactable organic compounds) from two unblocked metathesisable olefinic groups. It has been surprisingly found that for many situations where the reaction will not proceed under normal conditions, the performance of this reaction under microwave radiation conditions overcomes this problem and enables this reaction to proceed. Another strategy for improving the performance of the cross-metathesis which does not rely on microwave is outlined below. The advantages of the use of microwave irradiation applies particularly to the situation where the method is performed on a single reactable organic compound having a pair of unblocked complementary metathesisable groups, for the formation of an intramolecular dicarba bridge. In other cases, microwave radiation overcomes inefficient metathesis reactions that do not otherwise go to completion. Other details relating to the types of compounds that this method is particularly suited to are outlined in the detailed description.
- In a related aspect, in which it is desired to form a saturated dicarba bridge, the process involves a following step of subjecting the unsaturated dicarba bridge to hydrogenation (suitably homogeneous hydrogenation). Accordingly, in total, this second aspect provides a method for the synthesis of an organic compound with a saturated dicarba bridge, comprising:
-
- providing a reactable organic compound having a pair of unblocked complementary metathesisable groups, or two or more reactable organic compounds having between them a pair of unblocked complementary metathesisable groups,
- subjecting the reactable organic compound or compounds to cross-metathesis under microwave radiation conditions to form an organic compound with unsaturated dicarba bridge, and
- subjecting the unsaturated dicarba bridge to hydrogenation.
- The hydrogenation step is suitably homogeneous hydrogenation.
- According to one particularly preferred embodiment, the process enables the selective formation of multiple dicarba bridges. According to this embodiment, there is provided a method for the synthesis of an organic compound with a plurality of dicarba bridges, comprising:
-
- providing one or more reactable organic compounds having within the single compound, or between the multiple compounds, a first pair of complementary metathesisable groups which are unblocked, a second pair of complementary metathesisable groups, which are blocked and can be unblocked by an unblocking reaction or series of reactions specific to that second pair, and optionally further pairs of complementary methathesisable groups, which are blocked and can be unblocked by an unblocking reaction or series of reactions specific to each further pair,
- subjecting the reactable organic compound or compounds to cross-metathesis to form an organic compound with an unsaturated dicarba bridge across the first pair of complementary metathesisable groups, without cross-metathesis between the pair or pairs of blocked complementary metathesisable groups,
- subjecting the second pair of complementary metathesisable groups to the unblocking reaction or series of reactions specific to the second pair,
- subjecting the second pair of complementary metathesisable groups to cross-metathesis to form an organic compound with an unsaturated dicarba bridge across the second pair of complementary metathesisable groups, without cross-methathesis between any pair or pairs of complementary methathesisable groups that remain blocked, and
- if any complementary metathesisable groups remain, subjecting those groups to unblocking reactions specific to those pairs, followed by cross metathesis, wherein at least one of the cross-metathesis reactions is conducted under microwave radiation conditions.
- Preferably, the unblocking reaction specific to the second pair comprises cross-metathesis with a butadiene-free disposable olefin. 1,3-butadiene acts as a poison in the unblocking reaction, if it is present in the disposable olefin composition used in this reaction.
- In many circumstances it will be desirable to subject some or all of the unsaturated dicarba bridges formed by cross-metathesis to hydrogenation. This can be completed in stages following each cross-metathesis, or it may be conducted as a single hydrogenation step for converting all unsaturated dicarba bridges present at that point into saturated dicarba bridges (following two or more cross-metathesis reactions). By convenient selection of the appropriate time at which to perform the hydrogenation(s), it is possible for selected dicarba bridges to be saturated and for other dicarba bridges to remain unsaturated. The hydrogenation step(s) is/are suitably homogeneous hydrogenation.
- Thus, where all dicarba bridges are desired to be saturated, the process described above may comprise the further steps of:
-
- subjecting the unsaturated dicarba bridge formed between the first pair of complementary metathesisable groups to hydrogenation, and
- subjecting the unsaturated dicarba bridge formed between the second pair of complementary metathesisable groups to hydrogenation, wherein each homogenous hydrogenation is performed either separately or in the one step.
- According to one embodiment, the hydrogenation of the complementary methathesisable groups takes place immediately after cross-metathesis of that pair of complementary metathesisable groups. The hydrogenation is suitably a homogeneous hydrogenation.
- It is an option to perform each intramolecular cross-metathesis reaction under microwave radiation conditions.
- In the detailed description, a particularly suitable series of reactions appropriate to the formation of two and three dicarba bridges is described.
- The method of the present invention is particularly suited to the formation of peptides with dicarba bridges. In this event, the reactable compound, or one of the reactable compounds, is attached to a solid support. Suitable conditions for performing the reaction, taking into account the difficulties that are introduced as a result of conducting the reaction on a solid support, are described in the detailed description. It is noted however that compounds other than peptides can also suitably be prepared through a reactable compound which is attached to a solid support, using the microwave cross-metathesis reaction conditions.
- A strategy that is an alternative to microwave irradiation has been devised for improving the performance of a cross-metathesis between two complementary metathesisable groups (olefins) in the one organic compound.
- According to this embodiment, there is provided a method for the synthesis of an organic compound with a dicarba bridge, comprising:
-
- synthesising a reactable organic compound to contain a pair of unblocked complementary metathesisable groups, and a turn-inducing group in between the pair of complementary metathesisable groups, and
- subjecting the reactable organic compound to cross-metathesis to form a compound with an unsaturated dicarba bridge.
- If the target organic compound is to contain a saturated dicarba bridge, the compound is subjected to hydrogenation (suitably homogeneous hydrogenation).
- This method is particularly suited to the synthesis of peptides with dicarba bridges.
- The present invention also provides for a compound produced by the method of the invention. The compound may be a peptide with at least one dicarba bridge, or may be any other organic compound with a dicarba bridge.
-
FIG. 1 is a reaction scheme demonstrating some possible locations for the complementary metathesisable groups, in a peptide. -
FIG. 2 is a 1H n.m.r. spectrum for assessing binding between dienamide 57 and catalyst, forming a ruthenium-vinylalkylidene complex 73 (spectrum a), a new species 74 (spectrum b) after 60 minutes, and complex 74 (spectrum c) after 18 hours. -
FIG. 3 is a 1H n.m.r. spectrum ofcompounds -
FIG. 4 is a graph of catecholamine release from dicarba-conotoxims 118 and 119. -
FIG. 5 is a gas chromatogram trace for commercial trans 2-butene, showing trans 2-butene (A), cis 2-butene (B) andcatalyst poison 1,3-butadiene. - As described above, this application relates to the formation of organic compounds containing dicarba bridges.
- The term organic compound is used in its broadest sense to refer to organic, carbon-containing compounds, as opposed to inorganic compounds that are not based on carbon. To the extent that the method can be used to prepare organic ligands for organometallic compounds, this is also encompassed. Specific examples of organic compounds that the invention is particularly suited to are peptides.
- The term “peptide” is used in this specification in its broadest sense to refer to oligomers of two or more amino acids. The term “side chain” is used in the usual sense to refer to the side chain on the amino acid, and the backbone to the H2N—(C)x—CO2H (where x=1, 2 or 3) component, in which the carbon in bold text bears the side chain (the side chain being possibly linked to the amino nitrogen, as in the case of proline).
- One class of peptides of interest are the peptidomimetics—that is, a peptide that has a series of amino acids that mimics identically or closely a naturally occurring peptide, but with at least one dicarba bridge, and optionally one or more further differences, such as the removal of a cystine bridge, a change by up to 20% of the amino acids in the sequence, as non-limiting examples. Of particular interest are dicarba analogues of naturally-occurring disulfide-containing peptides, in which one or more of the disulfide bonds are replaced with dicarba bridges. These may also be classed as pseudo-peptides.
- The term “amino acid” is used in its broadest sense and refers to L- and D-amino acids including the 20 common amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (illustrated in the Appendix); and the less common amino acid derivatives such as homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids and α,α-di substituted amino acids, for example, cystine, 5-hydroxylysine, 4-hydroxyproline, α-aminoadipic acid, α-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine, ornithine, pipecolic acid, ortho, meta or para-aminobenzoic acid, citrulline, canavanine, norleucine, δ-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine and thyroxine; β-amino acids (as compared with the typical α-amino acids) and any amino acid having a molecular weight less than about 500. The term also encompasses amino acids in which the side chain of the amino acid comprises a metathesisable group, as described herein. Further, the amino acid may be a pseudoproline (ψPro).
- The amino acids may be optionally protected. The term “optionally protected” is used herein in its broadest sense and refers to an introduced functionality which renders a particular functional group, such as a hydroxy, amino, carbonyl or carboxyl group, unreactive under selected conditions and which may later be optionally removed to unmask the functional group. A protected amino acid is one in which the reactive substituents of the amino acid, or the amino group or carboxyl group of the amino acid are protected. Suitable protecting groups are known in the art and include those disclosed in Greene, T. W., “Protective Groups in Organic Synthesis” John Wiley & Sons, New York 1999, (the contents of which are incorporated herein by reference) as are methods for their installation and removal.
- Preferably the N-protecting group is a carbamate such as, 9-fluorenylmethyl carbamate (Fmoc), 2,2,2-trichloroethyl carbamate (Troc), t-butyl carbamate (Boc), allyl carbamate (Alloc), 2-trimethylsilylethyl (Teoc) and benzyl carbamate (Cbz), more preferably Fmoc.
- The carboxyl protecting group is preferably an ester such as an alkyl ester, for example, methyl ester, ethyl ester, t-Bu ester or a benzyl ester.
- The amino acids may be protected, for example, the carboxyl groups of aspartic acid, glutamic acid and α-aminoadipic acid may be esterified (for example as a C1-C6 alkyl ester), the amino groups of lysine, ornithine and 5-hydroxylysine, may be converted to carbamates (for example as a C(═O)OC1-C6 alkyl or C(═O)OCH2Ph carbamate) or imides such as thalimide or succinimide, the hydroxyl groups of 5-hydroxylysine, 4-hydroxyproline, serine, threonine, tyrosine, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine and thyroxine may be converted to ethers (for example a C1-C6 alkyl or a (C1-C6 alkyl)phenyl ether) or esters (for example a C═OC1-C6 alkyl ester) and the thiol group of cysteine may be converted to thioethers (for example a C1-C6 alkyl thioether) or thioesters (for example a C(═O)C1-C6 alkyl thioester).
- The term “dicarba bridge” is used broadly, unless the context indicates otherwise, to refer to a bridging group that includes the sequence —C—C—. This encompasses both the unsaturated (—C═C—) and saturated (—C—C—) dicarba sequence. The atoms directly attached to the carbon atoms of the dicarba sequence (—C—C—) are typically H, although further or alternative reactions can be performed to introduce substituents other than hydrogen onto the carbon atoms of the dicarba sequence of the dicarba bridge. Hydrogenated dicarba bridge refers to the specific case where the dicarba bridge is —CH2—CH2—. The term unsaturated hydrogen dicarba bridge is used to refer to —CH═CH—. This may be cis- or trans-in geometry.
- In addition to the dicarba sequence, the dicarba bridge may include any other series of atoms, typically selected from C, N, O, and P, although the atoms to either side of the dicarba sequence are preferably carbon, and with the proviso that the nitrogen atoms present in the compound during metathesis are not free amines (protected amines, such as carbamates, are acceptable). Thus, the dicarba bridge encompasses the following possible bridges, as illustrative examples:
- In IV, R1 and R2 are each independently selected from any divalent linking group. Such divalent linking groups should not be groups that poison the metathesis catalyst. Most free amines poison metathesis catalysts and therefore are preferably protected or avoided during methathesis.
- The dicarba bridge may form a bridge between two separate reactable organic compounds, to form an intermolecular bridge, or it may form a bridge between two points in a single reactable organic compound, so as to form an intramolecular bridge, otherwise known as a ring. It is particularly difficult to form intramolecular bridges, due to steric hindrance, and the need to bring the reactable (metathesisable) groups together. The use of microwave radiation in the cross-metathesis step has enabled this to occur, or occur more efficiently.
- “Reactable organic compound” is a term used to refer to the organic compound that is subjected to the reaction, as distinct from the target organic compound, to facilitate understanding of which “organic compound” is being referred to in the process. The “reactable” organic compound is therefore any compound that can be subjected to the reaction described, and using other terminology may be considered to be a starting material, an intermediate, a reagent or otherwise.
- In this specification, including the claims which follow, except where the context requires otherwise due to express language or necessary implication, the word “comprising” or variations such as “comprise” or “comprises” is used in the inclusive sense, to specify the presence of the stated features or steps but not to preclude the presence or addition of further features or steps.
- As used in the specification, the words “a”, “an” and “the” include the plural equivalents, unless the context clearly indicates otherwise. Thus, for example, reference to “an amino acid” includes one or more amino acids.
- The method for the formation of dicarba bridges involves the use of complementary pairs of metathesisable groups on a compound.
- Cross-metathesis is a type of metathesis reaction involving the formation of a single olefin bond across two unblocked, or reactive olefins, to form a new olefinic bridge spanning across the two reactive olefins. In a general sense, metathesis can be described as the mutual intermolecular exchange of alkylidene (or carbene) fragments between two olefins promoted by metal-carbene complexes. The cross-metathesis is conducted with a metathesis catalyst. There are many metathesis catalysts known in the art. Examples of suitable catalysts are the ruthenium catalysts, such as Grubbs' catalyst—first and second generation. For details of other suitable cross-metathesis catalysts, reference is made to Grubbs, R.H. Handbook of Metathesis; Wiley-VCH: New York, 2003; 1204 pages, 3 volumes, the entirety of which is incorporated by reference. New catalysts are being developed all the time, and any of these new cross-metathesis catalysts can be used. For additional information on this reaction, and appropriate conditions and catalysts for the performance of the reaction, reference is also made to Chatterjee et al, J. Am, Chem, Soc, 2003, 125, 11360-11370, the entirety of which is incorporated herein by reference.
- Ring-closing metathesis is a particular example of cross-metathesis where the two reactive olefins are on the one compound, so as to form an intramolecular bridge, or ring.
- For metathesis to occur between two alkylidenes (olefins), the alkylidenes must not be blocked by any steric or electronic blocking groups. A steric blocking group is any bulky group that sterically prevents the metathesis from taking place in the presence of a cross-metathesis catalyst. Examples of steric blocking groups on an olefin are alkyl. Prenylglycine is an example of an amino acid containing a dialkyl-blocked olefin side chain (specifically, dimethyl-blocked). Removal of one or both of the blocking groups unblocks the olefin, and enables the cross-metathesis to take place. It is noted that the pair of metathesisable groups that remain after unblocking need not be identical—a mono-substituted olefin (such as a mono-methylated olefin) and an unsubstituted olefin (being unsubstituted at the open olefinic end) can form a suitable pair of cross-metathesisable groups. The term “complementary” is used to indicate that the pair of unblocked metathesisable groups are not necessarily identical, but are merely complementary in the sense that cross-metathesis can take place across the two olefinic groups.
- Electronic blocking refers to the presence of a group on the reactable organic compound or compounds that modifies the electronic nature of the olefin group of the reactable organic compound (which would otherwise undergo cross-metathesis), so as to prevent that olefin group from undergoing cross-metathesis. An example of an electronic blocking group is a conjugated double bond—that is, a double bond located in an α-β relationship to the olefinic group that would otherwise undergo cross-metathesis. The α-β-unsaturation withdraws electrons away from the olefinic cross-metathesisable group, to cause electronic blocking preventing cross-metathesis from taking place.
- By using a combination of blocking mechanisms, a series of pairs of cross-metathesisable olefinic groups in the reactable organic compound or compounds can be designed, with different reaction conditions to effect selective unblocking of particular pairs. In this way, it becomes possible to regioselectively synthesise multiple dicarba bridges (inter and/or intramolecular) in compounds.
- It has been found that when the cross-metathesis reaction is performed under microwave reaction conditions, the reaction may take place in situations where the reaction would not otherwise take place—for instance, when the metathesisable groups are unblocked, but the arrangement, length or spatial orientation of the reactable organic compound prevents the metathesisable groups from being close enough to one another to enable the reaction to proceed. An alternative strategy is described in Section 3.4.2.
- The microwave reaction conditions involve applying microwave radiation to the reactable organic compounds in the presence of the cross-metathesis catalyst for at least part of the reaction, usually for the duration of the reaction. The microwave or microwave reactor may be of any type known in the art, operated at any suitable frequency. Typical frequencies in commercially available microwave reactors are 2.45 GHz, at a power of up to 500 W, usually of up to 300 W. The temperature of the reaction is preferably at elevated temperature, as a consequence of the microwave radiation, preferably at reflux, or around 100° C., as is appropriate in the case. The reaction is preferably performed in a period of not more than 5 hours, suitably for up to about 2 hours.
- There is a strategy that is an alternative to microwave irradiation that has been devised for improving the performance of a cross-metathesis between two complementary metathesisable groups (olefins) in the one organic compound.
- According to this embodiment, there is provided a method for the synthesis of an organic compound with a dicarba bridge, comprising:
-
- synthesising a reactable organic compound to contain a pair of unblocked complementary metathesisable groups, and a turn-inducing group in between the pair of complementary metathesisable groups, and
- subjecting the reactable organic compound to cross-metathesis to form a compound with an unsaturated dicarba bridge.
- If the target organic compound is to contain a saturated dicarba bridge, the compound is subjected to hydrogenation (suitably homogeneous hydrogenation).
- This method is particularly suited to the synthesis of peptides with dicarba bridges.
- Peptides are generally quite linear, as the component amino acids (especially when these are the 20 common amino acids, with exception of proline) and the backbone of the peptide, is linear. Proline, with the ring structure linking to the amino nitrogen atom, induces a turn or a bend in an otherwise linear peptide. This is a naturally-occurring turn-inducing group. This embodiment is particularly suited to those peptides that do not contain a naturally-occurring turn-inducing amino acid. In this case, a synthetic (non-naturally occurring) turn-inducing group is located in the compound—or in the amino acid sequence.
- Preferably the turn-inducing group is a turn-inducting amino acid or protein, and is preferably synthetic (non-naturally occurring). Examples of suitable synthetic turn-inducing amino acids are the pseudoprolines, including derivatives of serine, threonine and cysteine which have been derivatised to contain a cyclic group between the amino acid sidechain (via the —OH or —SH group), and the amino nitrogen atom. A typical derivatising agent is CH3—C(═O)—CH3, such that the turn-inducing amino acids are:
- After cross-metathesis, the pseudoproline(s) are converted back to the underivatised amino acid (serine, threonine or cysteine) by removal of the derivatiseing agent. The conditions for cleavage from a solid support will achieve this.
- According to the present invention, there is provided a method for the synthesis of a peptide with an intramolecular dicarba bridge, the method comprising:
-
- synthesising a peptide comprising a series of amino acids supported on a solid support, wherein two amino acids comprise a first pair of complementary metathesisable groups, and one amino acid between said amino acids comprising the first pair of complementary metathesisable groups in the series which is a turn-inducing amino acid, and
- subjecting the peptide to cross-metathesis to form a peptide with an unsaturated dicarba bridge between the amino acids bearing the metathesisable groups.
- The method may further comprise one or more of the following additional steps:
-
- subjecting the unsaturated discarba bridge to hydrogenation to form a peptide with a saturated intramolecular dicarba bridge;
- cleaving the peptide from the solid support.
- If the turn-inducing amino acid is one of pseudo-serine, pseudo-proline or pseudo-cysteine, then the method may further comprise the step of converting the pseudo-serine, pseudo-proline or pseudo-cysteine to serine, proline or cysteine, respectively.
- The process can be combined with the other preferred features described herein.
- Particularly for reactions conducted with the (or one of the) reactable organic compound(s) attached to a solid support such as a resin, the cross-metathesis is preferably performed in a solvent combination of a resin-swelling solvent, with a co-ordinating solvent for the catalyst. In resin-supported reactions, swelling of the resin is required to avoid “clumping”, but such solvents are not generally compatible with cross-metathesis catalysts. For example, polystyrene-based resins show optimal swelling in chlorinated solvents such as dichloromethane, however these solvents are not compatible with hydrogenation catalysts. The solvents react with such catalysts to compromise catalyst function—which in turn reduces the catalytic cycle (or turn-over number—TON), resulting in incomplete conversion. It was found that the addition of a small amount of a co-ordinating solvent for the catalyst, such as an alcohol (methanol, isopropanol, etc) which can co-ordinate into a vacant site of the catalyst to facilitate stability, overcame this problem. The co-ordinating solvent is suitably used in an amount of about 1-30%, for example constituting 10% of the solvent, by volume. The resin swelling agent may be any polar solvent known to swell the resin, such as dichloromethane. Other suitable solvents for a range of resins are as set out in Santini, R., Griffith, M. C. and Qi, M., Tet. Lett., 1998, 39, 8951-8954, the entirety of which is incorporated herein by reference.
- The (or one of the) reactable organic compound(s) is preferably attached to a solid support—especially in the case of peptide reactable organic compound(s). A plethora of solid supports are known and available in the art, and include pins, crowns, lanterns and resins. Examples are polystyrene-based resins (sometimes referred to as solid supports), including cross-linked polystyrene containing some divinylbenzene (eg 1%), functionalised with linkers (or handles) to provide a reversible linkage between the reactable organic compound (which may be a peptide sequence containing side-chains with cross-metathesisable groups) and the resin. Examples are the Wang resin, Rink amide resin, BHA-Gly-Gly-HMBA resin and 2-chlorotrityl chloride resin, which are all polystyrene-based. Other forms of solid supports that may not necessarily be characterised as resins can also be used.
- It has been surprisingly found that using the microwave reaction conditions, it is possible to have a higher solid support loading than is conventionally used in peptide synthesis on solid supports. Typical solid support loadings are at the 0.1 mmol/g level, but microwave radiation (optionally combined with solvent choice, as described above) overcomes the aggregation problems at higher solid support loadings, so that solid support loading at around 0.9 mmol/g (nine times higher) is achievable. As a consequence, one embodiment of the invention relates to the performance of the reaction at high solid support loadings—that is, at loadings of 0.2 mmol/g and above, such as 0.5 mmol/g and above.
- The product of the cross-metathesis reaction is a compound with an unsaturated dicarba bridge. If the target organic compound is to contain a saturated dicarba bridge, the process further comprises the step of subjecting the unsaturated dicarba bridge to hydrogenation (suitably homogeneous hydrogenation).
- Hydrogenation of the dicarba bridge is performed with a catalyst that is chemoselective for unblocked non-conjugated carbon-carbon double bonds, as distinct from other double bonds (such as carbon-oxygen double bonds in carbonyl groups and carboxylic acids, and blocked conjugated double bonds). One notable example of a suitable catalyst is Wilkinson's catalyst. Wilkinson's catalyst and catalysts like it are not asymmetric hydrogenation catalysts but however as this type of hydrogenation does not form a new chiral centre this is acceptable for this form of hydrogenation reaction. Although the use of asymmetric hydrogenation catalyst is not necessary in the hydrogenation of the dicarba bridge, asymmetric hydrogenation catalysts can nevertheless be used. Suitable catalysts are well known in the art, and include the range of catalysts described for this purpose in Ojima, I. Catalytic Asymmetric Synthesis; Wiley-VCH: New York, 2000; Second Edition,
Chapter 1, 1-110, the entirety of which is incorporated by reference. New catalysts having such properties are developed from time to time, and these may also be used. Further examples of suitable asymmetric hydrogenation catalysts are the chiral phosphine catalysts, including chiral phospholane Rh(I) catalysts. Catalysts in this class are described in U.S. Pat. No. 5,856,525. Such homogenous hydrogenation catalysts are tolerant of sulfide, and disulfide bonds, so that the presence of disulfide bonds and the like will not interfere with the synthetic strategy. The hydrogenation can be conducted at any temperature, such as room temperature or at elevated temperature. The reaction is typically conduced at elevated pressure, although if slower reaction times can be tolerated, the reaction can be performed at atmospheric pressure. - In other stages of the process in which hydrogenation is used as a strategy for unblocking complimentary methasisable groups, it may be beneficial for the hydrogenation catalyst used in those reactions to be asymmetric to stereoselectively form a new chiral centre. Nevertheless, if a racemic mixture can be tolerated, a catalyst such as Wilkinson's catalyst could be used.
- Homogeneous hydrogenation is used in its broadest sense to refer to catalytic hydrogenations conducted in one phase such as a liquid phase, where the liquid phase contains the substrate molecule/s and solvent. More than one solvent, such as organic/aqueous solvent combinations, or fluorous solvent combinations, non-aqueous ionic pairs, supercritical fluids, or systems with soluble polymers may also be employed. This is distinct from heterogeneous reactions, which involve more than one phase—as in the case of hydrogenations performed with solid-supported catalysts in a liquid reaction medium.
- The strategy for the formation of a dicarba bridge described above can be combined with other reaction steps for the formation of an organic compound with a dicarba bridge and a disulfide bridge, or for the formation of organic compounds with multiple dicarba bridges, optionally with disulfide bridges.
- To form a plurality of (i.e. two or more) dicarba bridges, it is necessary to include at appropriate locations in the reactive organic compound or compounds pairs of complementary metathesisable groups which are blocked or deactivated for the times when different pairs of metathesisable groups are being linked together, and unblocked or “activated” to enable reaction to occur between those pairs. Accordingly, for each bridge-forming pair, there needs to be an unblocking reaction available that will selectively unblock the required pairs.
- The first pair to be subjected to the cross metathesis and hydrogenation to form a saturated dicarba bridge need not be blocked during synthesis of the reactive organic compound or compounds. The compound with this pair of unblocked complementary metathesisable groups is then subjected to the reactions described above to form a dicarba bridge (saturated or unsaturated). Suitable groups for forming the first pair of complementary methathesisable groups which are not blocked are —CH═CH2— containing organic moieties, and —CH═CH—CH3— containing moieties. In the case of peptide synthesis, this may be provided by any amino acid containing the side chain —CH═CH2, optionally with any divalent linking group linking the carbon at the “open” end (the —CH═ carbon atom) to the amino acid backbone, such as an -alkylene-, -alkylene-carbonyl-, and so forth. Examples of —CH═CH2— containing amino acids and —CH═CH—CH3— containing amino acids are allyl glycine and crotyl glycine, respectively. Each of these amino acids contains the divalent linking group —CH2— between the alkylene and the amino acid (peptide) backbone.
- At the completion of that reaction, (and optionally after hydrogenation of the first dicarba bridge) the blocked second pair of complementary metathesisable groups, can be subjected to an unblocking reaction. This unblocking reaction involves cross-metathesis with a disposable olefin—which replaces the steric blocking groups on the olefin (metathesisable group) with ═CHR5, described further below.
- Suitable functional groups for forming the second pair of complementary metathesisable groups are di-blocked alkylenes, such as the group —CH═CR3R4, in which R3 and R4 are each independently selected from any blocking groups, such as alkyl, for example methyl. Again, there may be a divalent linking group between the —CH═ carbon atom, and the amino acid backbone, such as an alkylene group, for instance —CH2—. An example of an amino acid containing this group is prenyl glycine, or protected prenyl glycine.
- The unblocking reaction, or activation reaction, to convert the pair of di-blocked alkylenes into an unblocked alkylenes involves subjecting the blocked second pair of complementary metathesisable groups to cross-metathesis with a disposable olefin, to effect removal of the blocking groups (such as R3 and R4 in the example shown above).
- It will be understood that in this case, cross metathesis is used to replace the portion ═CR3H4 with another unblocked portion ═CH2 or ═CHR5, (in which R5 may be —H, functionalised alkyl or alkyl for instance) which is then “activated” or “unblocked” and ready for being subjected to cross-metathesis for the formation of a dicarba bridge, using the same techniques described above.
- The conditions for this activation-type of cross-metathesis are the same as described above for the dicarba bridge forming metathesis. It can be performed under microwave conditions, although it need not be, as the disposable olefin is a smaller molecule and less subject to the spatial constraints as larger reactable organic compounds and single reactable organic compounds in which intramolecular bridges are to be formed.
- The “disposable olefin” is suitably a mono-substituted ethylene (such as monoalkylated ethylene—such as propene, which is a mono-methylated ethylene), or a 1,2-disubstituted ethylene such as high purity 2-butene (cis, trans or a mixture). Previously, commercial 2-butene has been attempted to be used as the disposable olefin in this unblocking reaction, and the reaction is thus sometimes referred to as “butenolysis”. However, until now commercially available 2-butene (which is a mixture of cis- and trans-2-butene) has inexplicably not enabled the reaction to proceed. As detailed further below, a method has been found for overcoming this problematic reaction.
- The substituents of the substituted ethylene disposable olefin are substituents that do not participate in the reaction. Examples are alkyl or a functionalised (substituted) alkyl. The functional group of the functionalised alkyl is suitably a polar functional group, to assist with swelling of the solid support, and solubility. Examples are hydroxy, alkoxy, halo, nitrile and carboxylic acids/esters. One specific example is the di-ester functionalised
disposable olefin 1,4-diacetoxy-2-butene. - Thus the disposable olefin is suitable a 1,3-butadiene-free disposable olefin, or a 1,3-butadiene-free mixture of disposable olefin and is preferably 1,3-butadiene-free olefin or olefin mixture of one or more of the following olefins:
- wherein X and Y are each independently selected from the group consisting of —H, alkyl and alkyl substituted with one or more substituents selected from halo, hydroxy, alkoxy, nitrile, acid and ester.
- Preferably, at least one of X and Y is not H.
- Preferably, in the case of the alkyl substituents, the substituent is preferably on the carbon atom. Preferably the substituted alkyl is a substituted methyl. According to one embodiment, at least one of X and Y is a substituted alkyl, such as a substituted methyl. X and Y may be the same or different. The olefins may be cis or trans, or mixtures of both.
- For the synthesis of a peptide with an intramolecular dicarba bridge, the method may comprise:
-
- providing a peptide comprising a series of amino acids supported on a resin, wherein two amino acids comprise sidechains with a first pair of complementary metathesisable groups which may be blocked or unblocked;
- unblocking the first pair of complementary metathesisable groups, if said groups are blocked; and
- subjecting the peptide to cross-metathesis under microwave radiation conditions to form a peptide with an unsaturated dicarba bridge between the amino acids bearing the metathesisable groups.
- The method may further comprise the step of:
-
- subjecting the unsaturated dicarba bridge to hydrogenation (suitably homogeneous hydrogenation), to form a peptide with a saturated intramolecular dicarba bridge.
- Generally, the peptide will be a protected peptide (such as Fmoc protected). The amino acids can be any of the amino acids described earlier, but it is convenient for the synthesis of peptidomimetics for the amino acids to be selected from the 20 naturally-occurring amino acids, γ- and β-amino acids and from any cross-metathesisable group-bearing analogues thereof. An example of a cross-metathesisable group-bearing analogue is allyl glycine.
- The peptide may also be formed so as to have a disulfide bridge in addition to one or more dicarba bridges. According to this embodiment, the reactable peptide comprises two protected cysteine residues, and the method comprises deprotecting the cysteine residues and oxidising the cysteine residues to form a disulfide bridge. This may be performed at any stage, such as before the formation of the dicarba bridge(s) or after. This step can be combined with the processes described in the following for the formation of two or three dicarba bridges and a disulfide bridge. It is noted that the cysteine residues may be located on the first peptide, on the second peptide (when present) or on a third peptide.
- For the synthesis of a peptide with two intramolecular bridges, the method comprises:
-
- providing a first peptide comprising a series of amino acids supported on a resin, wherein two amino acids comprise sidechains with a first pair of complementary metathesisable groups and two amino acids comprise sidechains with a second pair of blocked complementary metathesisable groups,
- subjecting the peptide to cross-metathesis under microwave radiation conditions to form a peptide with an unsaturated dicarba bridge between the amino acids that bore the first pair of complementary metathesisable groups,
- unblocking the second pair of complementary metathesisable groups, and
- subjecting the peptide to cross-metathesis to form a peptide with an unsaturated dicarba bridge between the amino acids that bore the second pair of complementary metathesisable groups.
- As described previously, one or both unsaturated dicarba bridges formed between the amino acids that bore the first and second pairs of complementary metathesisable groups may be subjected to homogenous hydrogenation, separately or at the same time.
- For the synthesis of a peptide with the intramolecular bridge, and a second bridge which is an intermolecular, the method comprises:
-
- providing a first peptide comprising a series of amino acids supported on a resin, wherein two amino acids comprise sidechains with a first pair of complementary metathesisable groups which may be blocked or unblocked, and one amino acid comprises a sidechain with a second metathesisable group which may be blocked or unblocked, with the proviso that the metathesisable groups out of at least one of the first or the second metathesisable groups are blocked;
- (a) unblocking the first pair of complementary metathesisable groups, if said groups are blocked;
- subjecting the peptide to cross-metathesis under microwave radiation conditions to form a peptide with an unsaturated dicarba bridge between the amino acids bearing the first pair of complementary metathesisable groups, to form a peptide with an intramolecular dicarba bridge, and
- (b) contacting the first peptide with a second peptide comprising one amino acid with a metathesisable group complementary to the second metathesisable group on the first peptide;
- unblocking the second complementary metathesisable groups, if the second metathesisable groups are blocked;
- subjecting the peptide to cross-metathesis to form a peptide with an unsaturated dicarba bridge between the amino acids bearing the second pair of complementary metathesisable groups, to form a dicarba bridge between the amino acids that bore the second metathesisable groups,
wherein steps (a) and (b) are performed in either order, so as to form a peptide with an intermolecular bridge and an intramolecular bridge.
- The method may further comprise the step or steps of subjecting one or both of the products of step (a) and step (b) to hydrogenation (suitably homogeneous hydrogenation) to form a peptide with a saturated intramolecular dicarba bridge and/or a saturated intermolecular dicarba bridge.
- These methods may be combined with a third stage of bridge-formation, to form a peptide with three bridges, one two or three of which are intramolecular. This is achieved by providing a third pair of metathesisable groups in the first peptide, or one in the first peptide and one in the second or in a third peptide to be coupled to the first peptide through an intermolecular bridge, and then subjecting the third pair of metathesisable groups to unblocking to form the compound. In another alternative, a complimentary metathesisable group can be “added” to the first or second peptide through the addition of an amino acid or peptide fragment bearing the metathesisable group. This is illustrated in
FIG. 1 . - For the formation of a peptide with three intramolecular bridges, the method comprises:
-
- providing a first peptide comprising a series of amino acids supported on a resin, wherein two amino acids comprise sidechains with a first pair of complementary metathesisable groups, two amino acids comprise sidechains with a second pair of blocked complementary metathesisable groups and two amino acids comprise sidechains with a third pair of blocked complementary metathesisable groups,
- subjecting the peptide to cross-metathesis under microwave radiation conditions to form a peptide with an unsaturated dicarba bridge between the amino acids bearing the first pair of complementary metathesisable groups,
- optionally subjecting the unsaturated dicarba bridge to hydrogenation,
- unblocking the second pair of complementary metathesisable groups,
- subjecting the peptide to cross-metathesis to form a peptide with an unsaturated dicarba bridge between the amino acids that bore the second pair of complementary metathesisable groups,
- optionally subjecting the unsaturated dicarba bridge to hydrogenation,
- unblocking the third pair of complementary metathesisable groups,
- subjecting the peptide to cross-metathesis to form a peptide with an unsaturated dicarba bridge between the amino acids that bore the third pair of complementary metathesisable groups, and
- optionally subjecting the unsaturated dicarba bridge to hydrogenation.
- Each of these techniques for the synthesis of peptides with one or more intramolecular bridges may be combined with additional steps for the formation of one or more intramolecular disulfide bridges.
- In each of these techniques it is also preferred that the unblocking reaction specific to the second pair of complementary metathesisable groups comprise cross-metathesis with a 1,3-butadiene free disposable olefin.
- It will be appreciated that if a peptide sequence is added later through an intermolecular bridge, the corresponding metathesisable groups on that peptide need not be blocked—as they can be added to the reaction at the time of cross-metathesis, after the unblocking of the groups on the resin-supported peptide.
- The present invention also provides for a compound produced by the method of the invention. The compound may be a peptide with at least one dicarba bridge, or may be any other organic compound with a dicarba bridge. Salts, solvates, derivatives, isomers and tautomers are encompassed in this context.
- Possible products include the dicarba analogues of cystine-containing peptides. Dicarba analogues refers to peptides contain the same amino acid sequence as the native peptide, but with one or more of the bridged cysteine-amino acid residue pairs substituted with amino acids bearing a dicarba bridge. “Native” is a term used to refer to the natural or synthetic analogue of a natural peptide—to be distinguished from the dicarba analogue being synthesised. Bis- and higher dicarba analogues are of particular interest, in view of the difficulty in synthesising such compounds. Examples are the dicarba analogues of Conotoxin ImI presented in
FIG. 6.4 . These include the fully dicarba-substituted analogues (the final three compounds in that figure) and the partial dicarba analogues (identified as “hybrids” inFIG. 6.4 ). Other suitable terminology is the mono-dicarba analogues (in which one disulfide bridge is replaced with a dicarba bridge), and the bis-dicarba analogues (two replaced). Thus, the present application also relates to dicarba analogues of Conotoxin, including the bis-dicarba, cystino-dicarba and higher-dicarba analogues. - In
FIG. 6.4 , the residue between the bridges is represented as “Hag”—based on its synthesis via this amino acid, although the double bond of Hag is no longer present. In some cases the new bridge is unsaturated and bears a new double bond; in other cases the bridge is saturated. If the peptide was synthesised via another amino acid, such as crotyl glycine (Crt), Crt would appear in place of Hag. In fact, the peptides are identical irrespective of whether they were synthesised via one of these amino acids or the other, as the dicarba bridge is all that remains from those starting amino acids. Accordingly, the amino acid indicated in the formula for the peptide should not be read as limiting the peptide to one made specifically through that amino acid. Sub (representing the amino acid suberic acid, which has the cyclised side chain —(CH2)4—) could also have been used to represent the same peptide. - “Conotoxin” is used in its broadest sense to refer to the peptides or peptide fragments that are present in the venom of cone snails of the genus Conus (Conidae). All species which are encompassed within this genus [class] are contemplated, including the species Conus imperialis, Conus geographus, Conus textile, Conus amadis, Conus tulipa, Conus marmoreus, Conus lynceus, Conus armadillo, Conus geographus and so forth. The peptides within this class include natural and synthetic peptides, and derivatives of the naturally-occurring peptides and peptide fragments. Conotoxins are classified according to their receptor subtype specificity and the arrangement of disulfide bonds and resultant loop sizes. The paralytic components of the venom (the conotoxins) that have been the focus of recent investigation are the α-, ω- and μ-conotoxins. All of these conotoxins act by preventing neuronal communication, but each targets a different aspect of the process to achieve this. The α-conotoxins target nicotinic ligand gated channels, and the μ-conotoxins target the voltage-gated sodium channels and the ω-conotoxins target the voltage-gated calcium channels. Of particular interest here are the α-, χ- and ω-conotoxins, which contain two or three disulfide bridges, although μ-conotoxins, δ-conotoxins, κ-conotoxins π-conotoxins and conatokins are also relevant. The conotoxins are generally between 12 and 30 amino acid residues in length.
- The salts of compounds are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
- In addition, some of the compounds may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the invention.
- By “derivative” is meant any salt, hydrate, protected form, ester, amide, active metabolite, analogue, residue or any other compound which is not biologically or otherwise undesirable and induces the desired pharmacological and/or physiological effect. Preferably the derivative is pharmaceutically acceptable.
- The term “tautomer” is used in its broadest sense to include compounds which are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.
- The term “isomer” is used in its broadest sense and includes structural, geometric and stereo isomers. As the compounds that may be synthesised by these techniques may have one or more chiral centres, it is capable of existing in enantiomeric forms.
- The present applicant has synthesised amino acids that are particularly useful as they enable the formation of a dicarba bridge when included in a peptide sequence. These amino acids include prenyl glycine in which the amino group is protected with a base-removable carbamate-protecting group. A particular example of this compound is Fmoc-protected prenyl glycine. Fmoc-protected prenyl glycine is a protected, blocked olefin-containing amino acid, suitable for forming the “second” of the dicarba bridges in a peptide, and its synthesis is achieved through the use of a specific reagent.
- Fmoc-protected prenyl glycine requires preparation through the cross-metathesis of Fmoc-protected allyl glycine with 2-alkyl-2-butylene (such as 2-methyl-2-butylene) in the presence of a cross-metathesis catalyst. The reaction is not complete when isobutylene is used as the olefin in the reaction. The reaction is suitably conducted at a pressure above 5 psi—preferably at 8 psi or above—for instance at about 10 psi.
- This section describes a solution phase model study for the development of a methodology that enables the regioselective formation of dicarba isosteres of cystine bonds. We investigated a sequence of ruthenium-catalysed metathesis and rhodium-catalysed hydrogenation reactions of non-proteinaceous allylglycine derivatives to achieve high yielding and unambiguous formation of two dicarba bridges. This theory can also be applied to the synthesis of non-peptide compounds with 2 or 3 dicarba bridges.
- Our initial strategy planned to capitalise on the use of α-N-acyl-dienamide 57, a masked precursor to allylglycine derivatives.118,119 We devised a strategy involving a double metathesis-hydrogenation sequence (Scheme 4.1). This required a selective ring closing metathesis of allylglycine units in the presence of dienamide functionalities. Grubbs et al. have previously reported that selective cross metathesis can be accomplished with olefins of varying reactivity.130,182 Terminal olefins such as allylglycine undergo rapid homodimerisation with both Grubbs' catalyst120 and second generation Grubbs' catalyst,121 whereas the electron-deficient α-N-acyl-dienamide 57 should be considerably less reactive. Subsequent asymmetric hydrogenation of the dienamide moieties would lead to reactive allylglycine units which could undergo ring closing metathesis to produce the second carbocycle. The final step in this catalytic sequence involves hydrogenation of the unsaturated carbocycles, if required, to afford the saturated cystine isosteres.
- In order to validate the proposed strategy, we needed to show that: i) the dienamide 57 would not react under conditions required for the ring closing metathesis of allylglycine residues, ii) asymmetric hydrogenation of the dienamide 57 would proceed in a highly regioselective and stereoselective manner, iii) ring closing metathesis of the resulting allylglycine units would proceed in the presence of an unsaturated carbocycle (without resulting in mixed cross metathesis products), and iv) the unsaturated carbocycles could be reduced to afford saturated dicarba bridges. We therefore conducted a series of independent experiments that would serve as a model to the peptide system.
- The dienamide 57 was synthesised according to a literature procedure reported by Teoh et al.119 from a Homer-Emmons olefination of a phosphonate ester 39 and an α,β-unsaturated aldehyde 58 (Scheme 4.2).
- The phosphonate, methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39, was prepared in three steps from commercially available acetamide 34 and glyoxylic acid 41.
- A mixture of commercially available acetamide 34 and glyoxylic acid 41 was heated at reflux in acetone to give pure N-acetyl-2-hydroxyglycine 42 as a viscous yellow oil in quantitative yield. The 1H n.m.r. spectrum supported formation of the α-hydroxyglycine derivative 42 with the appearance of a methine (H2) doublet and broad amide (NH) doublet at δ 5.39 and δ 8.65 respectively. Spectroscopic data were in agreement with those reported in the literature.195
- Treatment of N-acetyl-2-hydroxyglycine 42 with a catalytic amount of concentrated sulfuric acid in methanol furnished methyl N-acetyl-2-hydroxyglycinate 43 in 60% yield. These reaction conditions converted the carboxylic acid to the methyl ester and the hydroxyl functional group to methyl ether.
- Modification of the reported work-up procedure led to a significantly improved yield to that reported in the literature (32%).196 The presence of two new methoxyl peaks in the 13C n.m.r. spectrum at δ 53.0 and δ 56.8 and the corresponding methyl singlets in the 1H n.m.r. spectrum at δ 3.47 and δ 3.82 supported formation of the desired product 43. Spectroscopic data were also in agreement with those reported in the literature.196
- The final step in the synthesis of methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)-acetate 39 involved reaction of methyl N-acetyl-2-hydroxyglycinate 43 with phosphorous trichloride to produce the intermediate α-chloro ester. Nucleophilic attack of the newly introduced chlorine substituent with trimethyl phosphite gave phosphonate ester 39 as a colourless solid in low yield (14%). The high solubility of the ester in water initially led to poor mass recovery. The use of continuous extraction partially overcame this problem and led to isolation of the product in satisfactory yield (44%).
- The 1H n.m.r. spectrum confirmed formation of the target compound 39 with the appearance of a doublet of doublets attributed to the methine (H2) proton coupling to the phosphorous (J=22.2 Hz) and amide proton (J=8.8 Hz). The 13C n.m.r. spectrum displayed similar behaviour with the methine (C2) peak appearing as a doublet with large coupling to the vicinal phosphorous atom (J=146.8 Hz). The melting point of the isolated solid (89-91° C.) was consistent with that reported in the literature (88-89° C.).197
- (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 was synthesised by Homer-Emmons olefination of methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39 with commercially available acrolein 58 in the presence of tetramethylguanidine (TMG) (Scheme 4.4). Hydroquinone was added to prevent polymerisation of acrolein 58 and was found to be critical to the success of this reaction. The reaction requires the addition of base to a solution of phosphonate 39 in tetrahydrofuran to generate the carbanion 45, which was then reacted with aldehyde 58 to afford the dienoate 57 as an off-white solid in 85% yield (Scheme 4.4).
- The 1H n.m.r. spectrum of the product supported formation of the dienamide 57 with the appearance of signals corresponding to a new terminal olefin. Doublets at δ 5.49 and δ 5.61 for H5-E and H5-Z respectively, and an olefinic methine (H4) multiplet at δ 6.47 were consistent with formation of dienamide 57. The melting point of the isolated solid (60-62° C.) was also in agreement with that reported in the literature (61-63° C.).119
- Our group have demonstrated that high regioselectivity and enantioselectivity can be achieved in the asymmetric hydrogenation of dienamide esters. In this case, hydrogenation of dienamide 57 was effected with Rh(I)—(S,S)-Et-DuPHOS under 30 psi of hydrogen in benzene for 3 hours (Scheme 4.5). Over-reduction of the terminal olefinic bond was minimal (<3% 59) under these mild conditions. The (S)-configuration was determined based on literature assignment for the same transformation118 and a comparative optical rotation sign to that reported in the literature for (2S)-methyl 2-N-acetylaminopent-4-enoate 21a.208
- Asymmetric hydrogenation of dienamide 57 was also performed with Rh(I)—(R,R)-Et-DuPHOS to facilitate enantiomeric excess assessment. The reaction proceeded in quantitative conversion and <5% over-reduced product was detected. Chiral GC indicated the reactions proceeded with excellent enantioselectivity (95% e.e.).
- 1H n.m.r. spectroscopy showed the replacement of an olefinic methine (H3) doublet at δ 7.05 with a methylene (H3) multiplet at δ 2.43-2.62. The 13C n.m.r. spectrum also displayed new methine (C2) and methylene (C3) peaks at δ 51.8 and δ 36.5 respectively. Spectroscopic data were in agreement with those reported in the literature.119
- Homodimerisation is a type of cross metathesis in which an olefin self-couples. Conveniently, the only byproduct is a low molecular weight volatile olefin which is most commonly ethylene (Scheme 4.6).
- The mechanism involves an intermolecular exchange of alkylidene fragments between the metal-carbene catalyst and the reacting olefin. An unstable metallocyclobutane intermediate then decomposes to release the homodimer and a volatile olefinic byproduct (Scheme 4.7).
- Quantitative homodimerisation of allylglycine derivative 21a was achieved using Grubbs' catalyst in dichloromethane heated at reflux (Scheme 4.8). Purification of the crude product by flash chromatography gave the target compound, (2S,7S)-
dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60, as a brown oil in 88% yield. - High resolution mass spectrometry confirmed formation of the desired
product 60 with the appearance of a molecular ion plus sodium peak at m/z 337.1375 for the expected molecular formula (C14H22N2NaO6). In addition, the 13C n.m.r. spectrum displayed a new olefinic methine (C4, 5) peak at δ 128.8, whilst the terminal and methine olefinic (C5 and C4) peaks of the starting material 21a were absent. - The solution phase dimerisation of the allylglycine unit 21a is analogous to ring closing metathesis of allylglycine sidechains in a peptide (
Step 1, Scheme 4.1). In order to regioselectively construct multiple dicarba bonds within a peptide, via the strategy shown in Scheme 4.1, the dienamide 57 must not react under the conditions used for cross metathesis of allylglycine units 21a (Scheme 4.8). - The dienamide 57 was therefore subjected to analogous dimerisation conditions to those described above for allylglycine 21a. 1H n.m.r. spectroscopy confirmed complete recovery of the starting olefin 57 with no evidence of the dimerised dienoate 61 (Scheme 4.9). These results were very encouraging and supported our postulate that the dienamide 57 would be electronically compromised and therefore inert to metathesis (
Step 1, Scheme 4.1). - Subsequent asymmetric hydrogenation of the dienoate 57 would activate the olefin to metathesis by producing a reactive allylglycine unit 21a (
Step 2, Scheme 4.1). This hydrogenation proceeds with excellent stereoselectivity (>95% e.e.) and regioselectivity (<3% over-reduction) (Section 4.1.1) as it relies on chelation of the asymmetric Rh(I)-catalyst to the enamide olefin and amide carbonyl group. The terminal C═C bond does not chelate to the catalyst and is therefore not reduced under these conditions. Similarly, the newly formed C═C bond, generated via cross metathesis inStep 1, would be inert to this catalyst. - In our strategy, the next step involved ring closing metathesis of allylglycine units in the presence of an unsaturated carbocycle (
Step 3, Scheme 4.1).The solution phase model study therefore required the dimerisation of allylglycine in the presence of an unsaturated dimer (Scheme 4.10). A differentially protected allylglycine derivative 62 was synthesised to facilitate unambiguous assessment of cross metathesis selectivity. - The benzoyl-protected allylglycine derivative 62 was prepared via catalytic asymmetric hydrogenation of the dienamide 63. The hydrogenation precursor 63 was synthesised by Horner-Emmons olefination of the phosphonate ester 64 which was isolated in three steps from commercially available benzamide 35 and glyoxylic acid 41 (Scheme 4.11).
- A mixture of commercially available benzamide 35 and glyoxylic acid 41 was heated at reflux in acetone to give pure N-benzoyl-2-hydroxyglycine 65 as a colourless solid in quantitative yield (Scheme 4.12). The 1H n.m.r. spectrum supported formation of the α-hydroxyglycine derivative 65 with the appearance of a methine (H2) doublet and broad amide (NH) doublet at δ 5.60 and δ 9.26 respectively. Spectroscopic data were in agreement with those reported in the literature.209
- Treatment of N-benzoyl-2-hydroxyglycine 65 with a catalytic amount of concentrated sulfuric acid in methanol furnished methyl N-benzoyl-2-methoxyglycinate 66 in 87% yield (Scheme 4.13). These reaction conditions converted the carboxylic acid to the methyl ester and the hydroxyl functional group to the methyl ether.
- The presence of two new methoxyl peaks in the 13C n.m.r. spectrum at δ 53.2 and δ 57.0 and the corresponding methyl singlets in the 1H n.m.r. spectrum at δ 3.54 and δ 3.85 supported formation of the desired product 66. Spectroscopic data were in agreement with those reported in the literature.209
- Reaction of methyl N-benzoyl-2-methoxyglycinate 66 with phosphorous trichloride and trimethyl phosphite in toluene at 70° C. gave the phosphonate ester 64 in 76% yield (Scheme 4.14). The appearance of a methine doublet of doublets (H2) at δ 5.47 was consistent with vicinal phosphorous coupling and was in agreement with data reported in the literature.210
- (2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 was synthesised by Horner-Emmons olefination of methyl 2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 with commercially available acrolein 58 in the presence of tetramethylguanidine (TMG) (Scheme 4.15). The reaction proceeded through a nucleophilic intermediate 67 which reacted with acrolein 58 to afford the dienoate 63 as colourless needles in 80% yield.
- The 1H n.m.r. spectrum displayed a new terminal olefin doublet of doublets at δ 5.50 and δ 5.64 corresponding to H5-E and H5-Z respectively in addition to a well-defined methine (H4) doublet of doublet of doublets at δ 6.56. Spectroscopic data were in agreement with those reported in the literature.211
- The final step in the synthesis of (2S)-methyl 2-N-benzoylaminopent-4-enoate 62 involved asymmetric hydrogenation of the dienamide 63.† Use of Rh(I)—(S,S)-Et-DuPHOS under 30 psi H2 in benzene gave the allylglycine derivative 62 with excellent enantioselectivit‡ (100% e.e., Scheme 4.16). Approximately 7% of the over-reduced product 68 was obtained under these conditions and attempts to separate allylglycine 62 from 68 were unsuccessful. The contaminated sample of allylglycine 62 was used in subsequent reactions as the presence of the inert impurity 68 would not interfere in the catalytic strategy. † The benzoyl-protected olefin 62 can also be prepared in two steps from commercially available L-allylglycine ((2S)-2-aminopent-4-enoic acid).‡ Asymmetric hydrogenation of the dienamide 63 with Rh(I)—(R,R)-Et-DuPHOS was performed in order to facilitate enantiomeric excess determination. Chiral GC confirmed that the (R)- and (S)-allylglycine derivatives 62 were produced in 100% e.e.
- Formation of allylglycine 62 was supported by 13C n.m.r. spectroscopy which showed the replacement of an olefinic methine (C3) peak with a new methylene signal at δ 36.8 and a methine (C2) peak at δ 52.1. Spectroscopic data were in agreement with those reported in the literature.212
- The benzoyl-protected allylglycine unit 62 was quantitatively homodimerised under general cross metathesis conditions using Grubbs' catalyst (Scheme 4.17). The loss of ethylene drives the metathesis reaction to completion.
- Purification by flash chromatography furnished (2S,7S)-
dimethyl 2,7-N,N′-dibenzoylaminooct-4-enedioate 69 as a pale brown solid in 82% yield. 1H n.m.r. spectroscopy supported synthesis of the dimer 69 with the replacement of terminal olefin peaks by a new methine (H4, 5) triplet at δ 5.49. The accurate mass spectrum also displayed a molecular ion plus sodium peak at m/z 461.1695 which is consistent with that expected for the molecular formula C24H26N2NaO6. - With the benzoyl-protected allylglycine 62 in hand and characterisation of its dimer 69 complete, we attempted the cross metathesis of allylglycine 62 in the presence of the unsaturated N-acetyl-protected allylgycine dimer 60 (
Step 3, Scheme 4.4). - Cross metathesis of allylglycine derivative 62 in the presence of
unsaturated dimer 60 proceeded with Grubbs' catalyst to afford dimer 69 (Scheme 4.18). No mixed cross metathesis product 70 was observed. However, use of the more reactive metathesis catalyst, second generation Grubbs' catalyst, did lead to a mixture of cross metathesis products, 69, 70 and recovereddimer 60. The complicated 1H n.m.r. spectrum did not allow the distribution of products to be quantified but mass spectrometry confirmed the presence ofhomodimers 60 and 69 and the mixed cross metathesis product 70. - These results indicated that in a peptide application of this strategy (
Step 3, Scheme 4.1), selective cyclisation of the allylglycine units will only be successful in the presence of Grubbs' catalyst and the use of the more reactive second generation Grubbs' catalyst must be avoided. With successful completion ofStep 3, we moved to the last step of the strategy (Step 4, Scheme 4.1). - The final step in the model sequence involved reduction of the
unsaturated dimers 60 and 69 to give the corresponding saturated dicarba bridges 71 and 72. Homogeneous hydrogenation ofdimers 60 and 69 with Wilkinson's catalyst, Rh(I)(PPh3)3Cl, under mild experimental conditions, gave the saturated diaminosuberic acid derivatives 71 and 72 in excellent yields (>99%) (Scheme 4.19). We employed a homogeneous catalyst in order to facilitate the on-resin application of this hydrogenation which would otherwise be complicated by the more commonly employed heterogeneous systems such as palladium on charcoal. - Formation of the saturated dimers 71 and 72 was supported by spectroscopic analysis which displayed new methylene proton (H3, 4) and carbon (C3, 4) signals in the 1H and 13C n.m.r. spectra respectively.
- These results looked very promising: We had successfully completed all four steps in our devised synthesis (Scheme 4.1). However, our attempts to dimerise allylglycine 21a in the presence of dienamide 57 were unsuccessful with both first and second generation Grubbs' catalysts (Scheme 4.20). The inclusion of dienamide 57 also hampered dimerisation of benzoyl-protected allylglycine 62 and led to complete recovery of the starting dienoate 57 and allylglycine unit 62.
- 1H n.m.r. binding studies between the catalyst and dienamide 57 (ratio of 1:1) showed that the dienamide 57 rapidly coordinated to the ruthenium centre forming a ruthenium-vinylalkylidene complex 73 (spectrum a in
FIG. 2 ). Within 60 minutes, complex 73 had diminished and anew ruthenium species 74 was generated (spectrum b inFIG. 2 ). The second species is postulated to involve coordination of the ester carbonyl group to the ruthenium centre and liberation of a tricyclohexylphosphine ligand 75. Formation of complex 74 was complete within 2 hours and was stable and unreactive over 18 hours (spectrum c in FIG. 2).213 Unfortunately, attempts to isolate this complex 74 were unsuccessful. - Furthermore, attempts to regenerate the dienamide 57 from the ruthenium-
carbonyl chelate 74 via reaction with ethyl vinyl ether and formation of the Fischer-type carbene complex,214 failed due to conjugate addition of liberated tricyclohexylphosphine 75 to the dienamide substrate 57. This highlighted the sensitivity of acrylate 57 to N- and P-based nucleophiles and potential problems that could arise during peptide synthesis, where piperidine is routinely used to facilitate Fmoc-cleavage from residues prior to coupling. - Although the dienamide 57 was unexpectedly reactive to Grubbs' catalyst, the proposed strategy showed potential. Solution phase experiments with Steps 2-4 (Scheme 4.21) were not problematic and indicated that multiple dicarba bond formation was indeed feasible via a modified strategy. The first step, however, required revision. We postulated that the presence of a substituent at the olefinic terminus of the dienamide substrate might impede binding to the metathesis catalyst and therefore allow the ring closing metathesis of the more reactive allylglycine sidechains to proceed.
- A revised strategy was investigated centering on the use of non-proteinaceous, terminally functionalised allylglycine units. This modified route involved: i) metathesis of allylglycine units in the presence of a terminal-phenyl substituted dienamide 76, and ii) subsequent hydrogenation of the dienamide 76 to yield a more reactive olefin 77 for the second ring closing metathesis (Scheme 4.22). We postulated that the presence of a phenyl substituent at the olefin terminus might impede binding of the metathesis catalyst and circumvent the problems experienced in the first strategy. The solution phase model studies of this revised strategy therefore commenced with the synthesis of the phenyl-substituted dienamide 76.
- The dienamide 76 was prepared according to a procedure by Burk et al.117 from a Horner-Emmons olefination of methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)-acetate 39 and commercially available trans-cinnamaldehyde 78 (Scheme 4.23). The phosphonate 39 was prepared in three steps from commercially available acetamide 34 and glyoxylic acid 41.
- The dienamide 76 was isolated as an off-white solid in 74% yield. Mass spectrometry supported formation of the dienoate 76 with a molecular ion plus proton peak at m/z 246.2 which is consistent with that expected for molecular formula C14H16NO3. Spectroscopic data were in agreement with those reported in the literature.117
- 1H n.m.r. binding studies of a 1:1 mixture of Grubbs' catalyst and dienamide 76 showed no ruthenium-vinylalkylidene formation. Hence, this suggested that the poor chelating properties of the modified dienamide 76 to Grubbs' catalyst should now facilitate high yielding homodimerisation of allylglycine 21a into its dimer 60 (Scheme 4.24).
- Surprisingly, homodimerisation of 21a to 60 was found to proceed but with poor conversion (28%). This suggested that the dienamide 76 was still capable of influencing the metathesis cycle. Hopeful that this would later be rectified through modification of metathesis conditions, we continued to investigate subsequent steps of the proposed strategy.
- Rh(I)-DuPHOS-catalysed asymmetric hydrogenation of dienamide 76 under mild conditions (75 psi H2) gave (2S)-methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 in 99% e.e. (Scheme 4.25). Formation of the desired phenyl-substituted enamide 77 was confirmed by spectroscopic analysis which was in agreement with literature data.117
- Disappointingly, cross metathesis of 77 using Grubbs' catalyst was unsuccessful. After 13 hours, 1H n.m.r. spectroscopy showed no conversion to the desired
homodimer 60. Conditions to facilitate the required cross metathesis were found, however, using a 5 mol % solution of second generation Grubbs' catalyst in dichloromethane (Scheme 4.26). A modest conversion (44%) to the expectedhomodimer 60 was achieved. In spite of this promising result, this chemistry was not investigated further since the requirement for the more reactive second generation Grubbs' catalyst would render the previously formed unsaturated carbocycle vulnerable to further cross metathesis. Mixed cross metathesis products would therefore result (Section 4.1.3.3). - Selective reduction of the first-formed unsaturated carbocycle prior to the second metathesis reaction would, however, eliminate the chance of mixed cross metathesis (
Step 2, Scheme 4.27). We therefore subjected the phenyl substituted diene 76 to the hydrogenation conditions previously developed for the hydrogenation of theunsaturated dimer 60. Unfortunately, these conditions resulted in a 1:4 mixture of olefin 77: saturated derivative 79 (Scheme 4.28). - This disappointing result is not without literature precedent. The rate of olefin reduction by Wilkinson's catalyst is profoundly influenced by steric hindrance about the C═C double bond, but related reductions involving styrene have previously shown that electronic effects override these steric effects and that the aromatic substituent enhances the rate of reduction.215,216
- The failure of this second strategy led to a final revision and the discovery of a strategy which would enable the selective hydrogenation of an unsaturated carbocycle in the presence of a deactivated but potentially metathesis-active olefin. We decided to capitalise on the slow reactivity of trisubstituted olefins to Wilkinson's hydrogenation and their reduced reactivity to metathesis. 1,1-Disubstituted olefins, for example, do not undergo homodimerisation and only react with more reactive olefins.130,182 This differential reactivity would therefore facilitate the cross metathesis of allylglycine units and subsequent hydrogenation without interference from the 1,1-disubstituted olefin residues. A simple transformation then renders the trisubstituted olefin more reactive to metathesis and facilitates the formation of the second carbocycle (Scheme 4.29).
- The
prenyl olefin 19 was prepared via asymmetric hydrogenation of the corresponding dienamide 20. Theprenylglycine derivative 19 was isolated in quantitative yield and excellent enantioselectivity (Scheme 4.30). - This
prenylglycine unit 19 was subjected to the hydrogenation conditions that quantitatively reduce thedimer 60 to the saturated analogue 71 (Scheme 4.19) and encouragingly, 94% of the startingenamide 19 was recovered (Scheme 4.31). This was a very promising result which prompted us to further investigate cross metathesis reactions involving thissubstrate 19. Furthermore, we envisaged that incorporation of thisunit 19 into a peptide via solid phase peptide synthesis (SPPS) would be straightforward. - Cross metathesis of allylglycine unit 21a into
dimer 60 in the presence of the prenyl enamide 19 proceeded smoothly with quantitative conversion (Scheme 4.32); the startingprenyl enamide 19 was recovered unchanged. - The reduced reactivity of
prenylglycine 19 enabled the dimerisation of allylglycine 21a and the selective hydrogenation of theresultant homodimer 60. The next step in the strategy involves activation of the dormant olefin 19 (Step 3, Scheme 4.29). This can be achieved by cross metathesis with ethylene via a more active ruthenium alkylidene. - The
prenyl compound 19 was subjected to ethenolysis to convert it to the more reactive allylglycine derivative 21a (Scheme 4.33). Exposure of 19 to 20 mol % of Grubbs' catalyst under an atmosphere of ethylene resulted in the recovery of the startingolefin 19. Use of the more reactive 2nd generation Grubbs' catalyst at higher reaction temperature (50° C.) and ethylene pressure (60 psi) still led to only poor conversions to 21a (<32%). - We postulated that this result may be due to the unstable nature of the in situ generated ruthenium-methylidene intermediate 48 at elevated temperature (50° C.),202-204 or unfavourable competition between the rising concentration of terminal olefins and 21a for binding to the ruthenium catalyst.217
- In order to circumvent this problem, the
prenyl enamide 19 was instead exposed to an atmosphere of cis-2-butene (15 psi) thereby facilitating the catalysis via the more stable ruthenium-ethylidene complex 49. Butenolysis of 19 in the presence of 5 mol % second generation Grubbs' catalyst gave the expected crotylglycine derivative 81 with quantitative conversion (Scheme 4.33). - (2S)-Methyl 2-N-acetylaminohex-4-enoate 81 was isolated as a brown oil in 84% yield after flash chromatography. The 1H n.m.r. spectrum showed the replacement of the olefinic methine (H4) triplet in the starting
prenyl compound 19 with new olefinic methine (H4, 5) multiplets at δ 5.49 and δ 5.24 respectively. Spectroscopic data were also in agreement with those reported in the literature.117,119 - Interestingly, the purity of the 2-butene was found to be critical to the success of the cross metathesis reaction. When butenolysis reactions were conducted with a less expensive, commercially available mixture of cis- and trans-2-butene, only a trace of the butenolysis product was detected. Gas chromatographic analysis of the isomeric butene mixture showed that it was contaminated with 2.6% butadiene while none was detected in the pure cis-2-butene.218 The addition of butadiene (2%) to cis-2-butene inhibited formation of the butenolysis product while a cis+trans mixture (30:70) of 2-butene, free of butadiene,† led to quantitative conversion to the expected cross metathesis product. These results strongly suggested that butadiene was poisoning the metathesis catalyst. Grubbs et al. have previously reported that butadiene can react with the ruthenium-benzylidene catalyst to produce a vinyl alkylidene which is inactive for acyclic metathesis reactions.219 † The cis+trans-2-butene mixture (30:70)free of butadiene was obtained by isomerisation of cis-2-butene with benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro[bis(3-bromo-pyridine)]ruthenium at −5° C.218
- This activated crotylglycine derivative 81 was readily cross metathesised to the expected
homodimer 60 with 5 mol % of second generation Grubbs' catalyst in dichloromethane (Scheme 4.34). Spectroscopic data were in agreement with those previously reported (Section 4.1.2). - Finally, an equimolar mixture of
olefins 62 and 19 was exposed to a tandem sequence of the previously described five homogeneous catalytic reactions: i) dimerisation of allylglycine 62, ii) hydrogenation of the resultant homodimer 69, iii) activation ofprenylglycine 19, iv) dimerisation of the activated crotylglycine derivative 81 and v) hydrogenation of theresultant homodimer 60. Solvent removal and subsequent 1H n.m.r. analysis was performed on the crude product mixture after each transformation. The catalytic sequence resulted in quantitative conversion of the reactive substrate in each step and ultimately yielded diaminosuberic acid derivatives 71 and 72 as the only isolated products in 84 and 70% yield respectively (Scheme 4.35). - In conclusion, these model studies demonstrate that through the combination of homogeneous catalysis and judicious selection of non-proteinaceous allylglycine residues of varying reactivity, a highly efficient, unambiguous and regioselective synthesis of dicarba analogues of multi-cystine containing peptides may be achievable. The methodology is also amenable to natural product and polymer synthesis or wherever selective carbon-carbon bond formation is required.
Section 6 investigates the application of this methodology to synthetic and naturally occurring peptides. - The selective formation of multiple dicarba bonds in complex molecules is a significant synthetic challenge. In
section 4, we devised a strategy for a solution phase regioselective synthesis of two dicarba bridges. This chapter describes a catalytic strategy for the regioselective construction of three dicarba bridges in solution by selective and successive metathesis-hydrogenation transformations. - In the preceding chapter we achieved regioselective C—C bond formation through the use of olefinic substrates possessing tuneable reactivity and highly chemo- and stereoselective catalysts. The varying reactivity of allylglycine and prenylglycine units towards metathesis and hydrogenation has been previously described (Chapter 4). We postulated that the steric and particularly electronic effects of a
prenylglycine dienoate 82 would render it inert to metathesis and Wilkinson's hydrogenation. Two dicarba bridges can therefore be constructed in the presence of this inert olefin (Scheme 5.1). The diene can then be activated in two simple steps, the first of which involves a catalytic asymmetric hydrogenation to give optically pure prenylglycine. We have already demonstrated the facile activation of the prenyl sidechain by cross metathesis with either ethylene or cis-2-butene to give the corresponding allyl- or crotylglycine′ derivative respectively. The resultant activated olefin can readily undergo homodimerisation to give an unsaturated dimer which can be reduced to afford the saturated dicarba bridge. The final product mixture would ultimately contain three different diaminosuberic acid derivatives where the selective C—C bond formation would represent the formation of a dicarba analogue of a tricystine-containing peptide (Scheme 5.1). In order to validate the proposed strategy we conducted a series of solution phase reactions. - A
metathesis triplet -
TABLE 5.1 Reaction Sequence for the Construction of Three Dicarba Bridgesa Step 1: CM-H Grubbs' catalyst Step 2: Wilkason's Hydrogena- tion Step 3: CM 2nd gen. Grubbs' catalystStep 4: CM- H 2nd gen. Grubbs' catalystStep 5: Wilkinson's Hydrogena- tion Step 6: Rh(I)- DuPHOS Hydrogena- tion Step 7: CM 2ndgen. Grubbs' catalystStep 8: CM- H 2nd gen. Grubbs' catalystStep 9: Wilkinson's Hydrogena- tion Substrates C═C C—C Act C═C C—C Act Act C═C C—C Products Sidechain Reactivity Summary of Activity ✓ ✓ — — — — — — — Terminal allylic olefin. No activation required. X X ✓ ✓ ✓ — — — — Trisubstituted olefin. Activated via CM with 2-butene. X X X X X ✓ ✓ ✓ ✓ Hindered extended acrylamide olefin. Activated via i) asymmetric hydrogenation and ii) CM with 2-butene. a ✓= Reactive olefin, X = Unreactive olefin, — = Unreactive dicarba bridge, Act = Olefin activation step, CM-H = Cross metathesis-homodimerisation, CM = Cross metathesis - Three different N-acyl protecting groups were employed to facilitate unambiguous assessment of cross metathesis selectivity. A mixture of a p-nitrobenzoyl-protected
allylglycine derivative 83, an acetyl-protectedprenylglycine unit 19 and a benzoyl-protectedprenylglycine dienamide 82 gave adequate separation of characteristic peaks in the 1H n.m.r. spectrum (FIG. 3 ) to enable reaction monitoring of Steps 1-9. Importantly, the protecting groups on the amino group do not affect the mechanistic course of the reaction sequence. - The solution phase studies therefore commenced with preliminary experiments on the
diene 82 to ensure it was inert to metathesis and Wilkinson's hydrogenation. - The
dienamide 82 was synthesised by Horner-Emmons olefination of methyl 2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 with commercially available 3-methyl-2-butenal 40 and tetramethylguanidine (TMG) (Scheme 5.2), as described for several dienamides in this application - Methyl 2-N-benzoylamino-5-methylhexa-2,4-
dienoate 82 was isolated as an off-white solid in 73% yield. Formation of theprenylglycine dienamide 82 was supported by 13C n.m.r. spectroscopy which displayed new olefinic methyl peaks at δ 19.3 and δ 27.1 respectively, in addition to characteristic olefinic methine (C3, 4) and quaternary (C2, 5) peaks. A molecular ion plus proton peak at m/z 260.1282 in the accurate mass spectrum was consistent with the molecular formula C15H18NO3 and also supported formation of thedienamide 82. - The
dienamide 82 was subjected to homodimerisation conditions with second generation Grubbs' catalyst (Scheme 5.3). 1H n.m.r. spectroscopy confirmed complete recovery of the startingolefin 82 with no evidence of the dimerised dienoate 84. This result supported our postulate thatdiene 82 is electronically and sterically compromised and therefore inert to metathesis. - Our proposed reaction sequence then required the reduction of an unsaturated dicarba bridge in the presence of a diene moiety (
Step 2, Scheme 5.1). Thedienamide 82 was therefore subjected to the hydrogenation conditions that quantitatively reduce unsaturated dimers to their saturated analogues (Wilkinson's catalyst, 50 psi H2). Encouragingly, the reduced prenyl compound 85 was not observed and the startingolefin 82 was recovered unchanged (Scheme 5.3). - Finally, the
diene 82 was exposed to metathesis conditions used to activateprenylglycine 19 by conversion to the crotyl derivative 81 (cis-2-butene, second generation Grubbs' catalyst, Scheme 5.3). Again 1H n.m.r. spectroscopy indicated that thedienamide 82 was inert to these conditions. The startingolefin 82 was recovered unchanged with no evidence of the potential cross metathesis product 86. - Activation of the
dienamide 82 was initiated with a Rh(I)-Et-DuPHOS-catalysed asymmetric hydrogenation to give the prenylglycine derivative 87 in excellent yield and enantioselectivity (100% e.e.) (Scheme 5.4). - The replacement of olefinic methine (H3, 4) proton peaks in the 1H n.m.r. spectrum with new methylene (H3) and olefinic (H4) multiplets at δ 2.52-2.76 and δ 5.08 confirmed formation of the prenylglycine residue 87. Over-reduction of the terminal double bond was not observed under these conditions.
- The second activation step involved treatment of the prenyl olefin 87 with 5 mol % second generation Grubbs' catalyst and cis-2-butene (15 psi) to yield the crotylglycine derivative 88 (Scheme 5.4). The reaction proceeded with quantitative conversion as indicated by 1H n.m.r. and 13C n.m.r. spectroscopic analysis. The accurate mass spectrum also displayed a molecular ion plus proton peak at m/z 248.1284 which is consistent with that expected for the molecular formula C14H18NO3.
- The third olefin in the metathesis triplet is the
allylglycine derivative 83. Reaction of the hydrochloride salt of allylglycine methyl ester 51 with p-nitrobenzoyl chloride 89 and triethylamine in a mixture of dichloromethane:diethyl ether gave the protectedallylglycine residue 83 in 99% yield (Scheme 5.5). - The 1H n.m.r. and 13C n.m.r. spectra supported formation of the protected
allylglycine 83 with the downfield shift of the methine (H2) doublet of triplets at δ 4.90 and the introduction of aromatic resonances at δ 7.95 (H2′,6′) and δ 8.30 (H3′,5′). - The
allylglycine derivative 83 was quantitatively dimerised with Grubbs' catalyst in dichloromethane heated at reflux (Scheme 5.6). Formation of the dimer 90 was supported by 1H and 13C n.m.r. spectroscopic analysis which displayed signals due to the new olefinic methine proton (H4, δ 5.49-5.53) and carbon (C4, 128.8) respectively. - The unsaturated dimer 90 was subjected to the previously described Wilkinson's hydrogenation conditions (50 psi H2, benzene, 4 hours). Unfortunately, under these conditions, the aromatic nitro substituents were reduced, thus providing a potential mechanism for poisoning of the metathesis catalyst. Fortunately, Jourdant et al. recently reported the selective reduction of an olefin in the presence of an aromatic nitro group.220 Homogeneous hydrogenation under 15 psi H2 in a mixture of tetrahydrofuran:tert-butanol (1:1) led to the selective reduction of the unsaturated dimer 90 without concomitant reduction of pendant aromatic nitro groups (Scheme 5.6).
- (2S,7S)-
Dimethyl 2,7-N,N′-di(p-nitrobenzoyl)aminooctanedioate 91 was isolated as an off-white solid in 67% yield. The replacement of olefinic peaks in the 1H n.m.r. spectrum with new methylene (H4, 6 and H3, 5) multiplets at δ 1.39-1.54 and δ 1.74-2.04 respectively confirmed formation of the saturated dimer 91. - An equimolar mixture of
olefins olefinic mixture allylglycine 83 to form an unsaturated dicarba bridge 90. Predictably, the more sterically hinderedolefin 19 and the electronically compromisedolefin 82 were unreactive under these reaction conditions. The resultant alkene 90 was then selectively hydrogenated in a mixture of tert-butanol:tetrahydrofuran (1:1) with Wilkinson's catalyst to afford the saturated dicarba bridge 91. Again,olefins - The next reaction in this catalytic sequence involved the activation of the
dormant prenyl olefin 19 via cross metathesis with cis-2-butene (butenolysis) to generate a more reactive crotylglycine derivative (Section 4.3.2). The mixture of 91, 19 and 82 was exposed to an atmosphere of cis-2-butene (15 psi) in the presence of 5 mol % second generation Grubbs' catalyst to afford the expected crotylglycine derivative 81 and a trace of the correspondinghomodimer 60. The activated olefin 81 was then quantitatively homodimerised to the expectedunsaturated dimer 60 with 5 mol % of second generation Grubbs' catalyst. Exposure of the newly formedolefin 60 to a hydrogen atmosphere and Wilkinson's catalyst resulted in quantitative conversion to the saturated dicarba bridge 71 (Section 4.1.4). Once again, the sterically and electronically compromisedolefin 82 remained a spectator over the three reactions used to form the second diaminosuberic acid derivative 71. - The remaining acrylate-
type olefin 82 was then used to form the final dicarba bridge. A double activation sequence was employed to render this remaining olefin reactive to homodimerisation. Homogeneous hydrogenation ofdienamide 82 using chiral Rh(I)—(S,S)-Et-DuPHOS catalyst gave (S)-configured prenylglycine derivative 87 in excellent enantioselectivity (100% e.e.), chemoselectivity and conversion. No evidence of over-reduction of the C4 carbon-carbon double bond was observed. The resulting prenyl olefin 87 was then converted to the crotylglycine analogue 88 via butenolysis. Exposure of this olefin to the previously described cross-metathesis and hydrogenation conditions then led to the formation of the final dicarba bond and the third diaminosuberic acid derivative 72 via alkene intermediate 69. The metathesis-hydrogenation sequence led to generation of three diamidosuberic acid esters 91, 71 and 72 in 67, 81 and 70% yields respectively. Significantly, residual catalyst and/or decomposition products did not compromise subsequent transformations and no other byproducts were isolated. This demonstrates the high chemoselectivity exhibited by each catalytic step. - A combination of homogeneous hydrogenation and metathesis reactions has enabled the highly efficient, stepwise chemo- and stereoselective formation of three identical dicarba C—C bonds in three different 2,7-diaminosuberic acid derivatives without purification of intermediates. This homogeneous catalytic methodology can be used widely in peptidomimetics and total product synthesis where multiple (preferably 3) C—C bonds and/or rings need to be selectively constructed.
- This section describes the application of the regioselective strategy developed in
section 4 to a series of peptides. A model synthetic pentapeptide was initially investigated. The results from this substrate led to the production of dicarba analogues of conotoxin ImI. - Linear peptides were synthesised via standard solid phase peptide synthesis (SPPS) methodology.221 This procedure involves the attachment of an N-Fmoc-protected amino acid to a solid support and the construction of the sequence from the C- to N-terminus Scheme 6.1. Peptide construction requires: i) Fmoc-deprotection of the resin-tethered amino acid under basic conditions, ii) activation of the incoming Fmoc-protected amino acid and iii) its subsequent coupling to the resin-tethered amino acid. The process is repeated until the desired peptide sequence is constructed. Conveniently, the use of orthogonally protected amino acids enables sequential Fmoc-deprotection and coupling without loss of acid-sensitive sidechain protecting groups.
- The choice of resin plays an important role in peptide synthesis. A plethora of polystyrene-based supports are commercially available. These resins are typically cross-linked polystyrene (PS) containing 1% divinylbenzene and are functionalised with linkers (or handles) to provide a reversible linkage between the synthetic peptide chain and the solid support.221 Several linkers commonly utilised in Fmoc-SPPS are presented in. Diagram 6.1. With the target peptide in mind, the appropriate resin-linker can be chosen to functionalise the C-terminus as a carboxylic acid, carboxamide, ester or alcohol. In addition, peptides can be cleaved under acidic or basic conditions where acid sensitive sidechain protecting groups can be retained or simultaneously deprotected during peptide cleavage. Importantly, the resin-linkers must be inert to metathesis and hydrogenation catalysis conditions.
- Construction of the linear peptides via solid phase methodology provides two options for the construction of dicarba bridges: The complete linear sequence can be cleaved from the resin and then subjected to metathesis and hydrogenation in solution. Alternatively, the regioselective catalytic sequence can be performed entirely on the resin-bound peptides.
- We have conducted an on-resin metathesis-hydrogenation sequence for the preparation of carbocyclic analogues of cystine-containing peptides. This strategy involves conventional solid phase peptide synthesis followed by on-resin ruthenium-catalysed ring closing metathesis and on-resin homogeneous rhodium-catalysed hydrogenation of the resultant unsaturated bridge (Scheme 6.2).
- The on-resin strategy, however, is compromised by decreased activity of the metathesis and hydrogenation catalysts in the heterogeneous system. Previous studies have shown that higher catalyst loadings and longer reaction times are required to achieve quantitative conversion on resin-bound substrates.141,142In addition, ring closing metathesis of peptidic substrates is highly sequence dependent due to the involvement of aggregation phenomena. We have found that peptide aggregation, resulting from interchain secondary structures, can lead to poor solvation of the peptidyl-resin, reduced reagent penetration and ultimately low reaction yields. Strategies had to be developed to address these problems.
- We have investigated the synthesis of bis-dicarba analogues of bicyclic peptides possessing two disulfide bonds. To achieve this aim we required the use of complimentary pairs of both allyl- and prenylglycine residues (although variations described above can be used). In order to transfer the solution phase methodology across to the solid phase, we needed to demonstrate that Fmoc-protected prenylglycine 92 could be i) synthesised and incorporated into a peptide sequence using standard SPPS protocol; that it was stable to peptide coupling and deprotection conditions, and iii) that it possessed analogous reactivity to its solution phase congener in the catalysis steps. We therefore decided to synthesise model peptides based on naturally occurring conotoxin peptides.171,172,225 Conotoxin ImI 93 (Ctx ImI) is a small dodecapeptide possessing two cystine bonds.173,174,226 A truncated sequence 94 of the Cys8-Ala9-Trp10-Arg11-Cys12 Ctx ImI domain was initially investigated. This sequence possesses two allylglycine residues which undergo ring closing metathesis to yield an unsaturated carbocycle 95. After establishing optimum conditions for the formation of the first dicarba bond, the sequence was modified to include a prenylglycine residue to facilitate the formation of a second dicarba linkage.
- The pentapeptide 94 was synthesised on Rink amide resin, a polystyrene-based solid support bearing an amine linker that generates a C-terminal carboxamide upon resin cleavage. (Diagram 6.1). Prior to attachment of the first amino acid, the resin was swollen in dichloromethane to increase surface availability of resin active sites towards the incoming C-terminal Fmoc-protected amino acid. Peptide construction began with attachment of non-proteinaceous Fmoc-L-allylglycine (Fmoc-Hag-OH) 96 to Rink amide resin (A, Diagram 6.1) and remaining resin active sites were capped with acetic anhydride. Fmoc-deprotection of resin-tethered allylglycine followed by coupling of the successive amino acid and repetition of these steps (B and C, Diagram 6.1) enabled chain elongation. After coupling the last amino acid, a small aliquot of peptidyl-resin was exposed to trifluoroacetic acid cleavage solution (D, Diagram 6.1) to liberate the peptide 94 as a colourless solid. The mass spectrum displayed a molecular ion peak at m/z 847.1 (M+H)+ which was consistent with the formation of the pentapeptide 94.
- After confirming that pentapeptide synthesis had been successful, ring closing metathesis of the fully-protected resin-tethered sample 94a was performed with 20 mol % Grubbs' catalyst in dichloromethane and 10% lithium chloride in dimethylformamide. Mass spectral analysis of a cleaved aliquot of peptide indicated that these conditions resulted in complete recovery of the linear peptide 94. Use of the more active second generation Grubbs' catalyst did, however, lead to unsaturated carbocycle 95 but cyclisation failed to go to completion (Scheme 6.3). The presence of molecular ion peaks at m/z 819.2 (M+H)+ and m/z 847.2 (M+Na)+ were consistent with the presence of the unsaturated carbocycle 95 and the linear peptide 94 respectively.
- We postulated that the peptide sequence itself may be responsible for the reduced ring closing metathesis yield. Pentapeptide 94 lacks a proline residue between the two allylglycine sidechains and hence the predominance of transoid peptide bonds would disfavour a close arrangement of the reacting terminal olefins. The inclusion of turn inducers in a peptide sequence can reduce peptide aggregation via the formation of cisoidal amide bonds.227-229 In addition, the resultant turn can position the reactive allylglycine sidechains in close proximity to each other and thus facilitate cyclisation. The peptide was therefore reconstructed to incorporate proline, a naturally occurring turn-inducing amino acid.
- The pentapeptide 97 was synthesised on Rink amide resin via the general SPPS methodology previously described. The peptide possessed an Ala→Pro replacement adjacent to the N-terminal allylglycine residue. Formation of the pentapeptide 97 was confirmed by mass spectrometry with the appearance of a molecular ion peak at m/z 873.2 (M+H)+.
- Ring closing metathesis of the fully protected resin-bound peptide 97a with Grubbs' catalyst (20 mol %) in dichloromethane and 10% lithium chloride in dimethylformamide led to recovery of the starting peptide 97 with only a trace of product 98 evident in the mass spectrum. Use of second generation Grubbs' catalyst (20 mol %), however, led to complete cyclisation (Scheme 6.4). The appearance of molecular ion peaks at m/z 845.1 (M+H)+ and m/z 867.1 (M+Na)+ in the mass spectrum confirmed formation of the unsaturated carbocycle 98. This result clearly demonstrates the influence of the turn-inducing proline residue on peptide conformation and reactivity.
- In conjunction with this study, we simultaneously assessed the role of the catalytic cycle in affecting ring closing metathesis yield. We postulated that the incomplete cyclisation of linear sequence 94 could be due to thermal decomposition of the ruthenium-methylidene intermediate 48. We therefore investigated synthesis of the crotylglycine-containing peptide, Fmoc-Crt-Ala-Trp-Arg-Crt-NH2 99, for which metathesis proceeds through the more stable ruthenium-ethylidene species 49.
- This initially required the synthesis of the crotylglycine derivative 100. Acid-promoted hydrolysis of (2S)-methyl 2-N-acetylaminohex-4-enoate 81 gave (2S)-2-aminohex-4-enoic acid hydrochloride salt 101. Fmoc-protection of amino acid 101 was performed according to the procedure described by Paquet et al. using N-fluorenylmethoxycarbonylamino-suecinimide (Fmoc-OSu) in aqueous sodium carbonate and acetone (Scheme 6.5).230
- 1H n.m.r. and 13C n.m.r. spectral analysis of the product confirmed the formation of (2S)-2-N-fluorenylmethoxycarbonylaminohex-4-enoic acid (Fmoc-Crt-OH) 100 with the downfield shift of the methine proton (H2) peak (δ 4.55) and the corresponding carbon signal (δ 52.3). In addition, the appearance of aromatic signals characteristic of the Fmoc-group supported product formation. Spectroscopic data were also in agreement with those reported in the literature.146
- With the Fmoc-protected crotylglycine derivative 100 in hand, we synthesised the linear peptide, Fmoc-Crt-Ala-Trp-Arg-Crt-NH2 99, on Rink amide resin using the SPPS methodology previously described. The mass spectrum displayed a molecular ion peak at tn/z 875.2 (M+H)+ corresponding to the linear peptide 99.
- Ring closing metathesis of the linear resin-tethered peptide 99a with second generation Grubbs' catalyst (20 mol %) in dichloromethane and 10% lithium chloride in dimethylformamide led to quantitative formation of the unsaturated carbocycle 95\ (Scheme 6.6). Note: RCM of the crotylglyeine-containing peptide 99 leads to the same unsaturated carbocycle 95 resulting from cyclisation of the allylglycine-containing sequence 94, i.e. Fmoc-c[Hag-Ala-Trp-Arg-Hag]-OH is identical to Fmoc-c[Crt-Ala-Trp-Arg-Crt]-OH.
- These studies revealed two successful strategies for the synthesis of a dicarba cyclic peptide: i) the inclusion of proline residues to induce a turn in the peptide backbone and ii) the use of crotylglycine to avoid a ruthenium-methylidene intermediate in the catalytic cycle. Many naturally occurring cyclic peptides possess proline residues in their primary sequences and this could be used to advantage in RCM reactions. On the other hand, if the target peptide does not possess a proline residue (or a residue which can temporarily act as a pseudo-proline), incorporation of a non-native proline residue to enhance RCM yield is likely to have significant structural and biological impact on the final peptide. In this case, the use of crotylglycine residues would be beneficial.
- The Melbourne-based pharmaceutical company Metabolic have a peptidic agent, AOD9604, currently undergoing clinical trials. AOD9604 143 is a peptide fragment derived from the C-terminus of human growth hormone (hGH) and is believed to be responsible for the lipolytic activity of hGH.267 This 16-residue peptide was derived from the parent anti-obesity drug AOD9401 144 by addition of a terminal tyrosine residue, and is known to induce lipolysis and fat oxidation in vitro in adipose tissue.267 Ng et al. report the synthesis of both of these peptides using standard solid phase peptide synthesis techniques.267,268
- The x-ray crystal structure of native hGH shows that the region of interest (residues 177-191) contains a disulphide bridge between residues 182 and 189. An alanine scan of AOD9401 showed that when cysteine was replaced by alanine a dramatic reduction in antilipogenic activity was observed.268 This suggests that the cystine bridge and the cyclic conformation of the peptide are vital for the activity of AOD9401 and related peptide analogues.268 Thus, we were interested in synthesising the dicarba analogue of AOD9604 using the technology developed and described herein to provide analogues with increased biological stability.
- The linear derivative of the carbocyclic analogue of AOD9401 was initially synthesised utilising natural amino acids, as well as the non-proteinaceous residue allylglycine in place of cysteine. Upon synthesis of the linear peptide 145, an aliquot was subjected to cleavage conditions to assess the success of the synthesis. Mass spectral analysis indicated the synthesis of linear Fmoc-protected AOD9401.
- At this point, it was established that AOD9604 would be a more suitable target molecule, and the additional amino acid residue was coupled to the parent AOD9401 molecule already synthesised. The presence of the linear Hag6-Hag13 containing derivative 146 was confirmed by mass spectral analysis.
- Ring closing metathesis, catalysed by second generation Grubbs' catalyst was employed to achieve cyclisation leading to the synthesis of unsaturated dicarba AOD9604 147. Initially, standard metathesis conditions were used, as perfected in the synthesis of somatostatin analogues. Lithium chloride was employed to decrease aggregation and 20 mol % catalyst loading was used to initiate the metathesis reaction. Mass spectral analysis post-TFA cleavage indicated the failure of cyclisation, with the only peaks indicative of linear Fmoc-protected starting material 146. This reaction was repeated a number or times, including with a higher boiling solvent, however, all attempts yielded solely uncyclised starting material.
- Deleterious hydrogen bonding in the linear peptide was suspected as the cause of this failed ring closure under standard metathesis conditions. Hence, microwave-accelerated ring closing metathesis of the same resin-tethered peptide was attempted. Similar catalytic conditions to previous attempts were employed, with dichloromethane as the solvent. The temperature was increased from 40° C. to 100° C. and the time decreased to just 10 h. Again, mass spectral analysis of cleaved material indicated the failure of the reaction.
- Attention was turned to the primary sequence of the peptide itself. It was identified that residues such as proline and glycine can induce turns in peptides, and thus facilitate N→C cyclisation of peptides. N-alkylated residues and D-amino acids can also achieve this. There is a lack of any turn-inducing amino acid residues (peptides) in the sequence of AOD9604, a potential contributing factor in the failure to cyclise.
- Proline is the only naturally occurring amino acid which is known to induce cis/trans isomerisation about a peptide bond, a feature known to induce a turn in the peptide backbone, often resulting in a reversal of the direction of the backbone. This has led researchers to develop alternatives to native proline, and numerous mimetics which produce proline-like cis-peptide bonds and reverse turns have been investigated.269
- Pseudoproline (ψPro) residues derived from naturally occurring serine, threonine and cysteine residues have gained popularity in recent years. Their formation is reversible; they are synthesised by a cyclocondensation reaction with an aldehyde or ketone and upon exposure to acidic conditions they revert to the parent amino acid.
- The incorporation of pseudoproline residues into peptide sequences increase the rate and yield of head to tail cyclisation (macrolactamisation). It was decided to incorporate a pseudoproline residue in the synthesis of the linear AOD analogue and to conduct the metathesis under microwave irradiation conditions. There are two serine residues in the sequence, and
serine 13 was chosen to be replaced by a pseudoproline residue. The incorporation of a pseudoproline residue is highly dependent on the adjacent residue attached to the amine of the pseudoproline. Pseudoprolines are incorporated into the peptide sequence as a dipeptide due to the ease of synthesis and stability. Adjacent toserine 9 is an arginine residue; this pseudoproline is not commercially available and is highly difficult to synthesise due to the bulky side chain and equally bulky protecting group necessary for peptide synthesis. - The linear peptide was again synthesised, this time with the dipeptide sequence -Ser(tBu)-Gly-replaced with the commercially available pseudoproline analogue. This residue reverts to the required dipeptide upon exposure to the acidic cleavage solution after the cyclisation step.
- The microwave-accelerated metathesis reaction was repeated using the resin-tethered, pseudoproline-containing peptide 146a. After 1 h, an aliquot of resin was exposed to cleavage conditions. Mass spectral analysis indicated the reaction had been successful, with the presence of a peak at m/z 1000.1 corresponding to the doubly charged adduct of the unsaturated dicarba product 147. This example clearly illustrates the importance of using turn-inducing residues when the metathesisable groups are not naturally proximate to facilitate high yielding ring closure.
- Finally, the carbocyclic peptide 147 was obtained with a 75% conversion from the linear parent moiety 146a. A large aliquot of resin was exposed to cleavage conditions, and purification via preparative HPLC yielded the desired peptide in 6% yield. The low yield was attributed to purification difficulties caused by lingering catalyst, despite treatment with DMSO prior to cleavage, a technique thought to destroy interaction between the catalyst and resin.
- Catalytic hydrogneation of the unsaturated AOD peptide 147 proved to be difficult. Exposure of the peptide to Wilkinson's catalyst and 90 psi of hydrogen for 4 days failed to achieve complete reduction of the peptide to the saturated AOD derivative 148. The two dicarba analogues 147 and 148, however, were readily separated from each other using preparative HPLC.
- Capitalising on the findings of the previous study (Section 6.2) we constructed another model peptide, Fmoc-Hag-Pro-Pre-Arg-Hag-OH 102, with a strategically placed proline residue. The synthetic pentapeptide 102 contains two types of metathesis active groups: Two allylglycine (Hag) residues and a less reactive prenylglycine unit (Pre). This linear sequence facilitates the regioselective construction of two dicarba bonds: An intramolecular metathesis reaction (RCM) of the allylglycine residues generates a carbocyclic ring and the remaining prenylglycine can be used to form an intermolecular dicarba bridge via cross metathesis (CM) with a second unsaturated molecule (Scheme 6.7).
- This second dicarba linkage could be used to attach the carbocyclic peptide to another peptide chain, a drug molecule, a solid support or a chelating heterocycle for the generation of radiopharmaceuticals.
- Synthesis of the peptide 102 firstly required the preparation of the Fmoc-protected prenylglycine derivative (Fmoc-Pre-OH) 92. Cross metathesis of Fmoc-protected allylglycine 96 with 2-methyl-2-butene in the presence of 5 mol % second generation Grubbs' catalyst gave the target (2S)-2-N-fluorenylmethoxycarbonylamino-5-methylhex-4-enoic acid 92 with quantitative conversion (Scheme 6.8).
- 1H n.m.r. spectroscopy confirmed formation of the trisubstituted olefinic amino acid 92 by the replacement of terminal olefinic peaks with a new methine multiplet (H4) at δ 5.11 and two methyl singlets at δ 1.63 and δ 1.73. These signals are consistent with the generation of a prenyl group. The accurate mass spectrum also displayed a molecular ion peak at m/z 388.1525 (M+Na)+ which was consistent with that required for 92. Unfortunately, purification of the product 92 from residual catalyst was difficult. We later found, however, that the crude amino acid 92 could be used without affecting subsequent SPPS procedures.
- The peptide 102 was synthesised on inexpensive, readily available Wang resin, a polystyrene-based solid support bearing a benzylic alcohol linker (
FIG. 6.1 ). The non-proteinaceous prenylglycine residue 92 was incorporated into the peptide sequence without complication. Formation of the pentapeptide 102 was confirmed by mass spectral analysis with the appearance of a molecular ion peak at m/z 813.5 (M+H)+ and an additional peak at m/z 831.5 (M+H2O+H)+. The latter peak was due to the acid-promoted hydration of the prenyl sidechain during peptide cleavage, leading to the alcohol 103. The hydration of the prenyl group under acidic conditions was not unexpected. During the acid-catalysed cyclisation of the simple prenylglycine derivative 19 to pseudo-proline 18, acid-mediated hydration yielded alcohol 47 as a minor byproduct. - After confirming the synthesis of the pentapeptide 102, the peptidyl-resin was subjected to the regioselective catalytic strategy outlined in
section 4. This is presented in Scheme 6.7. - The first step involved selective RCM of the allylglycine residues in the presence of the less reactive prenyl sidechain. RCM of the resin-tethered pentapeptide 102a was performed with 40 mol % second generation Grubbs' catalyst in dichloromethane and 10% lithium chloride in dimethylformamide and, as expected, incorporation of prenylglycine did not hinder cyclisation (Scheme 6.9). Mass spectral analysis of a cleaved aliquot of peptide confirmed formation of the unsaturated carbocycle 104 with the appearance of a molecular ion peak at m/z 785.4 (M+H)+. A peak at m/z 803.4 (M+H2O+H)+, corresponding to a hydrated prenyl sidechain in the cyclic product, was also evident. Importantly, prenylglycine remained inert to the metathesis conditions and no mixed cross metathesis products were observed.
- Attempts to decrease reaction time and catalyst loading led to incomplete reaction. We therefore decided that the high catalyst loading and extended reaction times could be tolerated in order to avoid the time consuming and poor yielding HPLC purification of mixtures resulting from non-quantitative cyclisation reactions. Decreasing peptide loading on the resin (from 0.9 to 0.3 mmolg−1) did, however, enable complete RCM with 10 mol % of second generation Grubbs' catalyst. This is probably due to the fact that the use of low substitution resins decreases the density of peptide chains on the solid phase and minimises aggregation. The reduced loading enhances resin solvation and reagent access and ultimately leads to improved reaction yields.
- Selective hydrogenation of the resin-bound unsaturated carbocycle 104a was performed under 80 psi of hydrogen with homogeneous Wilkinson's catalyst, Rh(I)(PPh3)3Cl, in a mixture of dichloromethane:methanol (9:1) (Scheme 6.10). This solvent system served a dual function in maintaining a swollen resin (dichloromethane) and participating in the catalytic cycle (methanol). After 22 hours, a small aliquot of peptide was cleaved and analysed by mass spectrometry. The appearance of peaks at m/z 787.3 (M+H)+ and m/z 805.4 (M+H2O+H)+ were consistent with formation of the saturated carbocycle 105. Importantly, the prenyl group remained stable to these reducing conditions which was consistent with the observed reactivity of prenylglycine in the solution phase model studies.
- So far, the application of the solution phase methodology to resin-bound peptide substrates was proceeding as expected. A need for longer reaction times and catalyst loadings was apparent, however, and highlighted the subtle differences between the two approaches. After selective ring closing metathesis, the remaining prenylglycine residue was employed for the formation of the second dicarba bond.
- Activation of the prenyl group was achieved via butenolysis of the resin-bound pentapeptide 105a. The peptide was exposed to an atmosphere of cis-2-butene (15 psi) and 40 mol % second generation Grubbs' catalyst in dichloromethane for 42 hours. This led to a mixture of the desired product 106 and the starting peptide 105. The reaction was unexpectedly and inexplicably slow compared to the analogous solution phase activation step. The recovered resin-peptide was therefore re-subjected to analogous butenolysis conditions which led to the formation of the target crotylglycine-containing peptide 106 (Scheme 6.11). Mass spectral analysis of the cleaved peptide displayed the product molecular ion peak at m/z 773.2 (M+H)+ and no evidence of the starting prenylglycine-containing peptide 105 was observed.
- High purity 2-butene was found to be critical for high turnovers in butenolysis reactions (when butane is the disposable olefin). For example, when butenolysis reactions were conducted on an unsaturated triglyceride (triolein) with commercially available and less expensive cis+trans-2-butene mixtures only traces of butenolysis products were detected, even with high catalyst loadings. GC analysis of the isomeric 2-butene mixture showed that it was contaminated with 2.6% of butadiene, while none of this impurity was found in the commercially available cis-2-butene. The addition of 1,3-butadiene (2%) to pure cis-2-butene gave a mixture that did not give cross-metathesis products with triolein while a cis+trans-2-butene mixture (30:70) free of 1,3-butadiene was found to give the same activity in butenolysis reactions as pure cis-2-butene. These results suggested that the 1,3-butadiene was acting as a poison in reactions employing commercial grade cis+trans-2-butene. This discovery is significant and previously unreported; a GC trace of commercially available trans 2-butene is contaminated with 1,3-butadiene (
FIG. 5 ), GC traces of cis+trans-2-butene mixtures show the same impurities. In conclusion, cis-, trans- and mixtures of cis+trans-2-butene can all be used in butenolysis (unblocking reactions with a disposable olefin) reactions but all must be 1,3-butadiene free. Later work with other disposable olefins shows that functionalisation of the C1 or C4 carbon atoms of 2-butene further improves turnover, especially for resin-based peptides. - A cross metathesis reaction between the activated-resin bound peptide 106a and crotylglycine derivative 81 was then performed. We decided to investigate microwave technology as a means of decreasing reaction time in the solid-phase approach. Microwave irradiation of a mixture of resin-tethered peptide 106a with 40 mol % second generation Grubbs' catalyst, excess crotylglycine 81 (˜50 equiv) in dichloromethane and 10% lithium chloride in dimethylformamide resulted in formation of the desired intermolecular dicarba linkage (Scheme 6.12). Mass spectrometry confirmed product formation 107 with the appearance of a molecular ion peak at m/z 902.3 (M+H)+.
- Wilkinson's hydrogenation of the unsaturated intermolecular bridge was achieved under conditions previously established (80 psi H2, dichloromethane:methanol (9:1), room temperature, 22 hours) to give the target peptide 108 containing two regioselectively constructed dicarba bridges (Scheme 6.13).
- The successful application of the solution phase methodology (section 4) to a resin-bound pentapeptide 102a led to selective construction of an intramolecular and an intermolecular dicarba bridge. Several important biologically active peptides, such as those within the insulin superfamily (insulin and relaxin), possess metabolically unstable inter- and intramolecular cystine bonds. This methodology can be applied to the regioselective construction of stable dicarba analogues of these peptides. We next examined the extension of this strategy to the construction of bicyclic peptides—as cystino-dicarba analogues and bis-dicarba analogues. The latter analogues require the formation of two intramolecular dicarba bridges via sequential ring closing metathesis reactions.
- Liskamp et al. recently reported the synthesis of a crossed alkene-bridge of the complex DE-bisthioether ring system of nisin, a lantibiotic that possesses five thioether bridges (as distinct to disulfide bridges—which are less stable) (Diagram 6.2).157
- A linear precursor 109 containing four identical allylglycine residues was subjected to a solution phase double ring closing metathesis reaction. The first cyclisation reaction yielded four out of a possible six mono-cyclic peptides. Successive ring closing metathesis under similar conditions ultimately yielded the target 1-4, 3-6-carbocyclic peptide 110 (72%) and a contaminating 1-3, 4-6-bicycle 111 (19%) (Scheme 6.14).
- These results suggest favourable pre-organisation of the linear peptide for generation of the target regioisomer.157 The selective synthesis of multiple bridges, however, is rarely so fortuitous.129,225,231,232 Indeed, in the synthesis of native conotoxin sequences, several topoisomers (ribbon, globule and beads) are obtained after oxidative folding.225,231,233 Multiple cystine formation usually requires an orthogonal protection strategy and sequential oxidation of cysteine residues.225 For this reason, we investigated the regioselective methodology developed in
section 4 for the synthesis of dicarba analogues of a native conotoxin sequence, Ctx ImI 93. - Conotoxins are venom components of cone snails (Conidae) and represent a group of small disulfide-rich peptides that act as potent and highly specific antagonists for different receptor targets.171-174 Conotoxins derive their receptor subtype specificity from the arrangement of their disulfide bonds and resultant loop sizes. For example, α-conotoxins, which contain two disulfide bonds in a 1-3, 2-4 arrangement (Diagram 6.3), target nicotinic acetylcholine receptors of vertebrates.234 χ-Conotoxins, on the other hand, possess a 1-4, 2-3 disulfide bond arrangement and are selective for noradrenaline transporters.
- The small size (typically between 10-40 amino acids), selectivity and potency of conotoxins make them ideal therapeutic candidates for clinical conditions such as pain, epilepsy, stroke and cancer.171,234 Recently Ziconotide, an ω-conotoxin, completed Phase III clinical trials for neuropathic pain whilst two new conotoxin analogues (ω-Ctx CVID and χ-Ctx MrIA) are in clinical trials for chronic pain.
- Conotoxins possess a rich diversity of amino acid residues and this, coupled with their potential as pharmaceutical agents, makes them challenging and interesting targets. We chose to examine α-conotoxin ImI 93 (Ctx ImI), a small cysteine rich dodecapeptide isolated from the vermivorous conus species Conus imperialis. 173,174,226 Its two intramolecular disulfide bonds form the hydrophobic core of the molecule and generate a constrained two loop structure which, together with a central proline residue, arrange three essential residues (Asp5, Arg7, Trp10) for selective interaction with complementary residues within the α7 neuronal nicotinic acetylcholine receptor.
- Interestingly, the structural and functional role of the disulfide bonds in these natural products is yet to be elucidated. Generation of dicarba-cystino hybrids of conotoxin ImI and ultimately bis-dicarba analogues allows the importance of the constituent bridges on the structure and activity of the peptide to be elucidated. We therefore investigated the application of the on-resin metathesis-hydrogenation sequence to generate a library of dicarba analogues of conotoxin ImI (Diagram 6.4).
- Metathesis catalysts display high functional group tolerance and homogeneous rhodium-based catalysts, unlike their heterogeneous counterparts, are not poisoned by sulfur-containing functionality. We decided to initiate our study with the synthesis of dicarba-cystino hybrids of Ctx ImI.
- Native α-conotoxins are amidated at their C-termini. Rink amide resin was therefore chosen to facilitate linear peptide construction and generate the required C-terminal carboxamide upon resin cleavage. The low loading (0.52 mmolg−1) of the Rink amide linker helps to reduce crowding and aggregation of peptide chains and reduces the likelihood of homodimerisation in the subsequent metathesis reaction. Standard SPPS using HATU-NMM activation and Fmoc-protected amino acids was used to construct the two linear peptides: [2,8]-Hag-[3,12]-Cys conotoxin ImI 112 and [2,8]-Cys-[3,12]-Hag conotoxin ImI 113. Both of these sequences possess two strategically placed non-proteinaceous L-allylglycine (Hag) residues to facilitate construction of the dicarba bridge. Intermediates were carried through without purification or characterisation up to the dodecapeptides 112 and 113. A sample of each linear peptide was obtained by cleavage from the resin and determined to be of >95% purity by reverse-phase-HPLC. Mass spectral analysis gave the molecular ion peak at m/z 1565.7 (M+H)+ and the corresponding doubly charged ion peak at m/z 783.5 [½(M+2H)]+. Both ions are consistent with the structures of the isomeric sidechain deprotected linear peptides 112 and 113.
- Ring closing metathesis was performed on resin-attached linear peptides to eliminate any potential problems arising from dimerisation and/or poor peptide solubility. Exposure of [2,8]-Hag Ctx ImI 112a to first generation Grubbs' catalyst (50 mol %) in dichloromethane at 50° C. for 72 hours gave only trace amounts (<10%) of cyclised product 114. The more reactive second generation Grubbs' catalyst was then used to improve the cyclisation yield (Scheme 6.15). While RCM progressed further (˜70%) with this catalyst, conditions could not be found to effect full cyclisation to 114. Change in solvent, concentration, catalyst loading, and reaction time had no positive effect on conversion. The addition of a chaotropic salt (lithium chloride in dimethylformamide) to the reaction mixture also had no effect on RCM yield. Similarly, RCM of a dicrotylglycine analogue of the primary sequence of 112, which avoids catalytic cycling through an unstable ruthenium-methylidene species, also failed to achieve complete conversion to the cyclic target 114.
- Construction of the isomeric [3,12]-unsaturated carbocyclic Ctx ImI 115 was found to be even more problematic. Exposure of the resin-bound peptide 113a to both first and second generation Grubbs' catalysts under a variety of experimental conditions failed to yield the unsaturated carbocycle 115. Possible reasons for the poor reactivity of this isomer 113 included the diminished influence of the proline residue in assisting formation of the larger carbocycle (28-membered ring) and the close proximity of the C-terminal allylglycine residue to the bulky Rink amide linker. The sequence was therefore reconstructed on BHA resin bearing a linear HMBA-Gly-Gly-linker. Cyclisation of the BHA resin-bound peptide was attempted in the presence of 20 mol % second generation Grubbs' catalyst and chaotropic salts. Unfortunately, mass spectral analysis of the product mixture again showed only the starting peptide 113.
- Microwave-assisted ring closing metathesis of isomeric linear peptides 112a and 113a provided both of the target carbocycles 114 and 115. In our study, a microwave reactor emitting a focused irradiation at 2.45 GHz with a maximum power of 300 W was used. Irradiation of a mixture of Rink amide-bound [2,8]-Hag-[3,12]-Cys Ctx ImI 112a and second generation Grubbs' catalyst (10 mol %) in dichloromethane containing 10% lithium chloride in dimethylformamide resulted in complete ring closure in only one hour (Scheme 6.16). Decreasing the catalyst loading (5 mol %) also led to quantitative conversion to the unsaturated carbocycle 114 after just two hours of microwave irradiation. Mass spectral analysis of the product mixture showed the required molecular ion peak at m/z 1537.7 (M+H)+ and the corresponding doubly charged ion at m/z 769.5 [½(M+2H)]+ for the unsaturated carbocyclic peptide 114 and no starting linear peptide 112.
- Similar reaction conditions also resulted in complete cyclisation of 113a to the isomeric [3,12]-dicarba analogue 115 (Scheme 6.17). Although a higher catalyst loading (20 mol %) was required, the reaction went to completion in one hour. The enhancement in RCM yield via this microwave-assisted approach is remarkable in light of the poor results obtained using conventional heating methods. It is considered that the results must be attributed to something beyond just more efficient heating. It has been postulated that another possible factor is that microwave radiation causes highly efficient disruption of peptide aggregation on the solid support. It is noted that the reaction of scheme 6.17 does not proceed without microwave irradiation.
- On-resin Fmoc-deprotection of the unsaturated carbocycles 114a and 115a followed by acid-mediated cleavage yielded the fully deprotected peptides 116 and 117. Aerial oxidation of 116 and 117 in 5% dimethylsulfoxide/aqueous ammonium carbonate (0.1 M, pH 8) then afforded the unsaturated cystino-dicarba Ctx ImI analogues 118 and 119 respectively (Scheme 6.18, Scheme 6.19). Each peptide was purified by reverse-phase HPLC (>99% purity) and isolated in 5% yield. These dicarba-analogues, and others based on native conotoxin sequences of pharmaceutical significance (e.g. dicarba-analogues of conotoxins extracted from Conus regius and Conus victoriae (ACV1)), see experimental section) are predicted to be biologically active due to their similarities to the disulfide, and are predicted to have in vivo stability due to the presence of the dicarba-bond.
- It is important to note that the isolation and purification of native conotoxin sequences from cone snail venom is a low yielding and tedious process.247 Recently, 200 mg of venom extract from five cone snails (Conus textile) was purified to yield 1.1 mg (560 nmol) of conotoxin ε-TxIX.248 Most references detailing the isolation of conotoxin molecules from venom, however, do not cite isolation yields. Synthesis of conotoxin molecules can also be low yielding where oxidative folding leads to several topoisomers.225,231,233,249-251 Extensive chromatography must be employed to isolate pure samples of the target peptide. Although the final purified yields of our dicarba-cystino conotoxin analogues 118 and 119 were low, separation conditions were not optimised and the scale of the reactions could be easily increased to afford larger quantities of pure peptide.
- Hydrogenation of resin-bound unsaturated carbocyclic peptides 114a and 115a was performed with Wilkinson's catalyst. This homogeneous catalyst is ideal for this transformation as it allows reduction to be performed on the resin, operates under mild reaction conditions and is highly tolerant of sulfur-containing functionality. Hence, rhodium-catalysed hydrogenation of resin-bound carbocycles 114a and 115a in dichloromethane:methanol (9:1) effected quantitative reduction of the olefin at room temperature and low hydrogen pressure (80 psi) (Scheme 6.20). Interestingly, the crude product from each of these reactions was obtained as a mixture of the cystine reduced (120, 121) and oxidised (122, 123) forms. It is important to note that the final targets 122 and 123 are isomeric with the unsaturated deprotected precursor peptides, 116 and 117 respectively. An analogous hydrogenation experiment spiked with linear diallyl conotoxin sequence 112, the precursor to the unsaturated carbocycle 114, showed a molecular ion consistent with the formation of the dipropyl sidechain-containing peptide 124.235 This mass spectral data strongly suggests that the catalyst is not poisoned by the trityl-protected cysteine residues and that the hydrogenation conditions needed for olefin reduction are uncompromised. Hence, the species contributing to the peak at m/z 769.5 [½(M+2H)]+ are likely to be the final isomeric cystino-dicarba Ctx ImI peptides 122 and 123.
- Further support for this hypothesis comes from the LC-MS traces of the product mixtures. In each case, a signal at tR=6.01 min (122) and tR=7.02 min (123) was observed. In comparison, the retention times for the isomeric unsaturated carbocycles 116 and 117, under identical chromatographic conditions, are 5.66 min and 6.64 min respectively. The saturated cystino-dicarba α-conotoxin analogues 122 and 123 are currently undergoing chromatographic purification and are being assessed for biological activity and in vivo stability. NMR spectroscopy will also be used to further confirm the structures of the isomeric conotoxin analogues 122 and 123.
- The regioselective on-resin methodology described in
section 4 was also applied to the synthesis of fully carbocyclic conotoxin ImI analogues. The catalytic sequence involves the selective RCM of reactive allylglycine units in the presence of dormant prenylglycine residues followed by selective hydrogenation of the resultant unsaturated carbocycle. Activation of the prenyl groups via butenolysis gives the active crotyl sidechains which can undergo the RCM-hydrogenation process to afford the target bicycles 125 and 126 (Scheme 6.21). - This study commenced with the construction of the linear isomeric conotoxin analogues, [2,8]-Hag-[3,12]-Pre conotoxin ImI 127 and [2,8]-Pre-[3,12]-Hag conotoxin ImI 128. Standard SPPS techniques employing Rink amide resin, HATU-NMM activation and Fmoc-protected amino acids facilitated synthesis of the peptides 127 and 128. Both of these sequences possess two strategically placed non-proteinaceous L-allylglycine (Hag) residues to facilitate the selective construction of the first carbocycle. The incorporation of two less reactive prenylglycine residues later enables the selective formation of the second carbocycle. During construction of the linear peptides, intermediates were carried through without purification or characterisation up to the dodecapeptides 127 and 128. As expected, the prenylglycine residues were incorporated without complication and mass spectral analysis gave doubly charged molecular ion peaks at m/z 805.6 [½(M+2H)]+ and 816.6 [½(M+Na+H)]+ which are consistent with the structures of the isomeric sidechain deprotected linear peptides 127 and 128. An additional peak at m/z 814.6 [½(M+H2O+2H)]+, corresponding to the acid-promoted hydration of a prenyl group, was also apparent in the spectrum.
- After confirming the successful synthesis of the linear peptides 127 and 128, ring closing metathesis of the resin-tethered peptides was performed using conventional heating methods. Exposure of peptide 127a to second generation Grubbs' catalyst (40 mol %) in dichloromethane and 10% lithium chloride in dimethylformamide at 50° C. for 40 hours gave the unsaturated carbocycle 129 (Scheme 6.22).
- Analogous RCM conditions for 128a, however, led to complete recovery of the linear peptide. These results highlight the influence of the peptide sequence on RCM success when microwave is not used. A derivative of the problematic sequence 128 was therefore constructed to elucidate the effect of a turn-inducer. A new peptide sequence 130 was synthesised possessing a Pro9 residue rather than the native Ala9 residue. Interestingly, the resultant solid-supported peptide 130a cyclised under the previously unsuccessful metathesis conditions (without microwave radiation) to give 131, but the RCM did not go to completion (Scheme 6.23). Unfortunately, LC-MS analysis did not enable separation of the linear 130 and cyclic 131 peptide and hence an estimation of reaction conversion could not be made.
- Microwave-assisted ring closing metathesis, however, provided expedient syntheses for both the target carbocycles 129 and 132. Microwave irradiation of a solution of Rink amide bound-peptide 127a and second generation Grubbs' catalyst (10 mol %) in dichloromethane containing 10% lithium chloride in dimethylformamide at 100° C. resulted in complete ring closure in only one hour (Scheme 6.24). Mass spectral analysis of the product mixture showed the required molecular ion with m/z 791.4 [½(M+2H)]+ for the unsaturated dicarba peptide 129 and no starting linear peptide 127.
- The resin-bound isomeric dicarba analogue 128a also completely cyclised in one hour with 20 mol % second generation Grubbs' catalyst using the same solvent system at 100° C. (Scheme 6.25).
- These results were very exciting and demonstrated the power of microwave energy to yield carbocycles that were unattainable by conventional heating methods. In addition, the prenyl sidechains remained inert to the microwave-accelerated metathesis conditions and no cross metathesis products were observed.
- Rhodium-catalysed hydrogenation of the resin-bound carbocycles 129a and 132a in dichloromethane:methanol (9:1) effected quantitative reduction of the unsaturated carbocycle at room temperature and low hydrogen pressure (80 psi) (Scheme 6.26 and Scheme 6.27). The mass spectra of cleaved peptides from both reactions displayed doubly charged molecular ion peaks at m/z 792.5 [½(M+2H)]+ and m/z 801.5 [½(M+H2O+2H)]+ confirming formation of the isomeric products 133 and 134. Importantly, the prenyl groups resisted hydrogenation and were now available for activation to facilitate construction of the second carbocycle.
- Activation of the prenyl sidechains involved butenolysis of the solid-supported peptides 133a and 134a. The peptide 133a was exposed to an atmosphere of cis-2-butene (15 psi) and a mixture of 40 mol % second generation Grubbs' catalyst and benzoquinone in dichloromethane for 38 hours (Scheme 6.28). Benzoquinone was added to the reaction mixture to reduce or eliminate the potential for olefin isomerisation. Mass spectral analysis of a cleaved aliquot of peptide confirmed formation of the target dicrotylglycine-containing peptide 135 with the appearance of a peak at m/z 778.4 [½(M+2H)]+. No starting prenyl-containing peptide 133 was observed, however, mass spectral data revealed low intensity, doubly charged higher homologue species separated by m/z+7 units. Under the above described metathesis conditions, the generated crotyl sidechain can isomerise to a terminal butenyl chain and then undergo secondary cross metathesis with cis-2-butene (Scheme 6.29). The products arising from this process of isomerisation-cross metathesis are consistent with the observed mass spectral data.
- Reaction conditions were modified to minimise this competing isomerisation reaction. These changes involved the addition of chaotropic salts and variation of catalyst loading and reaction time. This aim was realised, although to a small extend this was still accompanied by partially metathesised peptide 136 and starting material 133.
- Microwave-accelerated ring closing metathesis of the resin-tethered peptide 135a using second generation Grubbs' catalyst (20 mol %) in dichloromethane and 10% lithium chloride in dimethylformamide afforded the target peptide 140 in only one hour (Scheme 6.30). Preliminary LC-MS analysis was encouraging with the appearance of the required doubly charged molecular ion peak at m/z 750.4 [(M+2H)]+, corresponding to the bicyclic peptide 140. Interestingly, a very low intensity peak at m/z 764.4 was also evident which corresponded to the cyclic product of a contaminating isomerisation-butenolysis adduct. The Fmoc-deprotected product 125 is being purified and submitted for biological testing.
- Rhodium-catalysed hydrogenation of the resin-bound bicycle 140a was performed in dichloromethane:methanol (9:1) at room temperature under low hydrogen pressure (80 psi) (Scheme 6.31). Preliminary mass spectral and LC-MS data of the isolated residue confirm the formation of the saturated bicycle 126.
- The problem of isomerization experienced during activation of the diprenyl-conotoxin sequence 133a (see
FIG. 6.5 ) was subsequently reinvestigated. It was postulated that the highly non-polar ethylene and 2-butene, used for activation of the prenyl groups, could be incompatible with the polystyrene resin support. Resins, such as Wang and Rink amide, swell well in polar solvents; exposure to non polar solvents results in resin shrinkage, poor accessibility of reagents (ie catalysts) to reactive functionality and consequently poor conversion. A more polar derivative of ethylene and 2-butene, however, would achieve better resin swell and higher activation yields. Conseqeuntly, an investigation using 1,4-diacetoxy-cis-2-butene was initiated. This molecule has the advantage of being a liquid at ambient temperature while exhibiting greater polarity than either ethylene or butene. - Before 1,4-diacetoxy-cis-2-butene, or other like analogues (such as 1,4-dichloro-2-butene), could be used to activate prenyl-containing resin-tethered peptides, (i) its reactivity and ability to activate hindered olefins; (ii) compatibility with resin-tethered substrates; and (iii) its ability to form a reactive intermediate (i.e. allylic acetate) capable of a subsequent CM with a reactive type I olefin (i.e. to form subsequent intra/inter dicarba bonds) needed to be investigated. Steps (i) and (iii) were investigated using a simple small molecule derived from commercially available racemic allylglycine in three steps (Scheme 6.32).
- To generate the required type III olefin, the benzoyl-protected methyl ester of allylglycine 62 underwent a cross metathesis reaction with 2-methyl-2-butene (Scheme 6.32). The reaction proceeded at 50° C. in dichloromethane for 72 h in a pressurised vessel giving the prenylglycine derivative 87 in quantitative conversion after chromatographic purification.
- Next, the fully protected prenylglycine analogue 87 was subjected to standard cross metathesis conditions using an excess of 1,4-diacetoxy-cis-2-butene, in the presence of second generation Grubbs' catalyst (Scheme 6.32). After stirring at 50° C. overnight, the reaction was complete, as indicated by t.l.c. Column chromatography of the crude material yielded the activated molecule 141 in 57% yield.
- With activation of the prenylglycine derivative complete, the reactivity of the resultant molecule was assessed. Initially, homodimerisation was attempted; the molecule was subjected to standard cross metathesis conditions, in the presence of second generation Grubbs' catalyst at 50° C. (Scheme 6.33). Mass spectral analysis indicated both the desired product and starting material. Gas chromatographic analysis indicated the expected equilibrium statistical mixture of the desired
homodimer 69, 1,4-diacetoxy-2-butene and starting material 141. - To assess the reactivity of the activated moiety 141, cross metathesis with a type I olefin was investigated (Scheme 6.31). Standard cross metathesis conditions were applied, with a 6-fold excess of the type I olefin to avoid the statistical distribution of products and increase the yield of the desired peptide. The desired cross metathesis product 142 was obtained as a brown oil in 81% yield following purification via column chromatography. Spectroscopic analysis confirmed the presence of both the E- and Z-isomers, though individual NMR signals could not be assigned to a specific geometry.
- To assess the compatibility of 1,4-diacetoxy-cis-2-butene with resin-tethered substrates, a simple prenylglycine-containing dipeptide was subjected to a cross metathesis reaction with 1,4-diacetoxy-cis-2-butene (Scheme 6.34). This reaction showed complete conversion of the resin-tethered type II olefin to the corresponding type I olefin, indicating complete compatibility of 1,4-diacetoxy-cis-2-butene with resin-bound substrates. No isomerization of the double bond was observed leading to the conclusion that the extended reaction times needed during activation reactions using non-polar olefins is responsible for the competing isomerisation pathway.
- Despite the known activity of conotoxins as therapeutics, their multiple disulfide bond frameworks are known to be unstable under reducing conditions. Reduction or framework scrambling by thiol containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs.
- Incubation of native-Ctx ImI in human blood plasma has been shown to produce significant rearrangement of the disulfide framework (i.e. scrambling). Similarly, treatment of Ctx-IMI with glutathione, a reducing enzyme commonly found in blood plasma, results in complete scrambling of the disulfide framework in ˜6 hours. See for instance Armishaw, C. J., Daly, N. L., Nevin, S. T., Adams, D. J., Craik, D. J., Alewood, P. F., J. Biol. Chem., 2006, in press. Such scrambling or reduction is not possible with dicarba-Ctx IMI analogues; the dicarba linkage is completely inert to such reductants.
- In conclusion, the strategy developed can be used for the regioselective construction of multi-dicarba bond-containing peptides. The methodology was successfully applied to a model synthetic pentapeptide 102 and led to the regioselective construction of an intramolecular and intermolecular dicarba bridge. Similarly, a tandem metathesis-hydrogenation sequence provided dicarba-cystino analogues of naturally occurring conotoxin ImI 93. Here, a microwave accelerated ring closing metathesis provided cyclic peptides that were unattainable via conventional heating methods. A fully carbocyclic analogue of conotoxin ImI 140 was also synthesised. Although activation of the prenylglycine units with non-polar olefins (such as 2-butene and ethene) was significantly retarded on the resin-bound peptide 133a, butenolysis did lead to the desired activated crotyl sidechains. The selectivity of the methodology was maintained when investigated in the heterogeneous system. An intramolecular dicarba bridge was selectively constructed from allylglycine units in the presence of two prenyl olefins. A microwave-promoted RCM of the resin-bound crotyl-containing peptide ultimately afforded the desired bicycle 140, and reduction lead to conotoxin 126. The use on polar 2-butene analogues, such as 1,4-diacteoxy-2-butene, was found to be more compatible with polystyrene supports and led to improved resin swelling and activation yields. This was illustrated in both solution phase model studies and on solid-supported peptide substrates.
- Microwave reactions were carried out on a Personal Chemistry (now Biotage) Smith Synthesiser. The instrument produces a continuous focussed beam of microwave irradiation at 2.45 GHz with a maximum power delivery of 300 W, which reaches and maintains a selected temperature (100° C.). Reactions were performed in high pressure quartz microwave vessels fitted with self-sealing Teflon septa as a pressure relief device, that were crimped in place. The vessels contained magnetic stirrer beads and the pressure and temperature of each reaction was monitored continuously with an in built pressure transducer (located in the lid) and infrared pyrometer respectively. Reaction times were measured from the time the microwave began heating until the reaction period had elapsed (cooling periods were not inclusive).
- Peptides were synthesised in polypropylene Terumo syringes (10 mL) fitted with a polyethylene porous (20 μm) filter. Solid phase peptide synthesis (SPPS) was performed using a Visprep™ SPE DL 24-port model vacuum manifold supplied by Supelco. Coupling reactions and cleavage mixtures were shaken on a KS 125 basic KA elliptical shaker supplied by Labortechnik at 400 motions per minute. Cleaved peptides were centrifuged on a HermLe Z200A centrifuge supplied by Medos at a speed of 4500 cycles per minute.
- N,N′-Dimethylformamide (DMF) was supplied by Auspep and stored over 4 Å molecular sieves. Dichloromethane (DCM) was supplied by BDH and stored over 4 Å molecular sieves. Wang resin, Rink amide resin, piperidine and trifluoroacetic acid (TFA) were used as supplied by Auspep. Phenol was used as supplied by BDH. Diisopropylcarbodiimide (DIC), diisopropylethylamine (DIPEA), 4-(N,N′-dimethylamino)pyridine (DMAP), ethanedithiol (EDT), Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid), N-methylmorpholine (NMM), and thioanisole were used as supplied by Aldrich. (2S)-2-Aminopent-4-enoic acid (L-allylglycine, Hag) was used as supplied by Peptech. N-Fluorenylmethoxycarbonylaminosuccinimide (Fmoc-OSu), O-(7-azabenzo-triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-phosphate (HATU) and sidechain-protected Fmoc-amino acids were used as supplied by GL Biochem unless otherwise specified.
- Peptides were prepared using general Fmoc-SPPS methodology.142,221 Manual SPPS was carried out using fritted plastic syringes, allowing filtration of solution without the loss of resin. The tap fitted syringes were attached to a vacuum tank and all washings were removed in vacuo. This involved washing (or swelling) the resin in the required solvent for a reported period of time, followed by evacuation which allowed the removal of excess reagents before subsequent coupling reactions.
- In a fitted syringe, Wang resin was swollen with DCM (7 mL, 3×1 min, 1×60 min) and DMF (7 mL, 3×1 min, 1×30 min). DIC (3 equiv.) was added to a solution of protected amino acid, Fmoc-L-Xaa-OH, (3 equiv.) in DMF (3 mL). The activated amino acid solution was added to the swelled resin and shaken gently for 1 min. A solution of DMAP (0.3 equiv.) in DMF (1 mL) was added to the resin and the reaction mixture was shaken gently for the reported period of time. The mixture was then filtered and the resin-tethered amino acid was washed with DMF (7 mL, 3×1 min) to ensure excess reagents were removed. In order to prevent formation of deletion products, remaining resin active sites were capped with an anhydride solution (5% acetic anhydride, 1% NMM, 94% DMF) for 1 h. The mixture was filtered and the resin was washed with DMF (7 mL, 3×1 min) and deprotected with 20% piperidine in DMF (7 mL, 1×1 min, 2×10 min). After this deprotection step, the resin was washed with DMF (7 mL, 5×1 min) to remove traces of base prior to coupling the next amino acid.
- Subsequent amino acids were coupled using the following procedure:
- NMM (6 equiv.) was added to a solution of protected amino acid, Fmoc-L-Xaa-OH (3 equiv.) and HATU (2 equiv.) in DMF (3 mL) and shaken gently for 1 min. The activated amino acid solution was added to the resin-tethered amino acid and shaken gently for the reported period of time. The peptidyl-resin was then washed with DMF (7 mL, 3×1 min) and the Kaiser test255 was performed to monitor coupling success. Any incomplete reactions were repeated with extended reaction times (indicated in brackets). Upon negative test results for the presence of free amine, the resin-peptide was deprotected with 20% piperidine in DMF (7 mL, 1×1 min, 2×10 min) and washed again with DMF (7 mL, 5×1 min) to remove traces of base prior to coupling the next amino acid.
- The above procedure was repeated until the desired peptide sequence was constructed. Once complete, the resin was washed with DMF (7 mL, 3×1 min), DCM (7 mL, 3×1 min), MeOH (7 mL, 3×1 min), DCM (7 mL, 3×1 min), MeOH (7 mL, 3×1 min) and dried in vacuo for 1 h. A small aliquot of resin-tethered peptide was then exposed to a TFA cleavage solution (Section 7.3.3).
- In a fitted syringe, Rink amide resin was swollen with DCM (7 mL, 3×1 min, 1×60 min) and DMF (7 mL, 3×1 min, 1×30 min) and deprotected with 20% piperidine in DMF (7 mL, 1×1 min, 2×10 min) and washed again with DMF (7 mL, 5×1 min). NMM (6 equiv.) was added to a solution of a protected amino acid, Fmoc-L-Xaa-OH (3 equiv.) and HATU (2 equiv.) in DMF (3 mL) and shaken gently for 1 min. The activated amino acid solution was added to the resin and shaken gently for the reported period of time. The peptidyl-resin was washed with DMF (7 mL, 3×1 min) to ensure excess reagents were removed. Kaiser tests were performed to monitor coupling success and any incomplete reactions were repeated with extended reaction times (indicated in brackets). Upon negative test results for the presence of free amine, the resin-peptide was deprotected with 20% piperidine in DMF (7 mL, 1×1 min, 2×10 min) and washed again with DMF (7 mL, 5×1 min). This coupling procedure was repeated until the desired peptide sequence was constructed.
- The above procedure was repeated until the desired peptide sequence was constructed. Once complete, the resin was washed with DMF (7 mL, 3×1 min), DCM (7 mL, 3×1 min), MeOH (7 mL, 3×1 min), DCM (7 mL, 3×1 min), MeOH (7 mL, 3×1 min) and dried in vacuo for 1 h. A small aliquot of resin-tethered peptide was then exposed to a TFA cleavage solution (Section 7.3.3).
- The Kaiser test was performed in order to monitor coupling success by detecting the presence of resin-bound free amines.221,255 Two drops of 5% ninhydrin in EtOH, 80% phenol in EtOH and 2% v/v 0.001 M potassium cyanide in pyridine were added to pre-washed (EtOH) resin beads in a tube and the mixture was subsequently heated at 120° C. for 3-5 min. Blue colouration of the beads indicate the presence of free amines and provide evidence for an incomplete coupling reaction. It should be noted that this test cannot be performed after coupling asparagine, aspartic acid, serine and proline.221,256
- A small aliquot of the resin-peptide (˜1 mg) was suspended in a
cleavage solution 2 mL):90% TFA:5% thioanisole:2.5% EDT 2.5% water and phenol (1.6 g/5 mL of cleavage solution) and shaken gently for 1.5 h. The mixture was then filtered and the resin beads were rinsed with TFA (2×0.5 mL). The filtrate was concentrated with a constant stream of air to yield an oil. The peptide was precipitated with ice-cold Et2O (2 mL) and collected by centrifugation (3×10 min). The supernatant liquid was decanted and the resultant residue was collected and analysed by mass spectrometry. - The Ellman's test was performed in order to monitor reaction progress during thiol oxidation (cystine formation) by detecting the presence of free sulfhydryl groups.257 200 of a solution of Ellman's reagent in aqueous (NH4)2CO3 buffer (4 mg mL−1 in 0.1 M buffer) was added to 200 μL of the reacting peptide solution. An intense yellow colouration of the solution indicates the presence of free thiol groups and provides evidence for an incomplete oxidation reaction.
- Peptide synthesis was also performed on a CEM Liberty Peptide Synthesiser™ with a CEM Microwave Discover System™. Both systems were operated with the use of PepDriver software. The desired peptide sequence and methods were installed on PepDriver. The resin was weighed directly into a 50 mL centrifuge tube, DMF (5 mL) added, then the tube was screwed into position on the Liberty resin manifold. Amino acid solutions (0.2M in DMF) were prepared and installed onto the Liberty amino acid manifold. External reagents were prepared as described: A 0.45M solution of HOBt and HBTU in DMF was prepared as the activator reagent. A 20% v/v solution of piperidine in DMF was used at the deactivation reagent. Activator base reagent was prepared by making a 2M solution of DIEA in NMP. Delivery volumes of all external reagents were calibrated on the Liberty Peptide Synthesizer™ prior to use. The temperature of the Discover System™ was maintained via a fiber optic sensor located below the microwave cavity.
- For all automated synthesis, “B.01 Initial Deprotection” followed by “B.01 Extended Deprotection” cycles were used in the method. These deprotection cycles consisted of washing with DMF (1×7 mL), addition of the deprotection reagent (20% piperidine in DMF, 10 mL), followed by the “B.01 Initial Deprotection” microwave program. The peptidyl-resin was exposed to a temperature of 37° C. at a power of 37 watts for 2 min. The resin was then washed with DMF (12 mL) and a further 10 mL of the deprotection reagent was added followed by the “B.01 Extended Deprotection” cycle. The peptidyl-resin was exposed to a temperature of 75° C. at a power of 45 watts for 10 min. The resin was then washed with DMF (3×7 mL). Amino acid coupling cycles varied for each type of peptide and these are specified in each automated peptide synthesis description.
- Palladium on charcoal (Pd/C) with 10% Pd concentration was used as supplied by Aldrich and stored in a desiccator. Tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst, Rh(I)(PPh3)3Cl]) was used as supplied by Aldrich and stored under argon in a dry box. Asymmetric catalysts: (+)-1,2-Bis[(2S,5S)-2,5-diethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I) trifluoromethane-sulfonate ([(COD)Rh(I)—(S,S)-Et-DuPHOS]OTf, Rh(I)—(S,S)-Et-DuPHOS), (−)-1,2-bis[(2R,5R)-2,5-diethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I) tetra-fluoroborate ([(COD)Rh(I)—(R,R)-Et-DuPHOS]BF4, Rh(I)—(R,R)-Et-DuPHOS), (+)-1,2-bis [(2S,5S)-2,5-dimethylphospholano]benzene (1,5-cyclooctadiene)rhodium(I) trifluoromethanesulfonate ([(COD)Rh(I)—(S,S)-Me-DuPHOS] OTf, Rh(I)—(S,S)-Me-DuPHOS), (−)-1,2-bis[(2R,5R)-2,5-dimethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I)tetrafluoroborate ([(COD)Rh(I)—(R,R)-Me-DuPHOS]BF4, Rh(I)—(R,R)-Me-DuPHOS), and (+)-1,2-bis [(2R,5R)-2,5-dimethylphospholano]ethane(1,5-cyclooctadiene)rhodium(I) trifluoromethanesulfonate ([(COD)Rh(I)—(R,R)-Me-BPE]OTf, Rh(I)—(R,R)-Me-BPE) and bis(carboxylato)[2,2′-bis(diphenylphosphino)-(R)-1,1-binapthyl]ruthenium(II) ((S)—Ru—BINAP) were used as supplied by Strem Chemicals and stored under argon.
- Argon and hydrogen were supplied by BOC gases and were of high purity (<10 ppm oxygen). Additional purification was achieved by passage of the gases through water, oxygen and hydrocarbon traps.
- Benzene, MeOH, DCM, tBuOH and THF used in metal-catalysed hydrogenation reactions were degassed with high purity argon prior to use.
- Fischer-Porter shielded aerosol pressure reactors (100 mL) fitted with pressure gauge heads and stirrer beads were employed for hydrogenation reactions.
- A Fischer-Porter tube was charged with substrate, catalyst (substrate:catalyst, 50:1) and solvent (5-10 mL). The reaction vessel was connected to the hydrogenation manifold, evacuated and flushed with argon gas before being charged with hydrogen gas to the reported pressure. The reaction was stirred at the specified temperature for the reported period of time. The hydrogen gas was then vented, the catalyst removed via filtration through a Celite pad and the solvent evaporated under reduced pressure.
- In a dry box, a Fischer-Porter tube was charged with substrate, catalyst (substrate:catalyst, 100:1) and dry deoxygenated solvent (4-10 mL). The reaction vessel was assembled and tightly sealed within the dry box. The apparatus was connected to the hydrogenation manifold and purged three times using a vacuum and argon flushing cycle before being pressurised with hydrogen gas to the reported pressure. The reaction was then stirred at the specified temperature for the reported period of time. The hydrogen gas was vented and the solvent was evaporated under reduced pressure. Purification was achieved by flash chromatography (silica, EtOAc).
- For liquid substrates, a freeze-pump-thaw cycle was applied and the solution was transferred into a dry box and loaded into a Fischer-Porter tube as described above. The substrate was dissolved in MeOH or benzene in a Teflon-sealed vessel. The solution was frozen upon immersion in liquid nitrogen and opened to a vacuum source (high vacuum line ˜0.05 mm) to remove gases. The vessel was re-sealed and the solution was allowed to thaw before being frozen with liquid nitrogen again. This cycle was repeated until gas evolution was no longer observed during the thaw cycle.
- In a dry box, a Fischer-Porter tube was charged with substrate, Wilkinson's catalyst (substrate:catalyst, 50:1) and dry deoxygenated solvent (4-10 mL). The apparatus was connected to the hydrogenation manifold and purged three times using a vacuum and argon flushing cycle before being pressurised with hydrogen gas to the reported pressure. The reaction was then stirred at ambient temperature for the reported reaction time. The hydrogen gas was vented and the solvent was evaporated under reduced pressure. Purification was achieved by flash chromatography (silica, EtOAc).
- Hydrogenation experiments are described in the following format: substrate (mg), solvent (mL), catalyst, hydrogen pressure (psi), reaction temperature (° C.), reaction time (h), isolated yield (%), retention time (tR, GC/HPLC conditions) and enantiomeric excess (e.e.).
- Bis(tricyclohexylphosphine)(benzylidene)ruthenium(II) dichloride (Grubbs' catalyst), tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene](benzylidene)ruthenium(II) dichloride (second generation Grubbs' catalyst) and 1,3-bis(2,4,6-trimethylphenyl)-2-(imidazolidinylidene)dichloro-(o-iso-propoxyphenylmethylene)ruthenium(II) dichloride (second generation Hoveyda-Grubbs' second generation catalyst) were used as supplied by Aldrich and stored under nitrogen.
- Cis-2-butene (99%), cis+trans-2-butene (99%) 2-methylpropene (iso-butylene) and 2-methyl-2-butene were used as supplied by Aldrich. Ethylene was used as supplied by BOC gases.
- DCM and a solution of lithium chloride in DMF (0.4 M LiCl/DMF) used in metal-catalysed metathesis reactions were degassed with high purity argon prior to use.
- Schlenk tubes and microwave reactor vessels fitted with stirrer beads were employed for ring closing and cross metathesis reactions involving the use of solid or liquid (non-volatile) reactants. Fischer-Porter shielded aerosol pressure reactors (100 mL) fitted with pressure gauge heads and stirrer beads were employed for cross metathesis reactions involving gaseous (ethylene, cis-2-butene, iso-butylene) or volatile (2-methyl-2-butene) reactants.
- A Schlenk tube was charged with substrate(s), catalyst (5-40 mol %) and deoxygenated solvent (˜5 mL) under an inert (nitrogen or argon) atmosphere. The reaction mixture was stirred at 50° C. for the specified period of time. Metathesis reactions were terminated upon exposure to oxygen and volatile species were removed under reduced pressure. The crude product was purified by flash chromatography.
- A high pressure quartz microwave vessel was loaded with resin-tethered peptide, catalyst (5-40 mol %) and deoxygenated solvent (˜3-5 mL) under an inert (nitrogen and argon) atmosphere. The reaction mixture was irradiated with microwaves and stirred at 100° C. for the reported period of time. The mixtures were then filtered and washed with DMF (3 mL, 3×1 min), DCM (3 mL, 3×1 min), MeOH (3 mL, 3×1 min) and dried on the SPPS manifold for 1 h. A small aliquot of resin-peptide (˜1 mg) was then subjected to the TFA-mediated cleavage procedure (Section 7.3.3). The isolated peptide was analysed by mass spectrometry.
- Microwave-accelerated reactions were also performed on a CEM Discover System™. The instrument produces a continuous focused beam of microwave irradiation at a power delivery of 40 W. The temperature on the Discover System™ was monitored via an infra-red sensor located below the microwave cavity. Reactions were performed in a 10 mL high-pressure quartz vessel fitted with a self-sealing Teflon septa. The vessel was charged with the peptidyl-resin, degassed solvent (5 mL DCM and 0.2 mL 2M LiCl in DMF), 2nd generation Grubb's catalyst (20 mol %) in an inert environment. The reaction mixture was irradiated with microwave energy whilst being stirred at 100° C. for 1 hr, cooled to room temperature, then terminated upon exposure to oxygen. The peptidyl-resin was filtered through a fritted syringe and washed with DMF (5 mL, 3×1 min), DCM (5 mL, 3×1 min), DMF (5 mL, 3×1 min) then MeOH (5 mL, 3×1 min) and dried in vacuo for 30 min prior to cleavage and analysis.
- In a dry box, a Fischer-Porter tube was charged with substrate, catalyst (5-50 mol %) and deoxygenated solvent (˜5 mL). The reaction vessel was then evacuated and purged with ethylene, cis-2-butene or iso-butylene to the reported pressure. The reaction mixture was stirred at 50° C. for the specified period of time. Metathesis reactions were terminated upon exposure to oxygen and volatile species were removed under reduced pressure. The crude product was purified by flash chromatography.
- A Fischer-Porter tube was charged with substrate, catalyst (5 mol %), deoxygenated solvent (˜5 mL) and 2-methyl-2-butene. The reaction mixture was stirred at 50° C. for the specified period of time. Metathesis reactions were terminated upon exposure to oxygen and volatile species were removed under reduced pressure. The crude product was purified by flash chromatography.
- Metathesis experiments are described using the following format: substrate (mg), solvent (mL), catalyst (mg), reacting olefin (in the case of cross metathesis) reaction temperature (° C.), reaction time (h), percent conversion (%). Chromatographic purification conditions (isolated yield, %) are also listed.
- Hydrogenation and metathesis experiments performed on-resin were subjected to the conditions described above. Resin-based metathesis reactions were quenched with ethyl vinyl ether (0.5 mL, 5 min). The mixtures were then filtered, washed with DCM (3 mL, 3×1 min), MeOH (3 mL, 3×1 min) and dried on the SPPS manifold for 1 h. A small aliquot of resin-peptide (˜1 mg) was subjected to the TFA-mediated cleavage procedure (Section 7.33). The isolated peptide was analysed by mass spectrometry.
-
- The titled compound 42 was prepared according to a procedure described by Williams et al.195 A solution of acetamide 34 (6.10 g, 0.10 mol) and glyoxylic acid monohydrate 41 (10.60 g, 0.14 mol) in anhydrous acetone (150 mL) was heated at reflux for 18 h. The reaction mixture was evaporated under reduced pressure to afford the desired product 42 as a viscous yellow oil (13.75 g, 100%). Spectroscopic data indicated the crude product 42 did not require purification and was used directly in the subsequent reaction (Section 7.9.2). νmax (neat): 3317bs, 2974w, 1732s, 1668s, 1538s, 1379m, 1234w, 1112w, 1048m, 880m cm−1. 1H n.m.r. (300 MHz, D6-DMSO): δ 1.84 (s, 3H, CH3CO), 5.39 (d, J=8.7 Hz, 1H, H2), (8.65, bd, J=8.4 Hz, 1H, NH), two exchangeable protons (OH) not observed. 13C n.m.r. (75 MHz, D6-DMSO): δ 22.5 (CH3CO), 70.9 (C2), 169.4 (CONH), 171.5 (C1). Mass Spectrum (ESI+, MeOH): m/z 134.2 (M+H)+, C4H8NO4 requires 134.1. Spectroscopic data were in agreement with those reported in the literature.195
-
- The methyl ester 43 was prepared according to a procedure described by Legall et al.196 Concentrated H2SO4 (4.5 nit) was added to an ice-cooled solution of N-acetyl-2-hydroxyglycine 42 (13.69 g, 0.10 mol) in MeOH (150 mL). The solution was stirred for 2 d at room temperature then poured into an ice-cooled saturated NaHCO3 solution (400 mL). The mixture was extracted with EtOAc (3×250 mL) and the combined organic extract was dried (MgSO4) and evaporated under reduced pressure to yield the titled compound 43 as a yellow oil (9.57 g, 60%). Spectroscopic data indicated the crude product 43 did not require purification and was used directly in the subsequent reaction (Section 7.9.3). νmax (neat): 3334bm, 2955w, 1753s, 1671s, 1528m, 1439m, 1375m, 1221m, 1088m, 783w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.10 (s, 3H, CH3CO), 3.47 (s, 3H, OCH3), 3.82 (s, 3H, COOCH3), 5.55 (d, J=9.3 Hz, 1H, H2), 6.72 (bd, J=8.1 Hz, 1H, NH). 13C n.m.r. (75 MHz, CDCl3): δ 23.3 (CH3CO), 53.0 (OCH3), 56.8 (COOCH3), 78.3 (C2), 168.7 (CONH), 170.8 (C1). Mass Spectrum (ESI+, MeOH): m/z 184.1 (M+Na)+, C6H11NNaO4 requires 184.2. Spectroscopic data were in agreement with those reported in the literature.196
-
- The phosphinyl compound 39 was prepared according to a procedure described by Schmidt et al.197 Phosphorus (III) chloride (3.91 mL, 44.6 mmol) was added to a solution of methyl N-acetyl-2-methoxyglycinate 43 (7.19 g, 44.6 mmol) in toluene (100 mL) at 70° C. and the mixture was stirred at this temperature for 17 h. Trimethyl phosphite (5.27 mL, 44.7 mmol) was then added dropwise and the reaction mixture was left to stir for 2 h at 70° C. The mixture was evaporated under reduced pressure and the resultant oil was re-dissolved in DCM (100 mL) and washed with saturated NaHCO3 solution (3×100 mL). The organic extract was dried (MgSO4) and evaporated under reduced pressure to afford the product 39 as a colourless solid (1.46 g, 14%). The combined aqueous layers were extracted in a continuous extractor with DCM (150 mL) for 3 d. The organic layer was then evaporated under reduced pressure to give the product 39 as a colourless solid (3.21 g, 30%) (44% combined yield), m.p. 89-91° C. (lit.197 88-89° C.). Spectroscopic data indicated the crude product 39 did not require purification and was used directly in the subsequent reaction (Section 7.94). νmax (KBr): 3281m, 3050w, 2852w, 1749m, 1673m, 1540m, 1309m, 1287w, 1232w, 1133m, 1061m, 1028m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.08 (s, 3H, CH3CO), 3.80-3.85 (m, 9H, COOCH3, 2×P—OCH3), 5.23 (dd, J=22.2, 8.9 Hz, 1H, H2), 6.42 (d, J=8.8 Hz, 1H, NH). 13C n.m.r. (75 MHz, CDCl3): δ 22.7 (CH3CO), 50.0 (d, J=146.8 Hz, C2), 53.3 (COOCH3), 54.1 (d, J=6.8 Hz, P—OCH3), 54.2 (d, J=6.5 Hz, P—OCH3), 167.0 (d, J=2.2 Hz, CONH), 169.0 (d, J=6.0 Hz, C1). Mass Spectrum (ESI+, MeOH): m/z 262.1 (M+Na)+, C6H11NNaO4 requires 262.2.
-
- The dienamide 57 was prepared according to a procedure described by Teoh et al.119 Tetramethylguanidine (3.22 mL, 25.7 mmol) and hydroquinone (10.0 mg) were added to a solution of methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39 (4.63 g, 19.4 mmol) in THF (60 mL) at −78° C. After 15 min, acrolein 58 (1.55 mL, 23.2 mmol) was added and the mixture was stirred at −78° C. for 2 h and then warmed to 25° C. and allowed to react an additional 2 h. The reaction mixture was diluted with DCM (100 mL) and washed with dilute HCl solution (1 M, 2×80 mL), CuSO4 solution (1 M, 2×80 mL), saturated NaHCO3 solution (2×80 mL) and saturated NaCl solution (1×80 mL). The organic layer was dried (MgSO4) and evaporated under reduced pressure to give the desired dienamide 57 as an off-white solid (2.78 g, 85%), m.p. 60-62° C. (lit.119 61-63° C.). Spectroscopic data indicated the crude product 57 did not require purification and was used directly in the subsequent reaction (Section 7.11.2). νmax (KBr): 3277m, 3011m, 2955m, 1733s, 1655s, 1594m, 1518s, 1438m, 1377w, 1350w, 1250w, 1113s, 1016m, 994m, 950s, 768m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.16 (s, 3H, CH3CO), 3.81 (s, 3H, OCH3), 5.49 (d, J=9.9 Hz, 1H, H5-E), 5.61 (d, J=17.1 Hz, 1H, H5-Z), 6.47 (m, 1H, H4), 7.05 (d, J=11.1 Hz, 1H, H3), 7.07 (bs, 1H, NH). 13C n.m.r. (75 MHz, CDCl3): δ 23.6 (CH3CO), 52.7 (OCH3), 123.7 (C2), 125.2 (C5), 132.0 (C4), 132.9 (C3), 165.5, 168.9 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 170.2 (M+1V, C8H12NO3 requires 170.2. Spectroscopic data were in agreement with those reported in the literature.119
-
- The dienamide 57 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 (108 mg, 0.64 mmol), benzene (7 mL), Rh(I)—(S,S)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give a yellow oil (106 mg, 97%). 1H n.m.r. spectroscopy confirmed formation of the desired product 21a and the fully saturated compound, (2S)-methyl 2-N-acetylaminopentanoate 59 (δ 0.93 (t, J=7.3 Hz, 3H, H5), 1.25-1.44 (m, 4H, H3, 4)), in a 97:3 ratio respectively. GC: (2S)-21a tR=18.6 min (GC chiral column 50 CP2/XE60-SVALSAPEA, 100° C. for 1 min, 5° C. min−1 to 280° C. for 9 min), 95% e.e. [α]D 22+45.0° (c=0.76, CHCl3) containing 3% of 59 (lit.208 for (S)-21a [α]D 22+45.4° (c=3.57, CHCl3)). νmax (neat): 3278s, 3079w, 2955w, 1744s, 1657s, 1546m, 1438m, 1375m, 1275w, 1226w, 1151m, 997w, 924w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.00 (s, 3H, CH3CO), 2.43-2.62 (m, 2H, H3), 3.73 (s, 3H, OCH3), 4.67 (dt, J=11.6, 5.8 Hz, 1H, H2), 5.08 (m, 1H, H5-E), 5.14 (m, 1H, H5-Z), 5.67 (m, 1H, H4), 6.06 (bs, 1H, NH). 13C n.m.r. (100 MHz, CDCl3): δ 23.1 (CH3CO), 36.5 (C3), 51.8 (C2), 52.4 (OCH3), 119.2 (C5), 132.3 (C4), 169.9, 172.4 (C1, CONH). Mass Spectrum (EST+, MeOH): m/z 194.1 (M+Na)+, C8H13NNaO3 requires 194.2. Spectroscopic data were in agreement with those reported in the literature.119
-
- The dienamide 57 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 (40.0 mg, 0.24 mmol), benzene (5 mL), Rh(I)—(R,R)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give as a yellow oil (36.0 mg, 88%). 1H n.m.r. spectroscopy confirmed formation of the desired product 21a and the fully saturated compound, (2R)-methyl 2-N-acetylaminopentanoate 59 in a 95:5 ratio respectively GC: (2R)-21a tR=18.2 min (GC chiral column 50 CP2/XE60-SVALSAPEA, 100° C. for 1 min, 5° C. min−1 to 280° C. for 9 min), 95% e.e. [α]D 22−43.0° (c=0.47, CHCl3) containing 5% of 59. Spectroscopic data were in agreement with those previously reported for the (S)-enantiomer.
-
- The titled compound 65 was prepared according to a procedure described by Zoller et al.209 A mixture of benzamide 35 (5.00 g, 41.3 mmol) and glyoxylic acid monohydrate 41 (4.32 g, 46.9 mmol) in anhydrous acetone (70 mL) was heated at reflux for 19 h. The reaction mixture was evaporated under reduced pressure to afford the desired product 65 as a colourless solid (8.06 g, 100%), m.p. 198-200° C. (lit.209 200-201° C. (dec)) Spectroscopic data indicated the crude product 65 did not require purification and was used directly in the subsequent reaction (Section 7.11.4). νmax (KBr): 3310bs, 3058w, 1728s, 1646s, 1602w, 1582w, 1535s, 1491w, 1452w, 1315m, 1292w, 1254m, 1161m, 1097s, 1040m, 1002w, 957m, 805w, 770w, 728m, 692m, 654m, 609w, 515m, 483w cm−1. 1H n.m.r. (300 MHz, D6-DMSO): δ 5.60 (d, J=7.8 Hz, 1H, H2), 7.41-7.49 (m, 2H, H3′, 5), 7.55 (m, 1H, H4′), 7.86-7.92 (m, 2H, H2′, 6′), 9.26 (d, J=7.8 Hz, 1H, NH), two exchangeable OH protons not observed. 13C n.m.r. (75 MHz, D6-DMSO): δ 71.7 (C2), 127.6, 128.3, 131.7 (Arom CH), 133.7 (C1′), 166.0, 171.5 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 218.2 (M+Na)+, C9H9NNaO4 requires 218.2.
-
- The methyl ester 66 was prepared according to a procedure described by Zoller et al.209 Concentrated H2SO4 (2.0 mL) was added to an ice-cooled solution of N-benzoyl-2-hydroxyglycine 65 (8.05 g, 41.3 mmol) in MeOH (65 mL). The solution was stirred for 48 h at ambient temperature then poured into an ice-cooled saturated NaHCO3 solution (100 mL). The mixture was extracted with EtOAc (3×100 mL) and the combined organic extract was dried (MgSO4) and evaporated under reduced pressure to yield the titled compound 66 as a yellow oil (8.00 g, 87%). Spectroscopic data indicated the crude product 66 did not require purification and was used directly in the subsequent reaction (Section 7.11.5). νmax (neat): 3310bm, 2955m, 2837w, 1760s, 1651s, 1603w, 1580w, 1525s, 1488m, 1439m, 1338w, 1286w, 1226w, 1198w, 1147w, 1108m, 1022w, 924m, 850w, 803m, 778m, 692m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 3.54 (s, 3H, OCH3), 3.85 (s, 3H, COOCH3), 5.78 (d, J=9.0 Hz, 1H, H2), 7.22 (bd, J=9.0 Hz, 1H, NH), 7.42-7.51 (m, 2H, H3′, 5), 7.56 (m, 1H, H4′), 7.80-7.88 (m, 2H, H2′, 6′). 13C n.m.r. (75 MHz, CDCl3): δ 53.2 (OCH3), 57.0 (COOCH3), 78.8 (C2), 127.4, 128.9, 132.5 (Arom CH), 133.2 (C1′), 167.6, 168.7 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 224.2 (M+H)+, C11H14NO4 requires 224.2; m/z 246.3 (M+Na)+, C11H13NNaO4 requires 246.2. Spectroscopic data were in agreement with those reported in the literature.209
-
- The phosphinyl compound 64 was prepared according to a procedure described by Teoh et al.119 Phosphorus (III) chloride (3.15 mL, 36.0 mmol) was added to a solution of methyl N-benzoyl-2-methoxyglycinate 66 (8.00 g, 35.9 mmol) in toluene (70 mL) at 70° C. and the mixture was stirred at this temperature for 14 h. Trimethyl phosphite (4.25 mL, 36.0 mmol) was added dropwise and the reaction mixture was left to stir for 2 h at 70° C. At the end of the reaction period, the mixture was evaporated under reduced pressure and the resultant oil was re-dissolved in DCM (100 mL) and washed with saturated NaHCO3 solution (3×70 mL). The organic extract was isolated, dried (MgSO4) and evaporated under reduced pressure to afford the titled compound 64 as a colourless solid (8.21 g, 76%), m.p. 110-112° C. (lit.210 112-114° C.). Spectroscopic data indicated the crude product 64 did not require purification and was used directly in the subsequent reaction (Section 7.11.6). νmax (KBr): 3300m, 3248m, 3059w, 2958m, 2852w, 1737s, 1671s, 1638m, 1618w, 1602w, 1581w, 1541s, 1492m, 1432w, 1292s, 1235s, 1188w, 1152w, 1044s, 915m, 881w, 832m, 812w, 791w, 780w, 758m, 708m, 616w, 562m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 3.82-3.90 (m, 9H, COOCH3, 2×P—OCH3), 5.47 (dd, J=21.9, 9.0 Hz, 1H, H2), 6.97 (bd, J=7.8 Hz, 1H, NH), 7.43-7.49 (m, 2H, H3′, 5′), 7.56 (m, 1H, H4′), 7.82-7.87 (m, 2H, H2′, 6′). 13C n.m.r. (75 MHz, CDCl3): δ 50.6 (d, J=147.1 Hz, C2), 53.5 (COOCH3), 54.2 (d, J=6.8 Hz, P—OCH3), 127.4, 128.8, 132.3 (Arom CH), 133.1 (C1′), 166.9 (d, J=5.4 Hz, C1), 167.3 (d, J=2.0 Hz, CONH). Mass Spectrum (ESI+, MeOH): m/z 302.2 (M+H)+, C12H17NO6P requires 302.2; m/z 324.2 (M+Na)+, C12H16NNaO6P requires 324.2.
-
- The dienamide 63 was prepared according to a procedure described by Teoh et al.119 Tetramethylguanidine (4.35 mL, 34.7 mmol) and hydroquinone (12.0 mg) were added to a solution of methyl 2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 (7.79 g, 25.9 mmol) in THF (120 mL) at −78° C. After 30 min, acrolein 58 (2.10 mL, 31.4 mmol) was added and the mixture was stirred at −78° C. for 2 h and then warmed to 25° C. and allowed to react an additional 2 h. The reaction mixture was diluted with DCM (150 mL) and washed with dilute HCl solution (1 M, 2×100 mL), CuSO4 solution (1 M, 2×100 mL), saturated NaHCO3 solution (2×100 mL) and saturated NaCl solution (1×100 mL). The organic extract was dried (MgSO4) and evaporated under reduced pressure to give the crude product 63 as a waxy-brown solid. Purification by flash chromatography (SiO2, light petroleum:EtOAc:DCM, 3:2:2) furnished the pure enamide 63 as colourless needles (4.78 g, 80%), m.p. 138-141° C. (dec). (KBr): 3288bm, 2952m, 2361w, 1727s, 1652s, 1602w, 1580w, 1514s, 1484s, 1436w, 1257s, 1196w, 1074w, 1028w, 991w, 931w, 800w, 737m, 710m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 3.83 (s, 3H, OCH3), 5.50 (dd, J=10.0, 1.7 Hz, 1H, H5-K), 5.64 (dd, J=16.8, 1.7 Hz, 1H, H5-Z), 6.56 (ddd, J=17.1, 11.4, 10.2 Hz, 1H, H4), 7.14 (d, J=11.2 Hz, 1H, H3), 7.45-7.51 (m, 2H, H3′, 5′), 7.56 (m, 1H, H4′), 7.78 (bs, 1H, NH), 7.88-7.91 (m, 2H, H2′, 6′). 13C n.m.r. (75 MHz, CDCl3): δ 52.8 (OCH3), 123.6 (C2), 125.2 (C5), 127.6 (CT, 6′), 128.9 (C3′, 5′), 132.2, 132.2, 132.3 (C3, 4, 4′), 133.9 (C1′), 165.6, 165.8 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 232.1 (M+H)+, C13H14NO3 requires 232.3; m/z 254.2 (M+Na)+, C13H13NNaO3 requires 254.2. Spectroscopic data were in agreement with those reported in the literature.211
-
- The dienamide 63 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 (100 mg, 0.43 mmol), benzene (8 mL), Rh(I)—(S,5)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give a pale yellow oil (100 mg, 99%). 1H n.m.r. spectroscopy confirmed formation of the desired product 62 and the fully saturated compound, (2S)-methyl 2-N-benzoylaminopentanoate 68 (δ 0.95 (t, J=7.3 Hz, 3H, H5), 1.36-1.50 (m, 2H, H4), 1.90-1.96 (m, 2H, H3)), in a 93:7 ratio respectively. GC: (2S)-62 tR=27.0 min (GC chiral column 50 CP2/XE60-SVALSAPEA, 180° C. for 1 min, 2° C. min−1 to 210° C. for 20 min), 100% e.e. [α]D 22+49.3° (c=1.12, CHCl3) containing 7% of 68. νmax (neat): 3325bw, 3062w, 2955w, 2360w, 1743s, 1644s, 1603w, 1580w, 1538m, 1489m, 1438w, 1360w, 1268w, 1225w, 1159w, 1075w, 1028w, 925m, 802w, 714w, 668w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 2.63-2.73 (m, 2H, H3), 3.79 (s, 3H, OCH3), 4.91 (m, 1H, H2), 5.15 (m, 1H, H5-E), 5.18 (m, 1H, H5-Z), 5.75 (m, 1H, H4), 6.67 (bd, J=7.0 Hz, 1H, NH), 7.42-7.46 (m, 2H, H3′, 5′), 7.52 (m, 1H, H4′), 7.78-7.81 (m, 2H, H2′, 6′). 13C n.m.r. (100 MHz, CDCl3): δ 36.8 (C3), 52.1 (C2), 52.6 (OCH3), 119.5 (C5), 127.2 (C2′, 6′), 128.8 (C3′, 5′), 131.9 (C4), 132.4 (C4′), 134.1 (C1′), 167.0, 172.4 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 234.3 (M+H)+, C13H16NO3 requires 234.3; m/z 256.2 (M+Na)+, C13H15NNaO3 requires 256.3. Spectroscopic data were in agreement with those reported in the literature.212
-
- The dienamide 63 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 (100 mg, 0.43 mmol), benzene (8 mL), Rh(I)—(R,R)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give a yellow oil (93.8 mg, 93%). 1H n.m.r. spectroscopy confirmed formation of the desired product 62 and the fully saturated compound, (2R)-methyl 2-N-benzoylaminopentanoate 68, in a 91:9 ratio respectively. GC: (2R)-62 tR=26.4 min (GC chiral column 50 CP2/XE60-SVALSAPEA, 180° C. for 1 min, 2° C. min−1 to 210° C. for 20 min), 100% e.e. [α]D 22−49.7° (c=0.64, CHCl3) containing 9% of 68. Spectroscopic data were in agreement with those previously reported for the (S)-enantiomer.
-
- The dienamide 76 was prepared according to a procedure described by Burk et al.117 Tetramethylguanidine (0.70 mL, 5.58 mmol) was added to a solution of methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 64 (1.00 g, 4.18 mmol) in THF (50 mL) at −78° C. After 15 min, trans-cinnamaldehyde 78 (0.63 mL, 5.00 mmol) was added and the mixture was stirred at −78° C. for 2 h, warmed to 25° C. and allowed to react an additional 2 h. The reaction mixture was diluted with DCM (100 mL) and washed with dilute HCl solution (1 M, 2×75 mL), CuSO4 solution (1 M, 2×75 mL), saturated NaHCO3 solution (2×75 mL) and saturated NaCl solution (1×75 mL). The organic layer was dried (MgSO4) and evaporated under reduced pressure to give the crude product 76 as a waxy solid (0.87 g). Purification by recrystallisation from a mixture of light petroleum, EtOAc and DCM furnished the pure dienamide 76 as an off-white solid (0.76 g, 74%), m.p. 180-181° C. (lit.117 179-180° C.). νmax (KBr): 3263w, 1721s, 1662s, 1517s, 1439m, 1368m, 1286m, 1229s, 1192w, 1116m, 993m, 769w, 752m, 728w, 692m, 600w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.20 (s, 3H, CH3CO), 3.82 (s, 3H, OCH3), 6.89-6.91 (m, 2H, H3, 4), 7.01 (m, 1H, H5), 7.22 (bd, J obscured by residual CHCl3 peak, 1H, NH), 7.29-7.37 (m, 3H, H3′, 4′, 5′), 7.45-7.48 (m, 2H, H2′, 6′). 13C n.m.r. (100 MHz, CDCl3): δ 23.9, (CH3CO), 52.7 (OCH3), 123.0 (C2), 124.0, 127.5, 128.9, 129.2, 132.8, 140.2 (Arom CH, C3, 4, 5), 136.5 (C1′), 165.6, 168.7 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 246.2 (M+H)+, C14H16NO3 requires 246.3. Spectroscopic data were in agreement with those reported in the literature.117
-
- The dienamide 76 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 76 (28.0 mg, 0.11 mmol), MeOH (7 mL), Rh(I)—(S,5)-Et-DuPHOS, 90 psi, 22° C., 2 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give a yellow oil (27.2 mg, 96%). 1H n.m.r. spectroscopy confirmed formation of the desired product 77 and the fully saturated compound, (2S)-methyl 2-N-acetylamino-5-phenylpentanoate 79 (δ 1.60-1.87 (m, 4H, H3, 4), 2.48-2.53 (m, 2H, H5), 3.72 (s, 3H, OCH3), 4.65 (m, 1H, H2)), in a 91:9 ratio respectively. [α]D 22+90.0° (c=0.64, CHCl3) containing 9% of 79. νmax (neat): 3280bw, 3070m, 2960w, 2350w, 1745s, 1648s, 1605w, 1575w, 1550m, 1478m, 1440w, 1369w, 1270w, 1225w, 1153w, 1075w, 1028w, 925m, 805w, 720w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 2.02 (s, 3H, CH3CO), 2.64-2.78 (m, 2H, H3), 3.76 (s, 3H, OCH3), 4.77 (m, 1H, H2), 6.00-6.09 (m, 214, H4, NH), 6.45 (d, J=15.8 Hz, 1H, H5), 7.20-7.34 (m, 5H, Arom CH). 13C n.m.r. (100 MHz, CDCl3): δ 23.3 (CH3CO), 36.0 (C3), 52.1, 52.6 (C2, OCH3), 123.6, 126.4, 127.8, 128.7, 134.3 (Arom CH, C4, 5), 136.9 (C1′), 171.5, 172.5 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 248.1 (M+H)+, C14H18NO3 requires 248.2. Spectroscopic data were in agreement with those reported in the literature.117
-
- The dienamide 76 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (27.4 mg, 0.11 mmol), MeOH (5 mL), Rh(I)—(R,R)-Et-DuPHOS, 90 psi, 22° C., 2 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give a yellow oil (25.7 mg, 93%). 1H n.m.r. spectroscopy confirmed formation of the desired product 77 and the fully saturated compound, (2R)-methyl 2-N-acetylamino-5-phenylpentanoate 79 in a 87:13 ratio respectively. [α]D 22−89.8° (c=1.03, CHCl3) containing 13% of 79. Spectroscopic data were in agreement with those previously reported for the (S)-enantiomer.
-
- The preparation of (2Z)-methyl 2-N-acetylamino-5-methylhexa-2,4-dienoate 20 from the phosophonate 39 has been previously described (Section 7.9.4).
-
- The preparation of (2S)-methyl 2-N-acetylamino-5-methylhex-4-
enoate 19 via asymmetric hydrogenation of dienoate 20 has been previously described (Section 7.9.5). -
- The
dimer 60 was prepared via the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (95.0 mg, 0.56 mmol), DCM (4 mL), Grubbs' catalyst (91.0 mg, 0.11 mmol, 20 mol %), 50° C., 20 h, 100% conversion into 60. Purification by flash chromatography (SiO2, DCM:light petroleum:EtOAc, 1:1:1→10% MeOH:DCM) furnishedpure dimer 60 as a brown oil (76.7 mg, 88%). GC: tR (E/Z)=12.7 min, 12.8 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). [α]D 22+92.0° (c=0.004, CHCl3). νmax (neat): 3286bm, 2956m, 2931m, 2856w, 1742s, 1659s, 1542m, 1438m, 1375m, 1267m, 1220m, 1138w, 1017w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.04 (s, 6H, CH3CO), 2.40-2.50 (m, 4H, H3, 6), 3.74 (s, 6H, OCH3), 4.64-4.70 (m, 2H, H2, 7), 5.36-5.40 (m, 2H, H4, 5), 6.34 (bd, J=7.2 Hz, 2H, NH). 13C n.m.r. (100 MHz, CDCl3): δ 23.1 (CH3CO), 35.1 (C3, 6), 51.7 (C2, 7), 52.6 (OCH3), 128.8 (C4, 5), 170.3, 172.6 (C1, 8, CONH). HRMS (ESI+, MeOH). Found: m/z 337.1375 (M+Na)+, C14H22N2NaO6 requires 337.1376. Spectroscopic data were in agreement with those reported in the literature.264 -
- The dimer 69 was prepared via the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (49.0 mg, 0.21 mmol), DCM (5 mL), Grubbs' catalyst (34.6 mg, 42.1 μmol, 20 mol %), 50° C., 18 h, 100% conversion into 69. Purification by flash chromatography (SiO2, DCM:light petroleum:EtOAc, 1:1:1) gave pure dimer 69 as a pale brown solid (37.8 mg, 82%), m.p. 140-142° C. GC: tR (E/Z)=13.5, 13.9 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). [α]D 22+56.4°=0.27, CHCl3). νmax (KBr): 3322bm, 2953m, 2358w, 1742s, 1644s, 1603w, 1580w, 1538m, 1488m, 1436m, 1267w, 1218m, 1027w, 973w, 802w, 736m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.57-2.69 (m, 4H, H3, 6), 3.67 (s, 6H, OCH3), 4.85-4.98 (m, 2H, H2, 7), 5.49 (t, J=4.1 Hz, 2H, H4, 5), 6.86 (bd, J=7.4 Hz, 2H, NH), 7.40-7.44 (m, 4H, H3′, 5′), 7.48-7.52 (m, 2H, H4′), 7.81-7.83 (m, 4H, H2′, 6′). 13C n.m.r. (100 MHz, CDCl3): δ 35.2 (C3, 6), 52.5 (C2, 7), 52.6 (OCH3), 127.2 (C2′, 6′), 128.7 (C3′, 5′), 128.8 (C4, 5), 131.9 (C4′), 133.9 (C1′), 167.1, 172.4 (C1, 8, CONH). HRMS (ESI+, MeOH). Found: m/z 461.1695 (M+Na)+, C24H26N2NaO6 requires 461.1689.
- The dimer 69 was also prepared via the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-benzoylaminopent-4-enoate 142, DCM (5 mL), Grubbs' catalyst (20 mol %), 50° C., 20 h, 100% conversion into 69.
-
- The
olefinic mixture 62 and 60 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: - (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (37.0 mg, 0.16 mmol), (2S,7S)-
dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60 (29.5 mg, 93.9 μmol), DCM (3 mL), 2nd generation Grubbs' catalyst (13.5 mg, 15.9 μmol, 10 mol %), 50° C., 15 h. Spectroscopic data indicated the presence of the starting acetyl-allylglycine dimer 60, the benzoyl-allylglycine dimer 69 and additional peaks which mass spectrometry indicated could be attributed to the “mixed” cross metathesis product, (2S,7S)-dimethyl 2-N-acetylamino-7-N-benzoylaminooct-4-enedioate 70. 1H n.m.r. spectroscopic data for thehomodimers 60 and 69 were in agreement with those previously reported (Section 7.12.1 and Section 7.12.2). The heterodimer 70 was detected by mass spectrometry. Mass spectrum (ESI+, MeOH): m/z 337.2 (M+Na)+ 60, C14H22N2NaO6; m/z 399.3 (M+Na)+ 70, C19H24N2NaO6; m/z 461.2 (M+Na)+ 69, C24H26N2NaO6 requires 461.1689. - (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (37.0 mg, 0.16 mmol), (2S,7S)-
dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60 (30.0 mg, 95.5 μmol), DCM (4 mL), Grubbs' catalyst (26.1 mg, 31.7 μmol, 20 mol %), 50° C., 18 h, 100% conversion of 62 into dimer 69.Dimer 60 was recovered unchanged. Spectroscopic data fordimers 60 and 69 were in agreement with those previously reported (Section 7.12.1 and Section 7.12.2). No “mixed” cross metathesis product, heterodimer 70, was observed. -
- The dienamide 57 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 (33.0 mg, 0.20 mmol), DCM (3 mL), Grubbs' catalyst (34.0 mg, 41.3 μmol, 20 mol %), 50° C., 15 h, 0% conversion into dimer 61. The dienamide 57 did not react under these conditions. 1H n.m.r. spectroscopic data for the recovered dienamide 57 were in agreement with those previously reported (Section 7.11.1).
-
- The mixture of olefins 21a and 57 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (34.0 mg, 0.20 mmol), (2Z)-methyl 2-N-acetylaminopenta-2,4-dienoate 57 (33.6 mg, 0.20 mmol), DCM (4 mL), Grubbs' catalyst (16.3 mg, 19.8 μmol, 10 mol %), 50° C., 18 h. The 1H n.m.r. spectrum displayed peaks characteristic of the starting allylglycine derivative 21a and dienamide 57 but no peaks characteristic of expected
dimer 60. The mass spectrum displayed peaks attributed to the allylglycine derivative 21a and the tricyclohexylphosphine-dienamide conjugate addition adduct, (2S)-methyl 2-N-acetylamino-5-tricyclohexylphosphinylpent-2-enoate 143. Mass Spectrum (ESI+, DCM/MeOH): m/z 194.1 (M+Na)+ 21a, C8H13NNaO3; m/z 450.4 (M+)143, C26H45NO3P+. -
- The mixture of olefins 57 and 62 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (46.0 mg, 0.20 mmol), (2Z)-methyl 2-N-acetylaminopenta-2,4-dienoate 57 (33.4 mg, 0.20 mmol), DCM (4 mL), 2nd generation Grubbs' catalyst (16.8 mg, 19.8 μmol, 10 mol %), 50° C., 4.5 h. The reaction mixture was evaporated under reduced pressure to afford a dark brown oil (97.9 mg). The 1H n.m.r. spectrum displayed peaks characteristic of the starting allylglycine derivative 62, dienamide 57, traces of the target allylglycine dimer 69 and additional peaks which were difficult to characterise. Mass spectrometry displayed peaks attributed to the allylglycine derivative 62, dienamide 57, allylglycine dimer 69, dienamide dimer (2S,7S)-
dimethyl 2,7-N,N′-diacetylaminooct-2,4,6-trienedioate 61, “mixed” dienamide-allylglycine dimer (2S,7S)-dimethyl 2-N-acetylamino-7-N-benzoyl-aminoocta-2,4-dienedioate 144 and the tricyclohexylphosphine-dienamide conjugate addition adduct 143. - Mass Spectrum (ESI+, DCM/MeOH): m/z 256.1 (M+Na)+ 62, C13H15NNaO3; m/z 337.3 (M+Na)+ 61, C14H18N2NaO6; m/z 397.3 (M+Na)+ 144, C19H22N2NaO6; m/z 450.4 (M)+ 143, C26H45NO3P+; m/z 461.3 (M+Na)+ 69, C24H26N2NaO6 requires 461.5.
-
- In a dry box, a Teflon-sealed n.m.r. tube was charged with (2S)-methyl 2-N-acetylaminopenta-2,4-dienoate 57 (10.8 mg, 63.9 μmol), Grubbs' catalyst (50.7 mg, 61.6 μmol) and degassed deuterated DCM (CD2Cl2, 0.8 mL) at room temperature. The n.m.r. tube was shaken gently and reaction progress was monitored by 1H and 31P n.m.r. spectroscopy. Compounds were identified by the following diagnostic resonances: 1H n.m.r. (300 MHz, CD2Cl2): After 15 min: Grubbs' catalyst: δ 8.61 (d, J=7.6 Hz, 2H, ortho-Arom CH), 20.05 (s, 1H, [Ru]═CHPh); Ruthenium-dienamide complex 73: δ 7.96 (d, J=11.0 Hz, 1H, [Ru]═CH═CH), 20.11 (d, J=11.0 Hz, 1H, [Ru]═CH); Ruthenium-dienamide chelate 74 (trace): δ 15.20 (d, J=4.2 Hz, 1H, [Ru]═CH); Ratio of ruthenium complexes [Ru]═CHPh:73:74=1.0:1.0:<0.1. After 60 min: Grubbs' catalyst: δ 8.45 (d, J=7.6 Hz, 2H, ortho-Arom CH), 20.04 (s, 1H, [Ru]═CHPh); Ruthenium-dienamide complex 73: δ 7.96 (d, J=11.0 Hz, 1H, [Ru]═CH═CH), 20.10 (d, J=11.0 Hz, 1H, [Ru]═CH); Ruthenium-dienamide chelate 74: δ 6.73 (d, J=3.0 Hz, 1H, [Ru]═CH═CH), 15.19 (d, J=4.2 Hz, 1H, [Ru]═CH); Ratio of ruthenium complexes [Ru]═CHPh:73:74=3:1:1. After 120 min (no change after 18 h): Ruthenium-dienamide chelate 74: δ 6.71 (d, J=3.0 Hz, 1H, [Ru]═CH═CH), 15.19 (d, J=4.0 Hz, 1H, [Ru]═CH). 31P n.m.r. (300 MHz, CDCl3): δ Ruthenium-dienamide chelate 74:35.0; Grubbs' catalyst: 37.0; Ruthenium-dienamide complex 73: 38.8; Tricyclohexylphosphine oxide: 46.5.
-
- In a dry box, a Teflon-sealed n.m.r. tube was charged with (2S)-methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (10.0 mg, 40.8 μmol), Grubbs' catalyst (33.6 mg, 40.9 μmol) and degassed CD2Cl2 (0.8 mL) at room temperature. The n.m.r. tube was shaken gently and reaction progress was monitored by 1H n.m.r. spectroscopy. After 4 h, ruthenium-vinylalkylidene formation was not observed and only peaks corresponding to Grubbs' catalyst and the starting dienamide 76 were present.
-
- The mixture of olefins 21a and 76 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (18.1 mg, 0.11 mmol), (2Z)-methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (26.1 mg, 0.11 mmol), DCM (4.0 mL), Grubbs' catalyst (8.7 mg, 10.6 μmol, 10 mol %), 50° C., 18 h, 28% conversion of allylglycine 21a into 60. Dienamide 76 did not react under these conditions. n.m.r. spectroscopic data for dienamide 76,
dimer 60 and recovered allylglycine derivative 21a were in agreement with those previously reported (Section 7.11.8, Section 7.12.1 and Section 7.11.2 respectively). -
- The enamide 77 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions:
- (2S)-Methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 (59.3 mg, 0.24 mmol), DCM (10 mL), Grubbs' catalyst (19.8 mg, 24.1 μmol, 10 mol %), 50° C., 13 h, 0% conversion into
dimer 60. The starting enamide 77 was recovered. 1H n.m.r. spectroscopic data for olefin 77 were in agreement with those previously reported (Section 7.11.9). - (2S)-Methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 (59.3 mg, 0.24 mmol), DCM (7 mL), 2nd generation Grubbs' catalyst (10.2 mg, 12.0 μmol, 5 mol %), 50° C., 20 h, 44% conversion into
dimer 60. 1H n.m.r, spectroscopic data fordimer 60 were in agreement with those previously reported (Section 7.12.1). The stilbene byproduct 145 was observed in the 1H n.m.r. spectrum. 1H n.m.r. (300 MHz, CDCl3): 7.15 (s, 2H, CH═), 7.40 (m, 4H, Arom CH), 7.55 (m, 4H, Arom CH), ortho-Arom CH peaks masked by starting olefin 77. 1H n.m.r. spectroscopic data for stilbene 145 were in agreement with those reported in the literature.265 -
- The mixture of
olefins 21a and 19 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (12.7 mg, 74.2 μmol), (2S)-methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (14.5 mg, 72.9 μmol), DCM (4 mL), Grubbs' catalyst (11.5 mg, 14.0 μmol, 20 mol %), 50° C., 18 h, 100% conversion of 21a intodimer 60. 1H n.m.r. spectroscopic data fordimer 60 were in agreement with those previously reported (Section 7.12.1). Theprenylglycine derivative 19 was recovered unchanged. -
- The
prenylglycine derivative 19 was subjected to the conventional cross metathesis procedure (Section 7.5.4) with ethylene under the following conditions: - (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (11.0 mg, 55.2 μmol), ethylene (atmospheric pressure), Grubbs' catalyst (11.0 mg, 13.4 μmol, 20 mol %), DCM (4 mL), 22° C., 17h, 0% conversion into 21a. 1H n.m.r. spectroscopy indicated the starting hex-4-
enoate 19 was recovered. - (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (10.8 mg, 54.2 μmol, ethylene (60 psi), 2nd generation Grubbs' catalyst (9.3 mg, 11 μmol, 20 mol %), DCM (4 mL), 22° C., 19 h, 9% conversion into 21a.
- (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (24.3 mg, 0.12 mmol), ethylene (60 psi), 2nd generation Grubbs' catalyst (31.1 mg, 36.6 μmol, 30 mol %), DCM (5 mL), 50° C., 38 h, 32% conversion into 21a. Spectroscopic data for 21a and the recovered
prenylglycine derivative 19 were in agreement with those previously reported (Section 7.11.2 and Section 7.9.5 respectively). -
- The
prenylglycine derivative 19 was subjected to the conventional cross metathesis procedure (Section 7.5.4) with cis-2-butene under the following conditions: (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (16.2 mg, 81.4 μmol), DCM (5 mL), 2nd generation Grubbs' catalyst (3.5 mg, 4.1 μmol, 5 mol %), cis-2-butene (5 psi), 50° C., 14 h, 100% conversion into 81. Purification by flash chromatography (SiO2, light petroleum:DCM EtOAc:MeOH, 1:2:1:0.2) gave (2S)-methyl 2-N-acetylaminohex-4-enoate 81 as a brown oil (12.6 mg, 84%). GC: tR (E/Z)=4.2 min, 4.4 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). νmax(neat): 3284s, 2966w, 2954m, 2856w, 1747s, 1658s, 1547s, 1437s, 1375s, 1217m, 1142m, 1072w, 1016w, 968m, 848m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 1.60 (dd, J=6.3, 1.2 Hz, 3H, H6), 1.95 (s, 3H, CH3CO), 2.36-2.44 (m, 2H, H3), 3.67 (s, 3H, OCH3), 4.55 (dt, J=7.8 Hz, 5.9 Hz, 1H, H2), 5.24 (m, 1H, H5), 5.49 (m, 1H, H4), 6.17 (bd, J=6.4 Hz, 1H, NH). 13C n.m.r. (100 MHz, CDCl3): δ 18.1 (C6), 23.3 (CH3CO), 35.4 (C3), 52.1, 52.4 (C2, OCH3), 124.6, 130.2 (C4, 5), 169.7, 172.6 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 208.1 (M+Na)+, C9H15NNaO3 requires 208.1. Spectroscopic data were in agreement with those reported in the literature.117,119 - An analogous cross metathesis reaction was performed using a mixture of cis+trans-2-butene under the following conditions: (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (12.8 mg, 64.3 μmol), DCM (5 mL), 2nd generation Grubbs' catalyst (2.8 mg, 3.3 μmol, 5 mol %), trans+cis-2-butene (10 psi), 50° C., 16 h, <10% conversion into crotylglycine 81.
-
- The crotylglycine derivative 81 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-acetylaminohex-4-enoate 81 (17.0 mg, 91.9 μmol), DCM (4 mL), 2nd generation Grubbs' catalyst (4.2 mg, 5.0 μmol, 5 mol %), 50° C., 17 h, 100% conversion into
dimer 60. The solvent was evaporated under reduced pressure to give thehomodimer 60 as a brown oil (21.5 mg, 100% crude yield). Spectroscopic data fordimer 60 were in agreement with those previously reported (Section 7.12.1). -
- The prenylglycine derivative 87 was subjected to the conventional cross metathesis procedure (Section 7.5.5) with cis-1,4-diacetoxy-2-butene 141 under the following conditions: (2S)-Methyl 2-N-benzoylamino-5-methylhex-4-enoate 87 (170 mg, 0.65 mmol), DCM (10 mL), 2nd generation Grubbs' catalyst (16.5 mg, 0.03 mmol, 5 mol %), cis-1,4-diacetoxy-2-butene (5 psi), 50° C., 20 h, 100% conversion into 142. Purification by flash chromatography (SiO2, light petroleum:EtOAc, 1:1) gave (2S)-6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 142 as a dark brown oil (113 mg, 57%). νmax (neat): 3333.3s; 3056.4w; 3015.4w; 2943.6s; 1738.5s; 1661.5m; 1641.0s; 1605.1m; 1574.4m; 1533.3s; 1487.2m; 1435.9m; 1364.1, m; 1235.9, s; 1153.8, w; 1071.6, w; 1025.6, m; 969.2, m; 800.8, w; 717.9, m; 692.3,w cm−1. 1H NMR (400 MHz, CDCl3): δ 2.00, s, 3H, CH3; 2.67, m, 2H, H3; 3.77, s, 3H, OCH3; 4.49, d, J 4.7 Hz, 2H, H6; 4.89, q, J 5.8 Hz, 1H, H2; 5.68, t, J 5.2 Hz, 2H, H4, 5; 6.75, d, J 7.4 Hz, 2H, H4, 5; 7.42, t, J 7.2 Hz, 2H, H4′, 6′; 7.50, t, J 6.4 Hz, 1H, H5′; 7.78, d, J 7.1 Hz, 2H, H3′, 7′. 13C NMR (125 MHz, CDCl3): δ 20.92, CH3; 35.22, C3; 52.09, OCH3; 52.65, C2; 64.52, C6; 127.17, C3′, 7′; 128.70, C4′, 6′; 128.93, C5; 129.14, C4; 131.93, C5′; 133.93, C2′; 167.07, C1′; 170.80, C1″; 172.27, C1. Mass Spectrum (ESP, CH3CN): m/z 328.1 (M+Na+) C16H19NO5Na.
-
- 2-Acetylamino-7-benzoylamino-oct-4-enedioic acid dimethyl ester 143 was synthesised using standard solution-phase metathesis conditions (refer to Section 7.5.2): 6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 142 (50 mg, 0.16 mmol), dichloromethane (10 mL), second generation Grubbs' catalyst (5 mol %, 7 mg, 8 μmol), methyl-2-acetylamino-4-pentenoate 21a (168 mg, 0.98 mmol), 50° C., 18 h. The desired compound was obtained as a brown oil and purified via column chromatography (SiO2; EtOAc:hexane; 2:1). 1H NMR (500 MHz, CDCl3, mixture of isomers (1:1.2)): δ 1.95, s (major isomer) and 1.97, s (minor isomer), 3H, CH3; 2.42-2.70, m, 4H, H3, 6; 3.62, s (minor isomer), 3.64, s (major isomer), 3.78, s (minor isomer) and 3.79, s (major isomer), 6H, 2×OCH3; 4.63-4.66, m, 1H, H2; 4.85-4.91, m, 1H, H7; 5.35-5.49, m, 2H, 114, 5; 6.20, d, J 7.7 Hz (major isomer) and 6.34, d, J 7.5 Hz, 1H, NH (minor isomer); 6.87, t, J 7.55 Hz, 1H, NH; 7.44, t, J 7.1 Hz, 2H, H4′, 6′; 7.50, t, J 6.9 Hz, 1H, H5′; 7.84, t, J 7.9 Hz, 2H, H3′, 7′. 13C NMR (75 MHz, CDCl3, mixture of isomers (1:1.2)): δ 22.83, CH3; 34.84, 35.05, 35.38 and 35.73, C3, 6; 51.51 and 51.55, C2; 52.35, 52.46, 52.53, 52.60 and 52.66, C7, 2×OCH3; 127.18 and 127.22, C3′, 7′; 128.57 and 128.62, C4′, 6′; 128.88 and 129.00, C4, 5; 131.86 and 131.91, C5′; 133.71, C2′; 167.06, COPh; 170.03 and 170.11, COMe; 172.20, 172.21, 172.24 and 172.43, 2×COOMe. Mass Spectrum (ESI+, CH3OH): m/z 399.2 (M+Na+) C19H24N2O6Na.
-
- (2S,7S)-
Dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60 was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Dimer 60 (25.0 mg, 79.6 μmol), benzene (5 mL), Wilkinson's catalyst, 60 psi, 22° C., 4 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the resulting oil was purified by flash chromatography (SiO2, EtOAc) to afford the saturated product 71 as a brown oil (25.0 mg, 99%). GC: tR=14.4 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). νmax (neat): 3426bm, 3055w, 2932m, 2857w, 2360w, 1741s, 1666s, 1543w, 1438m, 1375w, 1266s, 1177w, 1120w, 896w, 738w, 702w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 1.30-1.40 (m, 4H, H4, 5), 1.82-1.90 (m, 4H, H3, 6), 2.02 (s, 6H, CH3CO), 3.74 (s, 6H, OCH3), 4.56-4.63 (m, 2H, H2, 7), 6.16 (bd, J=7.5 Hz, 2H, NH). 13C n.m.r. (100 MHz, CDCl3): δ 23.3 (CH3CO), 24.7 (C4, 5), 32.3 (C3, 6), 52.0 (C2, 7), 52.5 (OCH3), 170.0, 173.1 (C1, 8, CONH). HRMS MeOH). Found: m/z 339.1531 (M+Na)+, C14H24N2NaO6 requires 339.1532. -
- (2S,7S)-
Dimethyl 2,7-N,N′-dibenzoylaminoocta-4-enedioate 69 was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Dimer 69 (20.0 mg, 45.7 μmol, benzene (5 mL), Wilkinson's catalyst, 60 psi, 22° C., 4 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the resulting oil was purified by flash chromatography (SiO2, EtOAc) to afford the saturated product 72 as a brown oil (20.0 mg, 100%). GC: tR 17.2 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). νmax (neat): 3055m, 2986w, 2955w, 1741s, 1662s, 1603w, 1580w, 1518m, 1486m, 1438s, 1359w, 1286s, 1182m, 1120m, 1028w, 896m cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 1.35-1.53 (m, 4H, H4, 5), 1.80-2.02 (m, 4H, H3, 6), 3.78 (s, 6H, OCH3), 4.82 (dt, J=7.3, 5.4 Hz, 2H, H2, 7), 6.73 (bd, J=7.4 Hz, 2H, NH), 7.40-7.49 (m, 6H, H3′, 4′, 5′), 7.78-7.82 (m, 4H, H2′, 6′). 13C n.m.r. (100 MHz, CDCl3): δ 24.9 (C4, 5), 32.6 (C3, 6), 52.5, 52.7 (C2, OCH3), 127.2 (C2′, 6′), 128.6 (C3′, 5′), 131.9 (C4′), 134.1 (C1′), 167.2, 173.2 (C1, 8, CONH). HRMS (ESI+, MeOH). Found: m/z 463.1842 (M+Na)+, C24H28N2NaO6 requires 463.1845. -
- The dienamide 76 was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: (2Z)-Methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (11.5 mg, 46.9 μmol), benzene (5 mL), Wilkinson's catalyst, 50 psi H2, 22° C., 4 h, 99% yield (mass recovery) of a 1:4 mixture of 77:79 as a brown oil. GC: tR=10.8 min 79, 13.9 min 77 (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). 1H n.m.r. spectroscopic datafor olefin 77 were in agreement with those previously reported (Section 7.11.9). Hydrogenation of the mixture using identical conditions led to 100% conversion into 79 (41.2 mg, 100% crude yield). νmax (neat): 3262w, 3054m, 2956m, 1736s, 1676s, 1509m, 1438s, 1372w, 1265s, 1174w, 1120m, 1028w, 738s, 700w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 1.53-1.65 (m, 4H, H3, 4), 1.94 (s, 3H, CH3CO), 2.52-2.59 (rn, 2H, H5), 3.65 (s, 3H, OCH3), 4.59 (m, 1H, H2), 5.90 (bd, J=7.2 Hz, 1H, NH), 7.07-7.29 (m, 5H, Arom CH). 13C n.m.r. (75 MHz, CDCl3): δ 23.3 (CH3CO), 27.2 (C4), 32.3 (C3), 35.5 (C5), 52.1, 52.5 (C2, OCH3), 126.1, 128.5, 132.2 (Arom CH), 141.7, (Arom C), 169.9, 173.2 (C1, CONH). Mass Spectrum (ESI+, MeOH): m/z 272.2 (M+Na)+, C14H19NNaO3 requires 272.1.
-
- (2S)-Methyl 2-N-acetylamino-5-methylhex-4-
enoate 19 was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Hex-4-enoate derivative 19 (11.3 mg, 56.8 μmol), benzene (4 mL), Wilkinson's catalyst, 50 psi, 22° C., 4 h. At the end of the reaction period, the solvent was evaporated under reduced pressure to afford a brown oil (12.5 mg). 1H n.m.r. spectroscopy indicated the reaction gave only 6% conversion into the saturated product 80; 94% of the startingprenylglycine derivative 19 was recovered. 1H n.m.r. (300 MHz, CDCl3): Hexanoate 80: δ 0.87 (d, J=6.6 Hz, 6H, H6), 1.09-1.28 (m, 2H, H4), 1.54 (h, J=6.7 Hz, 1H, H5), 1.61-1.71 (m, 2H, H3), 2.03 (s, 3H, CH3CO), 3.75 (s, 3H, OCH3), 4.60 (dt, J=7.8 Hz, 5.5 Hz, 1H, H2), 5.96 (bd, J=7.8 Hz, 1H, NH). -
- A solution of p-nitrobenzoyl chloride 89 (1.21 g, 6.54 mmol) in a mixture of DCM:Et2O (2:1, 15 mL) was added dropwise to a stirred solution of methyl 2-aminopent-4-enoate hydrochloride 51 (0.98 g, 5.94 mmol) and Et3N (1.80 mL, 13.0 mmol) in a mixture of DCM:Et2O (2:1, 15 mL) at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 20 h. The mixture was acidified with dilute HCl solution (1 M, pH 2) and extracted with DCM (3×20 mL). The combined organic extract was washed with distilled water (20 mL), dried (MgSO4) and evaporated under reduced pressure to afford the titled
compound 83 as an off-white solid (1.63 g, 99%), m.p. 99-100° C. Spectroscopic data indicated thecrude product 83 did not require purification and was used directly in the subsequent reaction (Section 7.15.1). GC: tR=12.20 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). [α]D 22+29.9° (c=0.37, CHCl3). νmax (neat): 3293w, 2954m, 2839m, 1725s, 1641m, 1602w, 1538w, 1529w, 1456s, 1377s, 1256m, 1160m, 1118w, 1066w, 998m, 972m, 941w, 841m cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 2.65-2.76 (m, 2H, H3), 3.82 (s, 3H, OCH3), 4.90 (dt, J=5.6, 7.5 Hz, 1H, H2), 5.14-5.30 (m, 2H, H5), 5.75 (m, 1H, H4), 6.73 (bd, J=6.6 Hz, 1H, NH), 7.95 (d, J=7.7 Hz, 2H, H2′, 6′), 8.30 (d, J=7.6 Hz, 2H, H3′, 5′). 13C n.m.r. (75 MHz, CDCl3): δ 36.6 (C3), 52.4, 52.9 (C2, OCH3), 119.9 (C5), 124.0 (C3′, 5′), 128.4 (C2′, 6′), 132.0 (C4), 139.6 (C1′), 150.0 (C4′), 165.1 (C1), 172.1 (CONH). HRMS (ESI+, MeOH). Found: m/z 279.0977 (M+H)+, C13H15N2O5 requires 279.0981; m/z 301.0798 (M+Na)+, C13H14N2NaO5 requires 301.0800. -
- The preparation of (2S)-methyl 2-N-acetylamino-5-methylhex-4-
enoate 19 via asymmetric hydrogenation of the dienoate 20 has been previously described (Section 7.9.5). -
- The
dienamide 82 was prepared according to a procedure described by Teoh et al.118,119 Tetramethylguanidine (3.40 mL, 27.1 mmol) and hydroquinone (3.0 mg) were added to a solution of methyl 2-N-benzoylamino-2-(dimethoxyphosphinyl)-acetate 64 (6.10 g, 20.3 mmol) in THF (120 mL) at −78° C. After 30 min, 3-methyl-2-butenal 40 (2.40 mL, 24.9 mmol) was added and the mixture was stirred at −78° C. for 2 h, warmed to 25° C. and allowed to react an additional 16 h. The mixture was diluted with DCM (150 mL) and washed with dilute HCl solution (1 M, 2×70 mL), CuSO4 solution (1 M, 2×70 mL), saturated NaHCO3 solution (2×70 mL) and saturated NaCl solution (1×70 mL). The organic extract was dried (MgSO4) and evaporated under reduced pressure to give thecrude product 82 as a yellow oil (5.37 g). Purification by flash chromatography (SiO2, light petroleum:EtOAc, 2:1) furnished thepure dienamide 82 as an off-white solid (3.84 g, 73%), m.p. 98-99° C. GC: tR=11.00 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). νmax (KBr): 3286m, 2991w, 2948w, 1716s, 1649s, 1601w, 1579w, 1524s, 1489s, 1436m, 1389w, 1331m, 1286m, 1254s, 1207m, 1190w, 1162w, 1135w, 1087s, 1048w, 996w, 977w, 931w, 864m, 802m, 760m, 739m, 710s, 688w, 677w, 630w, 614w, 585w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 1.87 (s, 3H, H6), 1.91 (d, J=0.7 Hz, 3H, CH3C—), 3.78 (s, 3H, OCH3), 6.03 (d with fine splitting, J=11.9 Hz, 1H, H4), 7.41 (d, J=11.8 Hz, 1H, H3), 7.43-7.47 (m, 2H, H3′, 5′), 7.53 (m, 1H, H4′), 7.63 (bs, 1H, NH), 7.89-7.90 (m, 2H, H2′, 6′). 13C n.m.r. (100 MHz, CDCl3): δ 19.3 (CH3C═), 27.1 (C6), 52.5 (OCH3), 121.0 (C4), 121.2 (C5), 127.6 (C2′, 6′), 128.9 (C3′, 55, 129.9 (C3), 132.0 (C4′), 134.1 (C2), 147.2 (C1′), 166.0, 166.1 (C1, CONH). HRMS (ESI+, MeOH). Found: m/z 260.1282 (M+H)+, C15H18NO3 requires 260.1287; m/z 282.1099 (M+Na)+, C15H17NNaO3 requires 282.1106. -
- The dimer 90 was prepared via the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2S)-Methyl 2-N-(p-nitrobenzoyl)aminopent-4-enoate 83 (43.5 mg, 0.16 mmol), DCM (3 mL), Grubbs' catalyst (26.0 mg, 31.6 μmol, 20 mol %), 50° C., 14 h, 100% conversion into dimer 90. The reaction mixture was evaporated under reduced pressure to give the homodimer 90 as a brown oil (69.7 mg, 100% crude yield).
- The dimer 90 was also prepared and purified from an analogous reaction using 2nd generation Grubbs' catalyst under the following conditions: (2S)-Methyl 2-N-(p-nitrobenzoyl)aminopent-4-enoate 83 (86.3 mg, 0.31 mmol), DCM (4 mL), 2nd generation Grubbs' catalyst (13.6 mg, 16.0 μmol, 5 mol %), 50° C., 12 h, 100% conversion into dimer 90. Purification by flash chromatography (SiO2, light petroleum:EtOAc:DCM, 4:2:1) gave the pure dimer 90 as a pale brown solid (74.0 mg, 90%), m.p. 90-92° C. GC: tR (E/Z)=16.1 min, 16.2 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). [α]D 22+20.0° (c=0.21, CHCl3). νmax (neat): 3365m, 3057w, 2957w, 2854w, 1728s, 1667s, 1602m, 1525s, 1487m, 1437m, 1348s, 1267m, 1227m, 1174w, 1157w, 1110w, 1014m, 974m, 869m, 874m, 737s, 718s em′. 1H n.m.r. (400 MHz, CDCl3): δ 2.60-2.64 (m, 4H, H3, 6), 3.70 (s, 6H, OCH3), 4.88 (apparent q, J=5.9 Hz, 2H, H2, 7), 5.49-5.53 (m, 2H, H4, 5), 7.11 (bd, J=7.4 Hz, 2H, NH), 8.02 (d, J=8.7 Hz, 4H, H2′, 6′), 8.21-8.29 (m, 4H, H3′, 5′). 13C n.m.r. (100 MHz, CDCl3): δ 35.0 (C3, 6), 52.8, 52.8 (C2, 7, OCH3), 123.8 (C3′, 5′), 128.8, 128.9 (C2′, 6′, C4, 5), 139.2 (C1′), 149.9 (C4′), 165.2 (C1, 8), 172.1 (CONH). HRMS (ESI+, MeOH). Found: m/z 529.1560 (M+H)+, C24H25N4O10 requires 529.1571; m/z 551.1379 (M+Na)+, C24H24N4NaO10 requires 551.1390.
-
- The
dienamide 82 was subjected to the conventional cross metathesis procedure (Section 7.5.2) under the following conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (30.7 mg, 0.12 mmol), DCM (5 mL), 2nd generation Grubbs' catalyst (5.1 mg, 6.0 μmol, 5 mol %), 50° C., 15 h, 0% conversion into dimer 84. Thedienamide 82 was recovered unchanged. 1H n.m.r. spectroscopic data for the recovereddienamide 82 were in agreement with those previously reported (Section 7.14.3). -
- The
dienamide 82 was subjected to the conventional cross metathesis procedure (Section 7.5.4) with cis-2-butene under the following conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (39.3 mg, 0.15 mmol), DCM (5 mL), cis-2-butene (15 psi), 2nd generation Grubbs' catalyst (6.6 mg, 7.8 μmol, 5 mol %), 50° C., 14 h, 0% conversion into 86. Thedienamide 82 was recovered unchanged. 1H n.m.r. spectroscopic data for the recovereddienamide 82 were in agreement with those previously reported (Section 7.14.3). -
- The activation of
prenylglycine 19 via butenolysis (Section 7.5.4) to give the crotylglycine derivative 81 has been previously described (Section 7.12.13). -
- The
dienamide 82 was subjected to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (26.1 mg, 0.10 mmol), MeOH (5 mL), Rh(I)—(S,S)-Et-DuPHOS, 75 psi, 22° C., 3 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give the prenylglycine derivative 87 as a pale yellow oil (23.9 mg, 91%). HPLC: tR=6.20 min (Chiralcel OJ column, 1.0 mL min−1, detection at 254 nm, 5% EtOH:95% hexane). [α]D 22+53.0° (c=1.19, CHCl3). νmax (neat): 3334m, 2953w, 1744s, 1645s, 1603w, 1580w, 1538s, 1489m, 1437m, 1353w, 1274w, 1211w, 1175w, 1095w, 1031w, 736w, 714w, 693w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 1.61 (d, J=0.5 Hz, 3H, CH3C═), 1.71 (d, J=1.0 Hz, 3H, H6), 2.52-2.76 (m, 2H, H3), 3.77 (s, 3H, OCH3), 4.85 (dt, J=7.7, 5.5 Hz, 1H. H2), 5.08 (m, 1H, H4), 6.65 (bd, J=6.9 Hz, 1H NH), 7.41-7.47 (m, 2H, H3′, 5), 7.51 (m, 1H, H4′), 7.76-7.79 (m, 2H, H2′, 6′). 13C n.m.r. (75 MHz, CDCl3): δ 18.0 (CH3C═), 26.0 (C6), 30.9 (C3), 52.5, 52.6 (C2, OCH3), 117.6 (C4), 127.2 (C2′, 6′), 128.7 (C3′, 5′), 131.8 (C4′), 134.3 (C5), 136.8 (C1′), 167.0, 172.8 (C1, CONH). HRMS (ESI+, MeOH). Found: m/z 262.1441 (M+H)+, C15H20NO3 requires 262.1443; m/z 284.1256 (M+Na)+, C15H19NNaO3 requires 284.1263. -
- The
dienamide 82 was subjected, to the general asymmetric hydrogenation procedure (Section 7.4.3) under the following conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (80.1 mg, 0.31 mmol), MeOH (7 mL), Rh(I)—(R,R)-Et-DuPHOS, 75 psi, 22° C., 3 h. At the end of the reaction period, the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (SiO2, EtOAc) to give the prenylglycine derivative 87 as a yellow oil (78.2 mg, 97%). HPLC: tR=5.90 min (Chiralcel OJ column, 1.0 mL min−I, detection at 254 nm, 5% EtOH:95% hexane). [α]D 22−53.4° (c=0.98, CHCl3). Spectroscopic data were in agreement with those previously reported for the (S)-enantiomer. -
- The enamide 87 was subjected to the conventional cross metathesis procedure (Section 7.5.5) with cis-2-butene under the following conditions: (2S)-Methyl 2-N-benzoylamino-5-methylhex-4-enoate 87 (90.0 mg, 0.35 mmol), DCM (5 mL), cis-2-butene (15 psi), 2nd generation Grubbs' catalyst (14.6 mg, 17.2 μmol, 5 mol %), 50° C., 12 h, 100% conversion into 88. The reaction mixture was evaporated under reduced pressure to give the crotylglycine derivative 88 as a brown oil (101 mg, 100% crude yield). GC: tR (E/Z)=9.68 min, 9.93 min (GC Column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). νmax (neat): 3337bm, 3057w, 2954m, 2856w, 1743s, 1652s, 1603w, 1580w, 1532s, 1488m, 1438m, 1360w, 1266s, 1217w, 1180w, 1116w, 1031m, 969w, 896w, 801w, 738s, 638w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 1.66 (dd, J=6.4, 1.4 Hz, 3H, H6), 2.52-2.66 (m, 2H, H3), 3.77 (s, 3H, OCH3), 4.82 (apparent dd, J=7.6, 5.7 Hz, 1H, H2), 5.33 (m, 1H, H5), 5.63 (m, 1H, H4), 6.66 (bd, J=7.0 Hz, 1H, NH), 7.43 (t, J=7.0 Hz, 2H, H3′, 5′), 7.50 (m, 1H, H4′), 7.78 (d, J=7.1 Hz, 2H, H2′, 6′). 13C n.m.r. (100 MHz, CDCl3): δ 18.0 (C6), 35.4 (C3), 52.4, 52.5 (C2, OCH3), 124.5 (C5), 127.1 (CT, 6′), 128.6 (C3′, 5′), 130.2 (C4), 131.7 (C4′), 134.1 (C1′), 166.9, 172.5 (C1, CONH). HRMS (ESI+, MeOH). Found: m/z 248.1284 (M+H)+, C14H18NO3 requires 248.1287; m/z 270.1098 (M+Na)+, C14H17NNaO3 requires 270.1106.
-
- The dimerisation of crotylglycine 81 using the conventional cross metathesis procedure has been previously described (Section 7.12.14).
-
- The enamide 88 was subjected to the conventional cross metathesis procedure under the following conditions: (2S)-Methyl 2-N-benzoylaminohex-4-enoate 88 (89.6 mg, 0.36 mmol), DCM (5 mL), 2nd generation Grubbs' catalyst (15.3 mg, 18.0 μmol, 5 mol %), 50° C., 17 h, 100% conversion into dimer 69. The reaction mixture was evaporated under reduced pressure to afford the homodimer 69 as a brown oil (106 mg, 100% crude yield). Spectroscopic data for dimer 69 were in agreement with those previously reported (Section 7.12.2).
-
- The
dienamide 82 was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (47.0 mg, 0.18 mmol), benzene (5 mL), Wilkinson's catalyst, 50 psi, 22° C., 4 h. Thedienamide 82 was recovered unchanged. 1H n.m.r. spectroscopic data for the recovereddienamide 83 were in agreement with those previously reported (Section 7.14.3). -
- (2S,7S)-
Dimethyl 2,7-N,N′-di(p-nitrobenzoyl)aminoocta-4-enedioate 90 was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Dimer 90 (20.6 mg, 0.04 mmol), THF:tBuOH (1:1, 5 mL), Wilkinson's catalyst, 15 psi H2, 22° C., 14 h. At the end of the reaction period, the solvent was evaporated under reduced pressure to afford the product 91 as a brown oil. Purification by flash chromatography (SiO2, light petroleum:EtOAc:DCM, 1:1:1) gave the pure dimer 91 as an off-white solid (13.8 mg, 67%), m.p. 117-119° C. GC: tR=16.8 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min). νmax (neat): 3304w, 2932w, 1740s, 1637s, 1603m, 1528s, 1438w, 1348m, 1265s, 1109w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 1.39-1.54 (m, 4H, H4, 5), 1.74-2.04 (m, 4H, H3, 6), 3.81 (s, 6H, OCH3), 4.82 (dt, J=7.3, 5.4 Hz, 2H, H2, 7), 6.85 (bd, J=7.4 Hz, 2H, NH), 7.96 (d, J=8.8 Hz, 4H, H2′, 6′), 8.28 (d, J=8.7 Hz, 4H, H3′, 5′). 13C n.m.r. (100 MHz, CDCl3): δ 24.7 (C4, 5), 32.4 (C3, 6), 52.7, 52.9 (C2, OCH3), 124.0 (C2′, 6′), 128.5 (C3′, 5′), 139.5 (C1′), 150.0 (C4′), 165.3, 172.8 (C1, 8, CONH). HRMS (ESI+, MeOH). Found: m/z 553.1550 (M+Na)+, C24H26N4NaO10 requires 553.1547. - Peptide sequences are represented by structural diagrams and three-letter codes of constituent amino acids. Synthetic amino acids allylglycine, crotylglycine and prenylglycine are represented by Hag, Crt and Pre respectively. Procedures for the preparation of the Fmoc-protected olefinic amino acids: (2S)-2-N-Fluorenylmethoxy-carbonylaminopent-4-enoic acid (Fmoc-L-Hag-OH) 96, (2S)-2-N-fluorenylmethoxy-carbonylaminohex-4-enoic acid (Fmoc-L-Crt-OH) 100 and (2S)-2-N-fluorenyl-methoxycarbonylamino-5-methylhex-4-enoic acid (Fmoc-L-Pre-OH) 92, are detailed below.
-
- The allylglycine derivative 96 was prepared according to the procedure described by Paquet.230 Fmoc-OSu (14.60 g, 43.3 mmol) was added to stirred solution of L-allylglycine (5.00 g, 43.5 mmol) and NaHCO3 (18.20 g, 0.22 mol) in a mixture of acetone:water (200 mL). The resultant white suspension was stirred at room temperature and after 20 h, t.l.c. analysis (SiO2, light petroleum:EtOAc; 1:1) showed the absence of starting material. The reaction mixture was acidified with concentrated HCl (pH 2) and the acetone was removed under reduced pressure. The resultant suspension was extracted into DCM (3×75 mL) and the combined organic extract was washed with dilute HCl solution (1 M, 2×50 mL), water (2×50 mL), dried (MgSO4) and evaporated under reduced pressure to afford the titled Fmoc-amino acid 96 as a colourless solid (14.01 g, 96%), m.p. 137-138° C. (lit.266 134-136° C.). νmax (KBr): 3484s, 3198bs, 3085m, 2967m, 2923m, 1723s, 1644m, 1525s, 1478w, 1449s, 1396m, 1340m, 1233s, 1189s, 1099m, 1048s, 998w, 966w, 943m, 924w, 850m, 781m, 761s, 740m, 648w, 623m, 582m, 560w, 540m, 424w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 2.52-2.70 (2.34-2.49) (m, 2H, H3), 4.23 (t, J=6.9 Hz, 1H, H9′), 4.42 (4.30) (d, J=6.9 Hz, 2H, CH2O), 4.52 (m, 1H, H2), 5.13-5.23 (m, 2H, H5), 5.31 (5.87) (bd, J=7.8 Hz, 1H, NH), 5.75 (m, 1H, H4), 6.63 (bs, 1H, OH), 7.31 (td, J=7.4, 0.8 Hz, 2H, H2′, 7′), 7.38 (t, J=7.4 Hz, 2H, H3′, 6′), 7.52-7.63 (m, 2H, H1′, 8′), 7.76 (d, J=7.5 Hz, 2H, H4′, 5′), one exchangeable proton (OH) not observed. 13C n.m.r. (100 MHz, CDCl3): δ 36.7 (C3), 47.5 (C9′), 53.4 (C2), 68.1 (CH2O), 122.0 (C5), 120.1 (C2′, 7′), 125.4 (C3′, 6′), 127.9 (C1′, 8′), 128.0 (C4′, 5′), 131.1 (C4), 141.7 (C8′a, 9′a), 144.0 (C4′a, 4′b), 156.3 (OCONH), 176.4 (C1). Mass Spectrum (ESI+, MeOH): m/z 338.4 (M+H)+, C20H20NO4 requires 338.1; 360.3 (M+Na)+, C20H19NNaO4 requires 360.1. Spectroscopic data were in agreement with those reported in the literature.266
-
- A solution of (2S)-methyl 2-N-acetylaminohex-4-enoate 81 (1.30 g, 7.05 mmol) in dilute HCl (1 M, 8 mL) was heated at reflux for 21 h. The reaction mixture was evaporated under reduced pressure to give 2-aminohex-4-enoic acid hydrochloride salt (L-crotylglycine.HCl) 101 as a pale brown solid (1.17 g, 100%), m.p. 212-214° C. νmax (KBr): 3500bs, 2965m, 2358s, 1731s, 1651m, 1455m, 901m cm−1. 1H n.m.r. (300 MHz, CD3OD): δ 1.69 (d, J=5.3 Hz, 3H, H6), 2.51-2.74 (m, 2H, H3), 3.99 (m, 1H, H2), 5.42 (m, 1H, H5), 5.73 (m, 1H, H4), exchangeable protons (NH and OH) not observed. 13C n.m.r. (75 MHz, CD3OD): δ 18.7 (C6), 35.1 (C3), 48.7 (C2), 124.6 (C5), 133.6 (C4), 174.3 (C1). Mass Spectrum (ESI+, MeOH): m/z 130.1 (M+H)+, C6H12NO2 requires 130.1.
- 2-N-Fluorenylmethoxycarbonylaminohex-4-enoic acid 100 was prepared according to the procedure described by Paquet.230 Fmoc-OSu (2.36 g, 7.00 mmol) was added to a stirred suspension of L-crotylglycine.HCl 101 (1.16 g, 7.03 mmol) and NaHCO3 (2.95 g, 35.0 mmol) in a mixture of acetone:water (1:1, 30 mL) The resultant suspension was stirred at room temperature for 15 h. The reaction mixture was then acidified with concentrated HCl (pH 2) and the acetone was removed under reduced pressure. The resultant suspension was extracted into DCM (3×25 mL) and the combined organic extract was washed with dilute HCl solution (1 M, 2×25 mL), water (2×25 mL), dried (MgSO4) and evaporated under reduced pressure to afford the titled Fmoc-amino acid 100 as colourless solid (1.91 g, 78%), m.p. 119-121° C. (KBr): 3390bm, 3033m, 2961s, 2357w, 1730s, 1651w, 1505w, 1450w, 1395w, 850w cm−1. 1H n.m.r. (300 MHz, CDCl3): δ 1.67 (d, J=6.2 Hz, 3H, H6), 2.37-2.69 (m, 2H, H3), 4.23 (t, J=6.8 Hz, 1H, H9′), 4.42-4.48 (m, 3H, CH2O, H2), 5.30-5.37 (m, 2H, H5, NH), 5.61 (m, 1H, H4), 7.31 (td, J=7.2, 1.3 Hz, 2H, H2′, 7′), 7.34 (td, J=7.4, 1.5 Hz, 2H, H3′, 6′), 7.60 (d, J=7.3 Hz, 2H, H1′, 8′), 7.74 (d, J=7.0 Hz, 2H, H4′, 5′), one exchangeable proton (OH) not observed. 13C n.m.r. (75 MHz, CDCl3): δ 16.7 (C6), 34.1 (C3), 46.2 (C9′), 52.3 (C2), 66.2 (CH2O), 118.9 (C5), 123.0 (C2′, 7′), 124.6 (C3′, 6′), 125.2 (C1′, 8′), 127.5 (C4′, 5′), 129.7 (C4), 140.3 (C8′a, 9′a), 142.7 (C4′a, 4′b), 154.9 (OCONH), 175.0 (C1). Mass Spectrum (ESI+, MeOH): m/z 352.1 (M+H)+, C21H22NO4 requires 352.2, Spectroscopic data were in agreement with those reported in the literature.146
-
- The allylglycine derivative 96 was subjected to the conventional cross metathesis procedure with 2-methyl-2-butene (Section 0) under the following conditions: 2-N-Fluorenylmethoxycarbonylaminopent-4-enoic acid 96 (200 mg, 0.59 mmol), DCM (7 mL), 2′ generation Grubbs' catalyst (26.0 mg, 30.6 μmol, 5 mol %), 2-methyl-2-butene (1 mL, 10 psi), 50° C., 12 h, 100% conversion into 92. The reaction mixture was evaporated under reduced pressure to give the prenylglycine derivative 92 as a brown oil (245 mg, 100% crude yield). νmax (neat): 3426w, 3324w, 3066w, 2932m, 1716s, 1514m, 1478w, 1450m, 1378w, 1338m, 1265m, 1220w, 1106w, 1057m, 910m, 855w, 759w, 738s, 704w, 648w, 621 w cm−1. 1H n.m.r. (400 MHz, CDCl3): δ 1.63 (s, 3H, H6), 1.73 (s, 3H, CH3), 2.49-2.65 (m, 2H, H3), 4.23 (t, J=6.7 Hz, 1H, H9′), 4.40 (d, J=6.7 Hz, 2H, CH2O), 5.11 (m, 1H, H4), 5.41 (bd, J=7.5 Hz, 1H, NH), 7.31 (t, J=7.3 Hz, 2H, H2′, 7′), 7.40 (t, J=7.3 Hz, 2H, H3′, 6′), 7.58-7.66 (m, 2H, H1′, 8′), 7.76 (d, J=7.4 Hz, 2H, H4′, 5′), 9.22 (bs, 1H, OH). 13C n.m.r. (100 MHz, CDCl3): δ 18.1 (C6), 26.0 (CH3C═), 30.8 (C3), 47.3 (C9′), 53.8 (C2), 67.2 (CH2O), 117.5 (C4), 120.1 (C2′, 7′), 125.2 (C3′, 6′), 127.2 (Cr, 8′), 127.8 (C4′, 5′), 136.9 (C5), 141.4, 143.9 (Arom C), 156.1 (CONH), 176.2 (C1). HRMS (ESI+, MeOH). Found: m/z 388.1522 (M+Na)+, C22H23NNaO4 requires 388.1525. The product later crystallised on standing to give a pale brown solid, m.p. 109-111° C.
-
- The procedure described in Section 73.2.2 was used for the attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.1. The first coupling reaction was shaken for 14 h.
-
TABLE 7.1 Quantities of Reagents used in the Synthesis of Peptide 94 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 155 mg 0.11 Fmoc-L-Hag-OH 110 mg 0.33 HATU 83.0 mg 0.22 NMM 71.8 μl 0.65 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the pentapeptide 94. Quantities of the coupling agents HATU and NMM remained constant throughout the synthesis. The quantities of successive amino acids and their reaction durations are detailed in Table 7.2.
-
TABLE 7.1 Quantities of Amino Acids used in the Synthesis of Peptide 94 Amino Acid Mass (mg) Mote (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 211 0.33 5 Fmoc-L-Trp(Boc)-OH 171 0.32 3 Fmoc-L-Ala-OH 102 0.33 4.5 Fmoc-L-Hag-OH 110 0.33 20 *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the pentapeptide 94. Mass spectrum (ESI+, MeCN/H2O): m/z 847.1 (M+H)+, C45H55N10O7 requires 847.4.
-
- The procedure described in Section 7.3.2.2 was used for the attachment of the first amino acid, Fmoc-L-Crt-OH 100, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.3. The first coupling reaction was shaken for 3 h.
-
TABLE 7.3 Quantities of Reagents used in the Synthesis of Peptide 99 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 110 mg 0.08 Fmoc-L-Crt-OH 81.5 mg 0.23 HATU 58.6 mg 0.15 NMM 51.0 μl 0.46 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the pentapeptide 99. Quantities of the coupling agents HATU and NMM remained constant throughout the synthesis. The quantities of successive amino acids and their reaction durations are detailed in Table 7.4.
-
TABLE 7.4 Quantities of Amino Acids used in the Synthesis of Peptide 99 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)- OH 150 0.23 20 Fmoc-L-Trp(Boc)-OH 122 0.23 4 Fmoc-L-Ala-OH 72.0 0.23 2 Fmoc-L-Crt-OH 81.5 0.23 12 *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the pentapeptide 99. Mass spectrum (ESI+, MeCN/H2O): m/z 875.2 (M+H)+, C47H59N10O7 requires 875.4.
-
- The procedure described in Section 7.3.2.2 was used for the attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.5. The first coupling reaction was shaken for 14 h.
-
TABLE 7.5 Quantities of Reagents used in the Synthesis of Peptide 97 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 154 mg 0.11 Fmoc-L-Hag-OH 109 mg 0.32 HATU 82 mg 0.22 NMM 71.4 μl 0.65 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the pentapeptide 97. Quantities of the coupling agents HATU and NMM remained constant throughout the synthesis. The quantities of successive amino acids and their reaction durations are detailed in Table 7.6
-
TABLE 0.2 Quantities of Amino Acids used in the Synthesis of Peptide 97 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 210 0.32 5 Fmoc-L-Trp(Boc)-OH 170 0.32 3 Fmoc-L-Pro-OH 110 0.33 4.5 Fmoc-L-Hag-OH 109 0.32 22 *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the pentapeptide 97. Mass spectrum (ESI+, MeCN/H2O): m/z 873.2 (M+H)+, C47H57N10O7 requires 873.4; 895.1 (M+Na)+, C47H56N10NaO7 requires 895.4.
-
- The resin-bound peptide 94a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 94a (20.0 mg, 14.0 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), Grubbs' catalyst (2.3 mg, 2.8 μmol, 20 mol %), 50° C., 41 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated recovery of the starting linear peptide 94. Mass spectrum (ESI+, MeCN/H2O): m/z 847.2 (M+H)+ linear, C45H55N10O7.
- The resin-bound peptide 94a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 94a (37.0 mg, 25.9 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2nd generation Grubbs' catalyst (4.4 mg, 5.2 μmol, 20 mol %), 50° C., 41 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 0). Mass spectral analysis of the isolated residue confirmed the presence of both cyclic 95 and linear 94 peptides. Mass spectrum (ESI+, MeCN/H2O): m/z 819.2 (M+H)+ cyclic, C43H51N10O7 requires 819.4; m/z 847.2 (M+H)+ linear, C45H55N10O7.
- The resin-bound peptide 99a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 99a (32.8 mg, 23.0 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (4.0 mg, 4.7 μmol, 20 mol %), 50° C., 41 h, 100% conversion into 95. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 95.† Mass spectrum (ESI+, MeCN/H2O): m/z 819.2 (M+H)+ cyclic, C43H51N10O7 requires 819.4. † RCM of the crotylglycine-containing peptide 99 leads to the same unsaturated carbocycle 95 resulting from cyclisation of the allylglycine-containing sequence 94, i.e. Fmoc-c[Hag-Ala-Trp-Arg-Hag]-OH is identical to Fmoc-c[Crt-Ala-Trp-Arg-Crt]-OH.
-
- The resin-bound peptide 97a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 97a (26.4 mg, 18.5 DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), Grubbs' catalyst (6.1 mg, 7.4 μmol, 20 mol %), 50° C., 41 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated recovery of the starting linear peptide 97. Mass spectrum (ESI+, MeCN/H2O): m/z 873.2 (M+H)+ linear, C47H57N10O7.
- The resin-bound peptide 97a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 97a (36.0 mg, 25.2 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2nd generation Grubbs' catalyst (4.4 mg, 5.2 μmol, 20 mol %), 50° C., 41 h, 100% conversion into 98. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 98. Mass spectrum (ESI+, MeCN/H2O): m/z 845.1 (M+H)+, C45H53N10O7 requires 845.4; 867.1 (M+Na)+,C45H52N10NaO7 requires 867.4.
-
- The procedure outlined in Section 7.3.2.1 was used for the attachment of the first amino acid, Fmoc-Hag-OH 96, to Wang resin. Quantities of the resin and coupling reagents are presented in Table 7.7. The first coupling reaction was shaken for 14 h.
-
TABLE 7.7 Quantities of Reagents used in the Synthesis of Peptide 102 Reagent Mass (mg) or Volume (μl) Mole (mmol) Wang Resin 212 mg 0.19 Fmoc-L-Hag-OH 195 mg 0.58 DIC 90.6 μl 0.58 DMAP 7.1 mg 0.06 - The procedure outlined in Section 7.3.2.1 was also utilised for subsequent coupling reactions in the synthesis of the pentapeptide 102. Quantities of the coupling reagents HATU and NMM are tabulated (Table 7.8) and remained constant throughout the synthesis. The quantities of successive amino acids and their reaction durations are detailed in Table 7.9.
-
TABLE 7.8 Quantities of Coupling Reagents used in the Synthesis of Peptide 102 Coupling Reagent Mass (mg) or Volume (mL) Mole (mmol) HATU 147 mg 0.39 NMM 128 μl 1.16 -
TABLE 7.9 Quantities of Amino Acids used in the Synthesis of Peptide 102 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h) Fmoc-L-Arg(Pbf)-OH 376 0.58 2 Fmoc-L-Pre-OH 211 0.58 3 Fmoc-L-Pro-OH 196 0.58 6 Fmoc-L-Hag-OH 195 0.58 2 (1) *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the pentapeptide 102. Mass spectrum (ESI+, MeCN/H2O): m/z 813.6 (M+H)+, C43H57N8O8 requires 813.4; m/z 831.5 (M+H2O+H)+ 103, C43H59N8O9 requires 831.4; m/z 927.6 (M+TFA+H)+, C45H58F3N8O10 requires 927.4.
- The pentapeptide 102 was also synthesised on Wang resin (590 mg) with reduced loading (0.3 mmol g−1) using the procedured described above. The relative quantities of the Fmoc-amino acids and coupling agents remained constant throughout the synthesis: Wang resin:DIC:DMAP:Fmoc-amino acid:HATU:NMM, 1:3:0.3:3:2:6 equiv.
-
- The resin-bound peptide 102a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 102a (70.0 mg, 63.7 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (21.6 mg, 25.4 μmol, 40 mol %), 50° C., 42 h, 100% conversion into 104. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 104. Mass spectrum (ESI+, MeCN/H2O): m/z 785.4 (M+H)+, C41H53N8O8 requires 785.4; m/z 803.3 (M+H2O+H)+, C41H55N8O9 requires 803.4; m/z 899.4 (M+TFA+H)+, C43H54F3N8O10 requires 899.4.
- The resin-bound peptide 102a (synthesised on reduced loading Wang resin) was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 102a (97.0 mg, 29.1 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (2.5 mg, 2.9 μmol, 10 mol %), 50° C., 42 h, 100% conversion into 104. Mass spectral data of the isolated residue confirmed formation of the cyclic peptide 104 and were in agreement with those reported above.
-
- The resin-bound peptide 104a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 104a (350 mg, 0.32 mmol), DCM:MeOH (9:1, 8 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 22 h, 100% conversion into 105. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the saturated cyclic pentapeptide 105. Mass spectrum (ESI+, MeCN/H2O): m/z 787.2 (M+H)+, C41H55N8O8 requires 787.4; m/z 805.2 (M+H2O+H)+, C41H57N8O9 requires 803.4; m/z 901.3 (M+TFA+H)+, C43H56F3N8O10 requires 901.4.
-
- The resin-bound peptide 105a was subjected to the general cross metathesis procedure (Section 7.5.4) with cis-2-butene under the following conditions: Resin-peptide 105a (212 mg, 0.19 mmol), ICM (8 mL), 2nd generation Grubbs' catalyst (82 mg, 9.7 μmol, 50 mol %), cis-2-butene (15 psi), 50° C., 42 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the starting peptide 105 and the desired butenolysis product 106. The recovered resin-peptide was subjected to the same butenolysis conditions in order to drive the reaction to completion. After 42 h, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed quantitative conversion to the activated peptide 106. Mass spectrum (ESI+, MeCN/H2O): m/z 773.2 (M+H)+, C40H53N8O8 requires 773.4.
-
- The resin-bound peptide 106a was subjected to the general microwave-accelerated cross metathesis procedure (Section 7.5.3) under the following conditions: Resin-peptide 106a (20.0 mg, 18.0 μmol), DCM (4 mL), LiCl/DMF (0.4 M, 0.4 mL), 2nd generation Grubbs' catalyst (6.2 mg, 7.3 μmol, 40 mol %), (2S)-methyl 2-N-acetylaminohex-4-enoate 81 (70.0 mg, 0.38 mmol), 100° C., 2 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cross metathesis product 107. Mass spectrum (ESI+, MeCN/H2O): m/z 902.4 (M+H)+, C45H60N9O11 requires 902.4.
-
- The resin-bound peptide 107a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 107a (15.0 mg, 13.5 μmol), DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 22 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the reduced cyclic pentapeptide 108. Mass spectrum (ESI+, MeCN/H2O): m/z 904.4 (M+H)+, C45H62N9O11 requires 904.5.
-
- The resin-bound peptide Fmoc-Pre-Phe-Wang 144 was subjected to the microwave-assisted cross metathesis procedure (Section 7.5.3) with cis-1,4-diacetoxy-2-butene 141 under the following conditions: Resin (Wang)-peptide 144 (180 mg, 0.09 mmol), DCM (10 mL), 2nd generation Grubbs' catalyst (16 mg, 20 mol %), cis-1,4-diacetoxy-2-butene (96 mg, 0.56 mmol, 15 psi), 100° C., 1 h. At the end of the reaction period, the peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the desired dipeptide product 145 and no starting material. Mass spectral analysis of the isolated residue confirmed quantitative conversion to the activated peptide 145. Mass spectrum (ESI+, CH3OH): m/z 579.0 (M+Na) C32H32N2O7Na.
-
- The procedure described in Section 7.3.2 was used for the attachment of the first amino acid, Fmoc-L-Cys(Trt)-OH, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.10. The first coupling reaction was shaken for 14 h.
-
TABLE 7.10 Quantities of Reagents used in the Synthesis of Peptide 112 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 740 mg 0.39 Fmoc-L-Cys(Trt)-OH 676 mg 1.15 HATU 293 mg 0.77 NMM 255 μl 2.31 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the dodecapeptide 112. Quantities of the coupling agents HATU and NMM remained constant throughout the synthesis. The quantities of successive amino acids and their reaction durations are detailed in Table 7.11.
-
TABLE 7.11 Quantities of Amino Acids used in the Synthesis of Peptide 112 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 749 1.15 2.5 Fmoc-L-Trp(Boc)-OH 608 1.15 2.5 Fmoc-L-Ala-OH 360 1.16 14 Fmoc-L-Hag-OH 390 1.16 2.5 Fmoc-L-Arg(Pbf)-OH 750 1.16 2.5 Fmoc-L-Pro-OH 390 1.16 1 Fmoc-L-Asp(tBu)—OH 475 1.15 14 Fmoc-L-Ser(tBu)—OH 443 1.16 2.5 Fmoc-L-Cys(Trt)-OH 676 1.15 2.5 Fmoc-L-Hag-OH 390 1.16 2.5 Fmoc-L-Gly-OH 343 1.15 14 *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the dodecapeptide 112. Mass spectrum (ESI+, MeCN/H2O): m/z 783.5 [½(M+2H)]+, ½(C71H98N20O17S2) requires 783.3; m/z 1565.7 (M+H)+, C71H97N20O17S2 requires 1565.7. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=8.86 min.
-
- The resin-bound peptide was subjected to the general microwave-accelerated RCM procedure (Section 7.5.3) under the following conditions: Resin-peptide 112a (158 mg, 82.2 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2nd generation Grubbs' catalyst (7.0 mg, 8.2 μmol, 10 mol %), 100° C., 1 h, 100% conversion into 114. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 114. Mass spectrum (ESI+, MeCN/H2O): m/z 769.4 [½(M+2H)]+, ½(C69H94N20O17S2) requires 769.3; m/z 1537.7 (M+H)+, C69H93N20O17S2 requires 1537.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=8.59 min.
- An analogous microwave-accelerated RCM reaction using 5
mol % 2nd generation Grubbs' catalyst was performed: Resin-peptide 112a (80.2 mg, 42 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2nd generation Grubbs' catalyst (7.0 mg, 2.1 μmol, 5 mol %), 100° C., 2 h, 100% conversion into 114. Mass spectral data were in agreement with those reported above. -
- The Rink-amide bound peptide 114a (100 mg, 52.0 μmol) was swollen with DCM (3×1 min, 1×30 min) and DMF (3×1 min, 1×30 min) and deprotected with 20% piperidine/DMF (1×1 min, 2×20 min). The resin was then washed with DMF (5×1 min), DCM (3×1 min), MeOH (3×1 min) and dried on the SPPS manifold for 1 h. The Fmoc-deprotected peptidyl-resin (47.0 mg, 24.4 μmol) was subjected to the TFA-mediated cleavage procedure (Section 0). The residue was then lyophilised for 18 h to give the fully deprotected carbocyclic peptide 116 as a colourless solid (20.0 mg, 15.2 μmol). Mass spectrum (ESI+, MeCN/H2O): m/z 658.4 [½(M+2H)]+, ½(C54H84N20O15S2) requires 658.3; m/z 1315.6 (M+H)+, C54H83N20O15S2 requires 1315.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=5.63 min
- A sample of lyophilised peptide (10.1 mg, 7.7 μmol) was dissolved in an aqueous solution of (NH4)2CO3 (0.1 M, 80 mL) containing 5% DMSO (4 mL). The reaction was stirred at room temperature and monitored by the Ellman's test (Section 7.3.4). After 3 d, the reaction mixture was lyophilised and mass spectral analysis of the isolated residue confirmed formation of the cystine-oxidised peptide 118. The peptide was purified by RP-HPLC (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid) and the unsaturated [2,8]-dicarba-[3,12]-cystino conotoxin hybrid 118 was isolated as a colourless solid (1.8 mg, 5%) in >99% purity. Mass spectrum (ESI+, MeCN/H2O): m/z 657.4 [½(M+2H)]+, ½(C54H82N20O15S2) requires 657.3; m/z 668.3 [½(M+H+Na)]+, ½(C54H81N20NaO15S2) requires 668.3; m/z 1313.5 (M+H)+, C54H81N20O15S2 requires 1313.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=5.50 min.
-
- The resin-bound peptide 114a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 114a (285 mg, 0.15 mmol), DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 22 h. At the end of the reaction period, a small aliquot of peptidyl-resin was Fmoc-deprotected (20% piperidine/DMF, 1×1 min, 2×10 min) and washed with DMF (5×1 min), DCM (5×1 min), MeOH (5×1 min) and dried on the SPPS manifold for 1 h. The Fmoc-deprotected peptidyl-resin was then subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of a mixture of the cystine-oxidised 122 and reduced 120 form of the saturated product. Mass spectrum (ESI+, MeCN/H2O): m/z 658.6 [½(M+2H)]+ oxidised, ½(C54H84N20O15S2) requires 658.3; m/z 1315.7 (M+H)+ oxidised, C54H83N20O15S2 requires 1315.6; m/z 659.4 [½(M+2H)]+ reduced, ½(C54H86N20O15S2) requires 659.3; m/z 1317.8 (M+H)+ reduced, C54H85N20O15S2 requires 1317.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR (122)=6.01 min.
-
- The procedure described in Section 7.3.2.2 was used for the attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.12. The first coupling reaction was shaken for 14 h.
-
TABLE 7.12 Quantities of Reagents used in the Synthesis of Peptide 113 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 730 mg 0.38 Fmoc-L-Hag-OH 384 mg 1.14 HATU 289 mg 0.76 NMM 250 μl 2.27 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the dodecapeptide 113. Quantities of the coupling agents HATU and NMM remained constant throughout the synthesis. The quantities of successive amino acids and their reaction durations are detailed in Table 7.13.
-
TABLE 7.13 Quantities of Amino Acids used in the Synthesis of Peptide 113 Amino Acid Mass (mg) mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 739 1.14 2.5 Fmoc-L-Trp(Boc)-OH 600 1.14 2.5 Fmoc-L-Ala-OH 355 1.14 14 Fmoc-L-Cys(Trt)-OH 667 1.14 2.5 Fmoc-L-Arg(Pbf)-OH 740 1.14 2.5 Fmoc-L-Pro-OH 385 1.14 1 Fmoc-L-Asp(tBu)—OH 470 1.14 14 Fmoc-L-Ser(tBu)—OH 437 1.14 2.5 Fmoc-L-Hag-OH 385 1.14 2.5 Fmoc-L-Cys(Trt)-OH 667 1.14 2.5 Fmoc-L-Gly-OH 340 1.14 14 *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the dodecapeptide 113. Mass spectrum (ESI+, MeCN/H2O): m/z 783.5 [½(M+2H)]+, ½(C71H98N20O17S2) requires 783.3; m/z 1565.7 (M+H)+, C71H97N20O17S2 requires 1565.7. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=9.13 min.
-
- The resin-bound peptide 113a was subjected to the general microwave-accelerated RCM procedure (Section 7.5.3) under the following conditions: Resin-peptide 113a (840 mg, 0.44 mmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (74.3 mg, 87.5 μmol, 20 mol %), 100° C., 1 h, 100% conversion into 115. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 115. Mass spectrum (ESI+, MeCN/H2O): m/z 769.4 [½(M+2H)]+, ½(C69H94N20O17S2) requires 769.3; m/z 1537.7 (M+H)+, C69H93N20O17S2 requires 1537.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=8.99 min.
-
- The resin-bound peptide 115a (100 mg, 52.0 μmol) was swollen with DCM (3×1 min, 1×30 min) and DMF (3×1 min, 1×30 min) and deprotected with 20% piperidine/DMF (1×1 min, 2×20 min). The resin was then washed with DMF (5×1 min), DCM (3×1 min), MeOH (3×1 min) and dried on the SPPS manifold for 1 h. The Fmoc-deprotected peptidyl-resin (61.7 mg, 32.1 μmol) was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). The residue was then lyophilised for 18 h to give the fully deprotected carbocyclic peptide 117 as a colourless solid (15.1 mg, 11.5 μmol). Mass spectrum (ESI+, MeCN/H2O): m/z 658.4 [½(M+2H)]+, ½(C54H84N20O15S2) requires 658.3; m/z 669.4 [½(M+H+Na)]+, ½(C54H84N20O15S2) requires 669.4; m/z 1315.6 (M+H)+, C54H83N20O15S2 requires 1315.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=6.62 min.
- A sample of lyophilised peptide (11.2 mg, 8.5 μmol) was dissolved in an aqueous solution of (NH4)2CO3 (0.1 M, 80 mL) containing 5% DMSO (4 mL). The reaction was stirred at room temperature and monitored by the Ellman's test. After 3 d, the reaction mixture was lyophilised and mass spectral analysis of the isolated residue confirmed formation of the cystine-oxidised peptide 119. The peptide was purified by RP-HPLC (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid) and the unsaturated [2,8]-cystino-[3,12]-dicarba conotoxin hybrid 119 was isolated as a colourless solid (2.3 mg, 5%) in >99% purity. Mass spectrum (ESI+, MeCN/H2O): m/z 657.3 [½(M+2H)]+, ½(C54H82N20O15S2) requires 657.3; m/z 668.3 [½(M+H+Na)]+, ½(C54H81N20NaO15S2) requires 668.3; m/z 1313.6 (M+H)+, C54H31N20O15S2 requires 1313.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=4.46 min.
-
- The resin-bound peptide 119a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 119a (320 mg, 0.17 mmol), DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 22 h. At the end of the reaction period, a small aliquot of peptidyl-resin was Fmoc-deprotected (20% piperidine/DMF, 1×1 min, 2×10 min) and washed with DMF (5×1 min), DCM (5×1 min), MeOH (5×1 min) and dried on the SPPS manifold for 1 h. The Fmoc-deprotected peptidyl-resin was then subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of a mixture of the cystine-oxidised 123 and reduced 121 form of the saturated product. Mass spectrum (ESI+, MeCN/H2O): m/z 658.5 [½(M+2H)]+ oxidised, ½(C54H84N20O15S2) requires 658.3; m/z 1315.7 (M+H)+ oxidised, C54H83N20O15S2 requires 1315.6; m/z 659.3 [½(M+2H)]+ reduced, ½(C54H86N20O15S2) requires 659.3; m/z 1317.6 (M+H)+ reduced, C54H85N20O15S2 requires 1317.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR (123)=7.02 min.
-
Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Asn-Tyr-Asp-His-Pro-Glu-Ile-Cys-NH2 - The procedure described in Section 7.3.5 was used for the synthesis of Vc1.1 on Rink Amide resin (loading 0.52 mmol/g). Quantities of the resin, coupling reagents and amino acids are tabulated below:
-
Mole Quantity (mmol)/ Compound (mL/g) Volume Conc (M) Cycle Name Rink Amide 0.481 g 5 mL DMF 0.25 mmol — DIPEA 7.7 mL 22 mL NMP 2M — HBTU 6.827 g 36 mL DMF 0.45M — HOBt 2.432 g Fmoc-Arg- 0.389 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Asn- 0.358 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Asp- 0.494 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Cys- 0.703 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Glu- 0.255 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Gly- 0.178 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-His- 0.372 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ile- 0.212 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Pro- 0.405 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ser- 0.230 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Hag- 0.405 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Tyr- 0.276 3 mL DMF 0.2M B0.25-Single (ext.) OH - Resin washings and deprotection cycles were performed as described in Section 7.3.5. The amino acid, activator and activator base solutions were added to the resin, followed by the “B.01 Extended Coupling” cycle. The peptidyl-resin was exposed to a temperature of 75° C. with no power (0 watts) for 2 min, then at a temperature of 75° C., power at 25 watts for 10 min. The peptidyl-resin was then washed with DMF (3×10 mL).
- Following the final amino acid coupling, a small aliquot of the resin bound peptide was cleaved as described in Section 7.3.3 for mass spec analysis. Mass spectrum (ESI+, MeOH/H2O): m/z 592.8 (M+3H/3), m/z 1011.1 (M+2H/2), m/z 1039.5 ((M+tBu)+3H/3).
-
- The resin bound linear peptide was subjected to microwave RCM procedure outlined in Section 7.5.3. Peptidyl-resin (0.4810 mg, 0.25 mmol) and 2nd generation Grubb's catalyst (42.4 mg, 0.05 mmol) was weighted into a glass vial loaded with stirrer bar. In a drybox, DCM (5 mL) and LiCl/DMF (0.2 mL) were added and the vial was sealed. The reaction vessel was placed in the microwave for 1 hr at 100 C. A small aliquot of the resin bound peptide was subjected to TFA cleavage and analysed by mass spectroscopy. Mass spectrum (ESI+, MeOH/H2O): m/z 997.2 (M+2H/2), m/z 1011.0 (SM+2H/2). The same procedure was followed for ring closure of linear α-RgIA.
-
Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Arg-Tyr-Arg-Cys-Arg-N H2 - The procedure described in Section 7.3.5 was used for the synthesis of RgIA on Rink Amide resin (loading 0.52 mmol/g). Quantities of the resin, coupling reagents and amino acids are tabulated in the Table below:
-
Mole Quantity (mmol)/ Compound (mL/g) Volume Conc (M) Cycle Name Rink Amide 0.192 g 5 mL DMF 0.10 mmol — DIPEA 3.8 mL 11 mL 2M — NMP HBTU 2.655 g 18 mL 0.45M — HOBt 0.946 g DMF Fmoc-Arg- 1.427 g 11 mL 0.2M B0.25-Single (ext.) OH DMF Fmoc-Asp- 0.247 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Cys- 0.703 g 6 mL DMF 0.2M B0.25-Single (cys/ OH his ext.) Fmoc-Gly- 0.178 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Pro- 0.202 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ser- 0.230 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Hag- 0.405 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Tyr- 0.276 g 3 mL DMF 0.2M B0.25-Single (ext.) OH - Resin washings and deprotection cycles were performed as described in Section 7.3.5. The amino acid, activator and activator base solutions were added to the resin, followed by the “B.01 Extended Coupling” cycle. The peptidyl-resin was exposed to a temperature of 75° C. with no power (0 watts) for 2 min, then at a temperature of 75° C., power at 25 watts for 10 min. The peptidyl-resin was then washed with DMF (3×10 mL). However cysteine residues in RgIA have been known to be susceptible to racemisation at 75° C., therefore a different cycle was used for the coupling of this amino acid. Following the deprotection cycles, “B.01 Single Cys/His Extended” coupling cycle was included in the method for the coupling of cysteine. This involves exposure to a temperature of 50° C. with no power (0 watts) for 2 min, then at a temp of 50° C., power at 25 watts for 10 min. The peptidyl resin was then washed with DMF (3×10 mL).
- Following the final amino acid coupling, a small aliquot of the resin bound peptide was cleaved as described in Section 7.3.3 for mass spec analysis. Mass spectrum (ESI+, MeOH/H2O): m/z 595.5 (M+3H/3), m/z 614.3 ((M+tBu)+2H/2), m/z 892.6 (M+2H/2).
-
- The procedure described in Section 7.3.2.2 was used for the attachment of the first amino acid, Fmoc-L-Pre-OH 92, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.14. The first coupling reaction was shaken for 4 h.
-
TABLE 7.14 Quantities of Reagents used in the Synthesis of Peptide 127 Regent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 610 mg 0.32 Fmoc-L-Pre-OH 350 0.96 HATU 241 mg 0.63 NMM 210 μl 1.91 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the dodecapeptide 127. The quantities of successive amino acids and their reaction durations are detailed in Table 7.15.
-
TABLE 7.15 Quantities of Amino Acids used in the Synthesis of Peptide 127 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 617 0.95 12 Fmoc-L-Trp(Boc)-OH 502 0.95 2.5 Fmoc-L-Ala-OH 297 0.95 2.5 Fmoc-L-Hag-OH 321 0.95 2.5 Fmoc-L-Arg(Pbf)-OH 617 0.95 14 Fmoc-L-Pro-OH 321 0.95 4 Fmoc-L-Asp(tBu)—OH 392 0.95 2.5 Fmoc-L-Ser(tBu)—OH 365 0.95 2.5 Fmoc-L-Pre-OH 350 0.96 12 Fmoc-L-Hag-OH 322 0.95 2.5 Fmoc-L-Gly-OH 285 0.96 2.5 (1) *Note: Reaction times have not been optimised.
After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed fonnation of the dodecapeptide 127. Mass spectrum (ESI+, MeCN/H2O): m/z 805.6 [½(M+2H)]+, ½(C79H110N20O17) requires 805.4; m/z 814.6 [½(M+H2O+2H)]+, ½(C79H112N20O18) requires 814.4. -
- The Rink amide-bound peptide 127a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 127a (165 mg, 0.12 mmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (39.9 mg, 47.0 μmol, 40 mol %), 50° C., 40 h, 100% conversion into 129. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 129. Mass spectrum (ESI+, MeCN/H2O): m/z 791.4 [½(M+2H)]+, ½(C77H106N20O17) requires 791.4. m/z 800.5 [½(M+H2O+2H)]+, ½(C77H108N20O18) requires 800.4.
- The Rink amide-bound peptide 127a was subjected to the general microwave-accelerated RCM procedure (Section 7.5.3) under the following conditions: Resin-peptide 127a (127 mg, 66.0 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (5.6 mg, 6.6 μmol, 10 mol %), 100° C., 1 h, 100% conversion into 129. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 129. Mass spectrum (ESI+, MeCN/H2O): m/z 791.5 [½(M+2H)]+, ½(C77H106N20O17) requires 791.4.
-
- The resin-bound peptide 129a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 129a (130 mg, 91.0 μmol), DCM:MeOH (9:1, 6.5 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 24 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the selectively hydrogenated cyclic dodecapeptide 133. Mass spectrum (ESI+, MeCN/H2O): m/z 792.5 [½(M+2H)]+, %(C77H108N20O17) requires 792.4; m/z 801.4 [½(M+H2O+2H)]+, ½(C77H110N20O18) requires 801.4.
- † Act=Activated sidechain, i.e, crotylglycine (Crt) or allylglycine (Hag)
- The resin-bound peptide 133a was subjected to the general cross metathesis procedure with cis-2-butene (Section 7.5.4) under the following conditions:
- Resin-peptide 133a (76.0 mg, 55.5 μmol), DCM (5 mL), 2nd generation Grubbs' catalyst (24.0 mg, 28.3 μmol, 50 mol %), cis-2-butene (15 psi), benzoquinone (6.2 mg, 57.4 mol), 50° C., 38 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the desired butenolysis product 135. No starting material was evident, however, low intensity doubly charged higher homologue species separated by m/z+7 units were observed.
- Mass spectrum (ESI+, MeCN/H2O): m/z 778.4 [½(M+2H)]+ product, ½(C75H104N20O17) requires 778.4. Low intensity higher homologue species at m/z 785.4, 793.5, 800.4; Very low intensity peaks at m/z 807.8, 814.3, 821.3, 828.1, 835.7, 842.6, 849.8, 856.1, 863.6.
- Resin-peptide 133a (20.0 mg, 10.4 μmol), DCM (3 mL), LiCl/DMF (0.3 mL), 2nd generation Grubbs' catalyst (1.0 mg, 1.2 μmol, 10 mol %), cis-2-butene (20 psi), 50° C., 62 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the desired product 135, a partially metathesised peptide (mono-crotylglycine containing peptide) 136 and the starting peptide 133. Mass spectrum (ESI+, MeCN/H2O): m/z 778.4 [½(M+2H)]+ product, ½(C75H104N20O17) requires 778.4. m/z 785.5 [½(M+2H)]+ 136, ½(C76H106N20O17); m/z 792.4 [½(M+2H)]+ 133, ½(C77H108N20O17); m/z 801.4 [½(M+H2O+2H)]+ 133, ½(C77H110N20O18).
- Resin-peptide 133a (35.0 mg, 18 μmol), DCM (5 mL), LiCl/DMF (0.5 mL), 2nd generation Grubbs' catalyst (6.2 mg, 7.3 μmol, 40 mol %), cis-2-butene (20 psi), 50° C., 24 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the desired product 135, a partially metathesised peptide (mono-crotylglycine containing peptide) 136 and the starting peptide 133. Mass spectral data were consistent with those reported above (Method B).
- An analogous reaction was performed for 62 h under the following conditions: Resin-peptide 133a (30.2 mg, 15.7 μmol), DCM (5 mL), LiCl/DMF (0.5 mL), 2nd generation Grubbs' catalyst (6.2 mg, 7.3 μmol, 40 mol %), cis-2-butene (20 psi), 50° C., 62 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the desired product 135, a partially metathesised peptide 136 and the starting peptide 133. Mass spectral data were consistent with those previously reported (Method B).
- The resin-bound peptide 133a was subjected to the general cross metathesis procedure with ethylene (Section 7.5.4) under the following conditions:
- Resin-peptide 133a (42.0 mg, 21.8 mop, DCM (5 mL), LiCl/DMF (0.5 mL), 2nd generation Grubbs' catalyst (7.5 mg, 8.8 μmol, 40 mol %), ethylene (60 psi), 50° C., 62 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the starting peptide 133 and a partially metathesised peptide (mono-allylglycine containing peptide) 137. Mass spectrum (ESI+, MeCN/H2O): m/z 778.4 [½(M+2H)]+ 137, ½(C75H104N20O17); m/z 792.4 [½(M+2H)]+ 133.
- An analogous reaction was performed in the absence of the chaotropic salt (LiCl) under the following conditions: Resin-peptide 133a (68.0 mg, 35.4 μmol), DCM (5 mL), 2nd generation Grubbs' catalyst (12.0 mg, 14.1 μmol, 40 mol %), ethylene (60 psi), 50° C., 62 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of the starting peptide 133 and a partially metathesised peptide (mono-allylglycine containing peptide) 137. Mass spectral data were consistent with those reported above.
-
- The Rink amide-bound peptide 135a was subjected to the general microwave-accelerated RCM procedure (Section 7.5.3) under the following conditions: Resin-peptide 135a (20.0 mg, 14.0 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (2.4 mg, 2.8 μmol, 20 mol %), 100° C., 1 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). LC-MS analysis of the isolated residue supported formation of the bicyclic peptide 140. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): tR=9.18 min, m/z 750.4 [½(M+2H)]+, ½(C71H94N20O17) requires 750.4.
-
- The resin-bound peptide 140a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 140a (12.2 mg, 8.5 μmol), DCM:MeOH (9:1, 6.5 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 24 h. At the end of the reaction period, a small aliquot of peptidyl-resin was Fmoc-deprotected with 20% piperidine/DMF (1×1 min, 2×20 min). The resin was then washed with DMF (5×1 min), DCM (3×1 min), MeOH (3×1 min) and dried on the SPPS manifold for 1 h. The Fmoc-deprotected peptidyl-resin was then subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral and LC-MS data of the isolated residue were inconclusive. The mass spectrum and LC-traces did not display peaks due to the fully deprotected starting peptide 125 and the target saturated bicycle 126. Lack of material and time constraints did not allow us to investigate this chemistry further.
-
- The procedure described in Section 7.3.2.2 was used for the attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.16. The first coupling reaction was shaken for 12 h.
-
TABLE 7.16 Quantities of Reagents used in the Synthesis of Peptide 128 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 705 mg 0.37 Fmoc-L-Hag-OH 371 mg 1.10 HATU 280 mg 0.74 NMM 245 μl 2.22 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the dodecapeptide 128. The quantities of successive amino acids and their reaction durations are detailed in Table 7.17.
-
TABLE 7.17 Quantities of Amino Acids used in the Synthesis of Peptide 128 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 715 1.10 2.5 Fmoc-L-Trp(Boc)—OH 580 1.10 2.5 Fmoc-L-Ala-OH 343 1.10 2.5 Fmoc-L-Pre-OH 402 1.10 12 Fmoc-L-Arg(Pbf)-OH 715 1.10 2.5 Fmoc-L-Pro-OH 371 1.10 2.5 Fmoc-L-Asp(tBu)—OH 453 1.10 2.5 Fmoc-L-Ser(tBu)—OH 422 1.10 12 Fmoc-L-Hag-OH 371 1.10 2.5 Fmoc-L-Pre-OH 402 1.10 2.5 Fmoc-L-Gly-OH 328 1.10 2 (12) *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectra analysis of the isolated residue confirmed formation of the dodecapeptide 128. Mass spectrum (ESI+, MeCN/H2O): m/z 805.6 [½(M+2H)]+, ½(C79H110N20O17) requires 805.4; m/z 816.6 [½(M+Na+H)]+, ½(C79H111N20NaO18) requires 816.4.
-
- The resin-bound peptide 128a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 128a (19.7 mg, 10.2 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2nd generation Grubbs' catalyst (3.5 mg, 4.1 μmol, 40 mol %), 50° C., 40 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated recovery of the linear peptide 132. Mass spectrum (ESI+, MeCN/H2O): m/z 805.6 [½(M+2H)]+ linear, ½(C79H110N20O17).
- The resin-bound peptide 128a was subjected to the general microwave-accelerated RCM procedure (Section 7.5.3) under the following conditions: Resin-peptide 128a (550 mg, 0.29 mmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), generation Grubbs' catalyst (49.0 mg, 57.7 μmol, 20 mol %), 100° C., 1 h, 100% conversion into 132. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the cyclic peptide 132. Mass spectrum (ESI+, MeCN/H2O): m/z 791.4 [½(M+2H)]+, ½(C77H106N20O17) requires 791.4.
-
- The resin-bound peptide 132a was subjected to the general Wilkinson's hydrogenation procedure (Section 7.4.4) under the following conditions: Resin-peptide 132a (365 mg, 0.19 mmol), DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H2, 22° C., 19 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the selectively hydrogenated cyclic peptide 134. Mass spectrum (ESI+, MeCN/H2O): m/z 792.5 [½(M+2H)]+, ½(C77H108N20O17) requires 792.4; m/z 801.4 [½(M+H2O+2H)]+, ½(C77H110N20O18) requires 801.4.
-
- The resin-bound peptide 134a was subjected to the conventional cross metathesis procedure with cis-2-butene (Section 7.5.4) under the following conditions:
- Resin-peptide 134a (78.5 mg, 41 μmol), DCM (5 mL), 2nd generation Grubbs' catalyst (13.9 mg, 16 μmol, 40 mol %), cis-2-butene (15 psi), 50° C., 62 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of a mixture of peptides: the starting peptide 134, the desired product 138 and a partially metathesised peptide (mono-butenolysis product) 139. Mass spectrum (ESI+, MeCN/H2O): m/z 778.5 [½(M+2H)]+ product, ½(C75H104N20O17) requires 778.4; m/z 785.5 [½(M+2H)]+ 139, ½(C76H106N20O17); m/z 792.5 [½(M+2H)]+ 134, ½(C77H108N20O17).
- An analogous reaction in the presence of a chaotropic salt (LiCl) was performed under the following conditions: Resin-peptide 134a (60.1 mg, 31 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (10.6 mg, 12 μmol, 40 mol %), cis-2-butene (15 psi), 50° C., 62 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue indicated the presence of a mixture of peptides: the starting peptide 134a, the desired product 138 and a partially metathesised peptide 139. Mass spectral data were consistent with those reported above.
-
- The procedure described in Section 7.3.2.2 was used for the attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin. Quantities of the resin and coupling reagents HATU and NMM are presented in Table 7.18. The first coupling reaction was shaken for 5 h.
-
TABLE 7.18 Quantities of Reagents used in the Synthesis of Peptide 130 Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 400 mg 0.29 Fmoc-L-Hag-OH 295 mg 0.88 HATU 223 mg 0.59 NMM 193 μl 1.75 - The procedure outlined in Section 7.3.2.2 was also utilised for subsequent coupling reactions in the synthesis of the dodecapeptide 130. The quantities of successive amino acids and their reaction durations are detailed in Table 7.19.
-
TABLE 7.19 Quantities of Amino Acids used in the Synthesis of Peptide 130 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH 570 0.88 12 Fmoc-L-Trp(Boc)—OH 462 0.88 2 (1) Fmoc-L-Pro-OH 296 0.88 1 (12) Fmoc-L-Pre-OH 328 0.90 4 Fmoc-L-Arg(Pbf)-OH 570 0.88 2 (12) Fmoc-L-Pro-OH 296 0.88 2 (2) Fmoc-L-Asp(tBu)—OH 361 0.88 2 (2) Fmoc-L-Ser(tBu)—OH 336 0.88 2 (12) Fmoc-L-Hag-OH 296 0.88 4 Fmoc-L-Pre-OH 330 0.90 1 (2) Fmoc-L-Gly-OH 262 0.88 2 (1) *Note: Reaction times have not been optimised. - After the final amino acid coupling, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed formation of the dodecapeptide 130. Mass spectrum (ESI+, MeCN/H2O): m/z 818.6 [½(M+2H)]+, ½(C81H112N20O17) requires 818.4; m/z 827.6 [½(M+H2O+2H)]+, ½(C81H114N20O18) requires 827.4.
-
- The resin-bound peptide 130a was subjected to the conventional RCM procedure (Section 7.5.2) under the following conditions: Resin-peptide 130a (97.0 mg, 70.8 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2nd generation Grubbs' catalyst (24.1 mg, 28.4 μmol, 40 mol %), 50° C., 40 h. At the end of the reaction period, a small aliquot of peptidyl-resin was subjected to the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysis of the isolated residue confirmed the presence of both cyclic 131 and linear 130 peptides. Mass spectrum (ESI+, MeCN/H2O): m/z 804.5 [½(M+2H)]+ cyclic, ½(C79H108N20O17) requires 804.4; m/z 813.8 [½(M+H2O+2H)]+ cyclic, ½(C79H110N20O18) requires 813.4; m/z 818.7 [½(M+2H)]+ linear, ½(C81H112N20O17); m/z 827.3 [½(M+H2O+2H)]+ linear, ½(C81H114N20O18).
-
- Standard solution phase metathesis conditions (see section 7.5) were employed to synthesise 6-acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 from the cross metathesis of the corresponding
prenyl derivative 87 and 1,4-diacetoxy-cis-2-butene. The desired product was obtained as a dark brown oil following by column chromatography (SiO2; EtOAc:Hexane, 1:1). - N-Bzl-O-Me-prenylglycine (170 mg, 0.65 mmol), dichloromethane (10 mL), second generation Grubbs' catalyst (16.5 mg, 5 mol %, 0.03 mmol), 1,4-diacetoxy-cis-2-butene (671.5 mg, 3.9 mmol), 50° C., 20 h; 112.5 mg, 57%.
- GC: tR (E/Z)=12.96, 13.06 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min.)
- IR (film): 3333s; 3056w; 3015w; 2944s; 1739s; 1662m; 1641s; 1605m; 1574m; 1533s; 1487m; 1436m; 1364m; 1236s; 1154w; 1072w; 1026m; 969m; 801w; 718m; 692w cm−1.
- 1H NMR (400 MHz, CDCl3): δ 2.00, s, 3H, CH3; 2.67, m, 2H, H3; 3.77, s, 3H, OCH3; 4.49, d, J 4.7 Hz, 2H, H6; 4.89, q, J 5.8 Hz, 1H, H2; 5.68, t, J 5.2 Hz, 2H, H4, 5; 6.75, d, J 7.4 Hz, 2H, H4, 5; 7.42, t, J 7.2 Hz, 2H, H4′, 6′; 7.50, t, J 6.4 Hz, 1H, H5′; 7.78, d, J 7.1 Hz, 2H, H3′, 7′.
- 13C NMR (125 MHz, CDCl3): δ 20.9, CH3; 35.2, C3; 52.1, OCH3; 52.7, C2; 64.5, C6; 127.2, C3′, 7; 128.7, C4′, 6′; 128.9, C5; 129.1, C4; 131.9, C5′; 133.9, CT; 167.1, C1′; 170.8, C1″; 172.3, C1.
- Mass Spectrum (ES CH3CN): m/z 328.1 (M+Na+) C16H19NO5Na.
- HRMS (EI, CH3OH). found m/z 305.1263, C16H19NO5 requires 305.1263.
-
- 2,7-Bis-benzoylamino-4-octenedioic acid dimethyl ester was synthesised using standard solution phase metathesis conditions (refer to section 7.5). Due to the equilibrium generated in the reaction, a mixture of the homodimer 69 and the starting material 141 was obtained.
- 6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 (53.5 mg, 0.18 mmol), dichloromethane (10 mL), second generation Grubbs' catalyst (5 mol %, 7.4 mg, 8.8 μmol), 50° C., 18 h.
- GC: tR (1,4-diacetoxy-cis-2-butene)=3.28; (product E/Z)=13.18, 13.31 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min.). The mass spectrum was consistent with that previously described for this compound.
-
- 2-Acetylamino-7-benzoylamino-4-octenedioic acid dimethyl ester 142 was synthesised using standard solution-phase metathesis conditions (refer to section 7.5) from 6-acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 and methyl-2-acetylamino-4-pentenoate 121a. The desired compound was obtained as a brown oil, and purified via column chromatography (SiO2; EtOAc:Hexane; 2:1).
- 6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 (50 mg, 0.16 mmol), dichloromethane (10 mL), second generation Grubbs' catalyst (7 mg, 5 mol %, 8 μmol), methyl-2-acetylamino-4-pentenoate 142 (168 mg, 0.98 mmol), 50° C., 18 h, 48.6 mg, 81%.
- GC: tR (E/Z)=14.30, 14.50 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min−1 to 280° C. for 6 min.)
- 1H NMR (500 MHz, CDCl3, mixture of isomers (1:1.2)): δ 1.95, s (major isomer) and 1.97, s (minor isomer), 3H, CH3; 2.42-2.70, m, 4H, H3, 6; 3.62, s (minor isomer), 3.64, s (major isomer), 3.78, s (minor isomer) and 3.79, s (major isomer), 6H, 2×OCH3; 4.63-4.66, m, 1H, H2; 4.85-4.91, m, 1H, H7; 5.35-5.49, m, 2H, H4, 5; 6.20, d, J 7.7 Hz (major isomer) and 6.34, d, J 7.5 Hz, 1H, NH (minor isomer); 6.87, t, J 7.55 Hz, 1H, NH; 7.44, t, J 7.1 Hz, 2H, H4′, 6′; 7.50, t, J 6.9 Hz, 1H, H5′; 7.84, t, J 7.9 Hz, 2H, H3′, 7′.
- 13C NMR (75 MHz, CDCl3): δ 22.8, CH3; 34.8, 35.1, 35.4 and 35.7, C3, 6; 51.5 and 51.6, C2; 52.4, 52.5, 52.5, 52.6 and 52.7, C7, 2×OCH3; 127.2 and 127.2, C3′, 7′; 128.6 and 128.6, C4′, 6′; 128.9 and 129.0, C4, 5; 131.9 and 131.9, C5′; 133.7, C2; 167.1, COPh; 170.0 and 170.1, COMe; 172.2, 172.3, 172.3 and 172.4, 2×COOMe.
- Mass Spectrum (ESI+, CH3OH): m/z 399.2 (M+Na+) C19H24N2O6Na.
-
- The dipeptide, Fmoc-Pre-Phe-OH, was synthesised on pre-functionalised Fmoc-Phe-Wang resin (250 mg, 0.13 mmol) according to standard SPPS techniques (see section 7.3.2). Fmoc-prenylglycine (138 mg, 0.38 mmol) was coupled using HATU (144.5 mg, 0.38 mmol) and NMM (83.6 mL, 0.76 mmol). An aliquot of resin was subjected to cleavage condtions (see section 7.3.3) to assess reaction success.
- Mass Spectrum (ESI+, CH3OH): m/z 513.2 (M+H+), +), C31H33N2O5; 535.1 (M+Na+), C31H32N2O5Na.
-
- The resin-tethered dipeptide was subjected to microwave-assisted cross metathesis conditions (see section 7.3.2) with 1,4-diacetoxy-cis-2-butene. An aliquot of resin was subjected to cleavage condtions (see section 7.3.3) to assess reaction success.
- Resin-tethered dipeptide (180 mg, 0.09 mmol), second generation Grubbs' catalyst (15.3 mg, 20 mol %, 0.018 mmol), 1,4-diacetoxy-cis-2-butene (97 mg, 0.56 mmol), dichloromethane (10 mL), 100° C., 1 h, 100% conversion.
- Mass Spectrum (ESI+, CH3OH): m/z 557.2 (M+H+), +), C32H33N2O7; 579.2 (M+Na+), C32H32N2O7Na.
- The manual peptide synthesis procedure described in Section 7.3.2 was used for the synthesis of AOD9604 on Wang-Phe-Fmoc resin. Quantities of the resin and coupling reagents HATU and NMM are tabulated below. The quantities of successive amino acids are summarized below:
-
Mole Compound Quantity MW or Loading (mmol) Equivalents Wang-Phe-Fmoc 500 mg 0.52 mmol/g 0.25 1 Resin HATU 198 mg 380.23 0.52 2 NMM 172 μL 101.15 1.56 6 Quantity Mole Reaction Compound (mg) MW (mmol)/Eq. Time (hr) Fmoc-Gly-OH 232 297.14 0.78/3 2 Fmoc-Hag-OH 263 337.37 0.78/3 16 Fmoc-Gly-Ser(ψPro)- 331 424.5 0.78/3 2 OH Fmoc-Glu-OH 332 425.5 0.78/3 2 Fmoc-Val-OH 265 339.22 0.78/3 2 Fmoc-Ser-OH 299 383.4 0.78/3 2 Fmoc-Arg-OH 534 684.4 0.78/3 16 Fmoc-Hag-OH 263 337.37 0.78/3 2 Fmoc-Gln-OH 476 610.7 0.78/3 2 Fmoc-Val-OH 265 339.22 0.78/3 2 Fmoc-Ile-OH 276 353.24 0.78/3 2 Fmoc-Arg-OH 534 684.8 0.78/3 16 Fmoc-Leu-OH 276 353.24 0.78/3 2 Fmoc-Tyr-OH 358 459.5 0.78/3 2 - Following the final amino acid coupling, a small aliquot of the resin bound peptide was cleaved as described in Section 7.3.3 for mass spec analysis. Mass spectrum (ESI+, MeOH/H2O): m/z 676.5 (M+3H/3), m/z 1014.6 (M+2H/2).
- The procedure described in Section 7.5.3 was used for the synthesis of AOD9604 on Wang-Phe-Fmoc resin. Quantities of the resin, coupling reagents and amino acids are tabulated below:
-
Mole Quantity (mmol)/ Compound (mL/g) Volume Conc (M) Cycle Name Wang-Phe- 0.481 g 5 mL DMF 0.25 mmol — Fmoc DIPEA 7.7 mL 22 mL NMP 2M — HBTU 6.827 g 36 mL DMF 0.45M — HOBt 2.432 g Fmoc-Arg- 1.427 g 11 mL DMF 0.2M B0.25-Double OH (ext.) Fmoc-Hag- 1.289 g 11 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Gln- 0.737 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Glu- 0.425 g 5 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Gly- 0.357 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ile- 0.424 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Leu- 0.353 g 5 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-ψPro- 0.562 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ser- 0.460 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Tyr- 0.551 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Val- 0.747 g 11 mL DMF 0.2M B0.25-Single (ext.) OH - Resin washings and deprotection cycles were performed as described in Section 7.5.3. The amino acid, activator and activator base solutions were added to the resin, followed by the “B.01 Extended Coupling” cycle. The peptidyl-resin was exposed to a temperature of 75° C. with no power (0 watts) for 2 min, then at a temperature of 75° C., power at 25 watts for 10 min. The peptidyl-resin was then washed with DMF (3×10 mL). Most amino acids are programmed with the “B0.25-Single (Extended)” coupling cycle, which concludes at this point. However arginine requires and “B0.25-Double (Extended)” coupling cycle as several AOD deletion products have been produced in the past. This involves two “B0.1 Extended Coupling” cycle programs.
- Following the final amino acid coupling, a small aliquot of the resin bound peptide was cleaved as described in Section 7.3.3 for mass spec analysis. Mass spectrum (ESI+, MeOH/H2O): m/z 689.1 (M+3H/3), m/z 1014.2 (M+2H/2).
- The resin bound peptide 146 was subjected to microwave RCM procedure outlined in section 7.5.3. Peptidyl-resin (0.9088 mg, 0.475 mmol) and 2nd generation Grubb's catalyst (80 mg, 0.095 mmol) was weighted into a glass vial loaded with stirrer bar. In a drybox, DCM (5 mL) and LiCl/DMF (0.2 mL) were added and the vial was sealed. The reaction vessel was placed in the microwave for 1 hr at 100 C. A small aliquot of the resin bound peptide was subjected to TFA cleavage and analysed by mass spec to show the target unsaturated AOD 147. Mass spectrum (ESI+, MeOH/H2O): m/z 1000.3 (SM+2H/2), m/z 1014.2 (M+2H/2). The crude peptide was purified by reverse phase HPLC.
- A 150 mL glass hydrogenation vessel with plastic shield was loaded with the peptidyl-resin (0.9168 mg, 0.476 mmol) and stirrer bar. In an inert atmosphere, Wilkinson's catalyst (22 mg, 0.024 mmol) and 10 mL solvent (DCM:MeOH, 9:1) was added. The vessel was sealed with a rubber 0 ring and fitted with a pressure regulator. The vessel was purged with argon then hydrogen to a pressure of 90 psi and reacted at r.t. for 4 days. The reaction was terminated upon exposure to oxygen and the resin was washed with DCM (5 mL, 3×1 min), DMF (5 mL, 3×1 min) then MeOH (5 mL, 3×1 min) and dried in vacuo for 30 min prior to cleavage and mass spec analysis. Mass spec analysis showed conversion to the saturated cyclic peptide 148 (ESI+, MeOH/H2O): m/z 1000.9 (M+2H/2), m/z 1015.6 (SM+2H/2). The crude peptide was purified by reverse phase HPLC.
- Bovine adrenal chromaffin cells can be used to test for the activity of α-CTX ImI at neuronal-type nicotinic receptors. These cells are of two types, adrenaline- and noradrenaline-containing, and possess the neuronal-type nicotinic receptor subtypes α3β4 and α7. When stimulated with nicotine, these cells release adrenaline and noradrenaline which can be measured. Native α-CTX ImI peptides inhibit the nicotine-stimulated release of these neurotransmitters by interacting with the α3β4-receptor subtype.
- Dicarba-conotoxins 118 and 119 were assayed in quadruplicate in multiwells (6×4) containing monolayer cultures of bovine adrenal chromaffin cells as described by Broxton et al. (Loughnan, M., Bond, T., Atkins, A., Cuevas, J., Adams, D. J., Broxton, N.M., Livett, B. G., Down, J. G., Jones, A., Alewood, P. F., Lewis, R. J. J. Biol. Chem., 1998, 273 (25), 15667-15674.)X The response of the cells to these peptides was tested at two
peptide concentrations 1 uM and 5 uM. The cells were stimulated with nicotine (4 uM) for 5 min at room temp (23C) and the amount of catecholamine release (noradrenaline and adrenaline) was measured over a 5 period and expressed as a % of the initial cellular content of these amines. This was performed in the presence and absence (control) of dicarba-conotoxin peptides. The chromaffin cells also leak small amounts of catecholamine over the measurement period, so a ‘basal release’ (no nicotine added) measurement is also recorded. -
FIG. 4 shows catecholamine release from dicarba-conotoxins 118 and 119. Basal release was measured at 0.72% (and is subtracted from other measurements). Nicotine stimulation alone released (6.85-0.72)=6.13% of the noradrenaline in the cells, but only (4.15-0.72)=3.43% in the presence of 5 uM of [2,8]-cystino-[3,12]-dicarba conotoxin 119. This represents 55.97% of the release produced by nicotine alone, or a 44% inhibition of release (see Table). The % inhibition of release of adrenaline (41%) was similar to that for noradrenaline (44%). This inhibition was found to be concentration related: A 1 uM sample of dicarba-conotoxin 119 produced only a 21.6% inhibition of noradrenaline release and a 16.5% inhibition of release of adrenaline. - Data for [2,8]-dicarba-[3,12]-cystino conotoxin 118 is also shown in
FIG. 4 and Table 8.1. This data shows that these dicarba-analogues are biologically active and possess activity profiles analogous to the native conotoxin sequences. -
TABLE 8.1 Catecholamine release for dicarba-conotoxins 118 and 119 1st 2nd 3rd 4th mean SEM n % control % inhibition Noradrenaline Release BASE 0.68 0.61 0.66 0.93 0.72 0.07 4 NICOTINE (4 uM) 6.62 6.89 6.99 6.89 6.85 0.08 4 100.000 119 (1 uM) 4.89 5.75 5.73 5.73 5.52 0.21 4 78.400 21.600 119 (5 uM) 3.22 4.58 3.99 4.81 4.15 0.35 4 55.971 44.029 118 (1 uM) 4.52 6.29 7.00 7.34 6.29 0.63 4 90.859 9.141 118 (5 uM) 4.19 5.86 4.32 5.51 4.97 0.42 4 69.384 30.616 Adrenaline Release BASE 0.38 0.28 0.31 0.34 0.33 0.02 4 NICOTINE (4 uM) 4.31 4.52 4.52 4.48 4.46 0.05 4 100.000 119 (1 uM) 3.31 3.67 4.14 3.98 3.77 0.18 4 83.420 16.580 119 (5 uM) 2.32 2.95 2.76 3.02 2.76 0.16 4 58.888 41.112 118 (1 uM) 2.94 4.40 4.46 4.09 3.97 0.35 4 88.260 11.740 118 (5 uM) 2.55 3.63 2.62 3.31 3.03 0.26 4 65.306 34.694 - Peptides samples (0.25 mM) were dissolved in a solution containing either 0.25 mM reduced glutathione, 12.3 μM reduced thioredoxin (Promega, Madison, Wis.) or 0.5 mM human serum albumin (Sigma, Madison, Wis.) in 100 mM phosphate buffer+1 mM EDTA, pH 7.4 (300 μL) and incubated at 37 C. Thioredoxin was reduced by treating the oxidised form with 0.9 equivalents of dithiothreitol for 15 minutes immediately prior to use. Aliquots (30 μL) were taken at various time intervals, quenched with extraction buffer consisting of 50% aqueous acetonitrile, 100 mM NaCl, and 1% TFA (30 μL) and analysed by reverse-phase-HPLC. The ratio of the degradation product to the tested peptide sample was determined by measuring the peak height, and compared against the peak height results for the HPLC of the corresponding natural or native peptide. The product was considered to have improved stability if the comparative HPLC test results showed less degradation product after 6 hours of contact with one of the agents (reduced glutathione, reduced thioredoxin or human serum albumin).
- Whole human blood containing 1% EDTA was centrifuged at 14,000 rpm for 30 minutes. The supernatant was then transferred to an Eppendorf tube and centrifuged for an additional 30 min at 14,000 rpm. Peptide samples were dissolved in plasma (200 μL) to an initial peptide concentration of approximately 0.25 mM. Aliquots (30 μL) were removed at various time intervals and quenched with extraction buffer (30 μL). The aliquot was then vortexed, diluted with additional water (60 μL) and chilled in an ice bath for 5 minutes prior to centrifuging at 14,000 rpm for 15 minutes. The supernatant was then analysed by RP-HPLC. The stability of the peptide sample was assessed by comparing the ratio of the peak heights representing the tested peptide, and the degradation products, against a sample of the corresponding natural or native peptide not containing the dicarba bridge or bridges. The product was considered to have improved stability in human blood plasma if the comparative HPLC test results showed less degradation product after 6 hours of contact.
- It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.
-
- 1. Maggon, K.
Drug Discovery Today 2005, 10, 739-742. - 2. Giannis, A.; Kolter, T. Angew. Chem., Int. Ed. Engl. 1993, 32, 1244-1267.
- 3. Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bös, M.; Cook, C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; Kahn, M.; Madison, V. S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall, J.; Vincent, G. P.; Voss, M. E. J. Med. Chem. 1993, 36, 3039-3049.
- 4. Fix, J. A. Pharm. Res. 1996, 13, 1760-1764.
- 5. Fletcher, M. D.; Campbell, M. M. Chem. Rev. 1998, 98, 763-795.
- 6. Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M. Curr. Med. Chem. 2002, 9, 811-822.
- 7. Seebach, D.; Overhand, M.; Kuhlne, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913-941.
- 8. Seebach, D.; Ciceri, P. E.; Overhand, M.; Jaun, B.; Rigo, D.; Oberer, L.; Hommel, U.; Amstutz, R.; Widmer, H. Helv. Chim. Acta 1996, 79, 2043-2065.
- 9. Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071-13072.
- 10. Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.; Xialolin, H.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381-384.
- 11. Iverson, B. Nature 1997, 385, 113-115.
- 12. Kimmerlin, T.; Seebach, D. J. Pept. Res. 2005, 65, 229-260.
- 13. Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodivers. 2004, 1, 1211-1239.
- 14. Arvidsson, P. I.; Ryder, N. S.; Weiss, H. M.; Hook, D. F.; Escalante, J.; Seebach, D. Chem. Biodivers. 2005, 2, 401-420.
- 15. Baldauf, C.; Hofmann, H.-J.; Günther, R. Helv. Chim. Acta 2003, 86, 2573-2588.
- 16. Li, P.; Roller, P. P. Curr. Top. Med. Chem. 2002, 2, 325-341.
- 17. Garzone, P. D.; Colburn, W. A.; Mokotoff, M. E. Pharmacokinet. Pharmacodyn. 1991, 3, 116-127.
- 18. Gorske, B. C.; Jewell, S. A.; Guerard, E. J.; Blackwell, H. E. Org. Lett. 2005, 7, 1521-1524.
- 19. Isabel, M.; Perez-Paya, E.; Messeguer, A. Comb. Chem. High Throughput Screen. 2005, 8, 235-239.
- 20. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500.
- 21. Hyrup, B.; Nielsen, P. E. Bioorg. Med. Chem. 1996, 4, 5-23.
- 22. Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem. Int. Ed. 1998, 37, 2796-2823.
- 23. Peptide Nucleic Acids; Egholm, M.; Nielsen, P. E., Eds.; Horizon Scientific Press: England, 1999.
- 24. Dean, D. A. Adv. Drug Delivery Rev. 2000, 44, 81-95.
- 25. Romanelli, A.; Saviano, M.; Pedone, C. Recent Res. Dev. Org. Chem. 2004, 8, 237-254.
- 26. Kumar, V. A.; Ganesh, K. N. Acc. Chem. Res. 2005, 38, 404-412.
- 27. Guichard, G.; Benkirane, N.; Zeder-Lutz, G.; Van Regenmortel, M. H. V.; Briand, J. P.; Muller, S. Proc. Natl. Acad. Sci. USA 1994, 91, 9765-9769.
- 28. Chorev, M. Biopolymers 2005, 80, 67-84.
- 29. An, S. S. A.; Lester, C. C.; Peng, J.-L.; Li, Y.-J.; Rothwarf, D. M.; Welker, E.; Thannhauser, T. W.; Zhang, L. S.; Tam, J. P.; Scheraga, H. A. J. Am. Chem. Soc. 1999, 121, 11558-11566.
- 30. Arnold, U.; Hinderaker, M. P.; Köditz, J.; Golbik, R.; Ulbrich-Hofmann, R.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 7500-7501.
- 31. Tang, W.; Zhang, X. Chem. Rev. 2003, 103, 3029-3069.
- 32. Zsigmond, A.; Balatoni, I.; Notheisz, F.; Hegednes, C.; Bakos, J. Catalysis Lett. 2005, 101, 195-199.
- 33. Burk, M. J.; Feaster, J. E.; Nugent, W. A.; Harlow, R. L. J. Am. Chem. Soc. 1993, 115, 10125-10138.
- 34. Burk, M. J. J. Am. Chem. Soc. 1991, 113, 8518-8519.
- 35. Burk, M. J.; Feaster, J. E. J. Am. Chem. Soc. 1992, 114, 6266-6267.
- 36. Robinson, A. J.; Lim, C. Y.; Li, H.-Y.; He, L.; Ma, P. J. Org. Chem. 2001, 66, 4141-4147.
- 37. Robinson, A. J.; Stanislawski, P.; Mulholland, D. J. Org. Chem. 2001, 66, 4148-4152.
- 38. Juaristi, E. Enantioselective Synthesis of β-Amino Acids; Wiley-VCH: New York, 1997.
- 39. The Organic Chemistry of β-Lactams; Georg, G. I., Ed.; Verlag Chemie: New York, 1993.
- 40. Juaristi, E.; Quintana, D.; Escalante, J. Aldrichim. Acta 1994, 27, 3-11.
- 41. Ondetti, M. A.; Engel, S. L. J. Med. Chem. 1975, 18, 761-763.
- 42. Abele, S.; Seebach, D. Eur. J. Org. Chem. 2000, 1-15.
- 43. Borman, S. Chem. Eng. News 1997, 75, 32-35.
- 44. Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180.
- 45. Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015-2022.
- 46. Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.; Hintermann, T.; Jaun, B. Helv. Chim. Acta 1997, 80, 2033-2038.
- 47. Seebach, D.; Abele, S.; Gademann, K.; Guichard, G.; Hintermann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V. Helv. Chim. Acta 1998, 81, 932-982.
- 48. Appella, D. H.; Christianson, L. A.; Klein, D. A.; Richards, M. R.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 7574-7581.
- 49. Claridge, T. D. W.; Goodman, J. M.; Moreno, A.; Angus, D.; Barker, S. F.; Taillefumier, C.; Watterson, M. P.; Fleet, G. W. J. Tetrahedron Lett. 2001, 42, 4251-4255.
- 50. Rueping, M.; Schreiber, J. V.; Lelais, G.; Jaun, B.; Seebach, D. Helv. Chim. Acta 2002, 85, 2577-2593.
- 51. Matthews, J. L.; Overhand, M.; Kühnle, F. N. M.; Ciceri, P. E.; Seebach, D. Liebigs Ann. 1997, 1371-1379.
- 52. Chung, Y. J.; Christianson, L. A.; Stanger, H. E.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 10555-10556.
- 53. Krauthäuser, S.; Christianson, L. A.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1997, 119, 11719-11720.
- 54. Seebach, D.; Abele, S.; Gademann, K.; Jaun, B. Angew. Chem. Int. Ed. 1999, 38, 1595-1597.
- 55. Langenhan, J. M.; Guzei, I. A.; Gellman, S. H. Angew. Chem. Int. Ed. 2003, 42, 2402-2405.
- 56. Syud, F. A.; Stanger, H. E.; Mortell, H. S.; Espinosa, J. F.; Fisk, J. D.; Fry, C. G.; Gellman, S. H. J. Mol. Biol. 2003, 326, 553-568.
- 57. Seebach, D.; Matthews, J. L.; Meden, A.; Wessels, T.; Baerlocher, C.; McCusker, L. B. Helv. Chim. Acta 1997, 80, 173-182.
- 58. Hintermann, T.; Seebach, D. Chimia 1997, 51, 244-247.
- 59. Seebach, D.; Abele, S.; Schreiber, J. V.; Martinoni, B.; Nussbaum, A. K.; Schild, H.; Schulz, H.; Hennecke, H.; Woessner, R.; Bitsch, F. Chimia 1998, 52, 734-739.
- 60. Frackenpohl, J.; Arvidsson, P. I.; Schreiber, J. V.; Seebach, D.
Chem Bio Chem 2001, 2, 445-455. - 61. Schreiber, J. V.; Frackenpohl, J.; Moser, F.; Fleischmann, T.; Kohler, H.-P. E.; Seebach, D.
Chem Bio Chem 2002, 3, 424-432. - 62. Wiegand, H.; Wirz, B.; Schweitzer, A.; Camenisch, G. P.; Perez, M. I. R.; Gross, G.; Woessner, R.; Voges, R.; Arvidsson, P. I.; Frackenpohl, J.; Seebach, D. Biopharm. Drug Dispos. 2002, 23, 251-262.
- 63. Gademann, K.; Ernst, M.; Hoyer, D.; Seebach, D. Angew. Chem. Int. Ed. 1999, 38, 1223-1226.
- 64. Gademann, K.; Kimmerlin, T.; Hoyer, D.; Seebach, D. J. Med. Chem. 2001, 44, 2460-2468.
- 65. Nunn, C.; Rueping, M.; Langenegger, D.; Schuepbach, E.; Kimmerlin, T.; Micuch, P.; Hurth, K.; Seebach, D.; Hoyer, D. Naunyn-Schmiedeberg's Arch Pharmacol 2003, 367, 95-103.
- 66. Takashiro, E.; Hayakawa, I.; Nitta, T.; Kasuya, A.; Miyamoto, S.; Ozawa, Y.; Yagi, R.; Yamamoto, I.; T., S.; Nakagawa, A.; Yabe, Y. Bioorg. Med. Chem. 1999, 7, 2063-2072.
- 67. Arvidsson, P. I.; Ryder, N. S.; Weiss, H. M.; Gross, G.; Kretz, O.; Woessner, R.; Seebach, D.
Chem BioC hem 2003, 4, 1345-1347. - 68. White, J. D.; Hong, J.; Robarge, L. A. J. Org. Chem. 1999, 64, 6206-6216.
- 69. Arndt, F.; Eistert, B.; Partale, W. Ber. Dtsch. Chem. Ges. 1927, 60, 1364-1370.
- 70. Leggio, A.; Liguori, A.; Procopio, A.; Sindona, G. J. Chem. Soc., Perkin Trans. 1 1997, 1969-1971.
- 71. Marti, R. E.; Bleicher, K. H.; Bair, K. W. Tetrahedron Lett. 1997, 38, 6145-6148.
- 72. Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998, 81, 187-206.
- 73. Named Organic Reactions; Laue, T.; Plagens, A., Eds.; Wiley: Chichester, 2000.
- 74. Yang, H.; Foster, K.; Stephenson, C. R. J.; Brown, W.; Roberts, E. Org. Lett. 2000, 2, 2177-2179.
- 75. Lubell, W. D.; Kitamura, M.; Noyori, R. Tetrahedron:
Asymmetry 1991, 2, 543-554. - 76. Zhu, G.; Chen, Z.; Zhang, X. J. Org. Chem. 1999, 64, 6907-6910.
- 77. Heller, D.; Holz, J.; Drexler, H.-J.; Lang, J.; Drauz, K.; Krimmer, H.-P.; Börner, A. J. Org. Chem. 2001, 66, 6816-6817.
- 78. Holz, J.; Stürmer, R.; Schmidt, U.; Drexler, H.-J.; Heller, D.; Krimmer, H.-P.; Börner, A. Eur. J. Org. Chem. 2001, 4615-4624.
- 79. Yasutake, M.; Gridnev, I. D.; Higashi, N.; Imamoto, T. Org. Lett. 2001, 3, 1701-1704.
- 80. Heller, D.; Holz, J.; Komarov, I. V.; Drexler, H.-J.; You, J.; Drauz, K.; Börner, A. Tetrahedron:
Asymmetry 2002, 13, 2735-2741. - 81. Heller, D.; Drexler, H.-J.; You, J.; Baumann, W.; Drauz, K.; Krimmer, H.-P.; Börner, A. Chem. Eur. J. 2002, 8, 5196-5203.
- 82. Lee, S.; Zhang, Y. J. Org. Lett. 2002, 4, 2429-2431.
- 83. Peña, D.; Minnaard, A. J.; de Vries, J. G.; Feringa, B. L. J. Am. Chem. Soc. 2002, 124, 14552-14553.
- 84. Tang, W.; Zhang, X. Org. Lett. 2002, 4, 4159-4161.
- 85. Zhou, Y.-G.; Tang, W.; Wang, W.-B.; Li, W.; Zhang, X. J. Am. Chem. Soc. 2002, 124, 4952-4953.
- 86. Holz, J.; Monsees, A.; Jiao, H.; You, J.; Komarov, I. V.; Fischer, C.; Drauz, K.; Börner, A. J. Org. Chem. 2003, 68, 1701-1707.
- 87. Jerphagnon, T.; Renaud, J.-L.; Demonchaux, P.; Ferreira, A.; Bruneau, C. Tetrahedron: Asymmetry 2003, 14, 1973-1977.
- 88. Tang, W.; Wang, W.; Chi, Y.; Zhang, X. Angew. Chem. Int. Ed. 2003, 42, 3509-3511.
- 89. Wu, J.; Chen, X.; Guo, R.; Yeung, C.-H.; Chan, A. S. C. J. Org. Chem. 2003, 68, 2490-2493.
- 90. Lee, H.; Park, J.; Kim, B. Y.; Gellman, S. H. J. Org. Chem. 2003, 68, 1575-1578.
- 91. Beddow, J. E.; Davies, S. G.; Smith, A. D.; Russel, A. J. Chem. Commun. 2004, 2778-2779.
- 92. Seebach, D.; Schaeffer, L.; Gessier, F.; Bindschädler, P.; Jäger, C.; Josien, D.; Kopp, S.; Lelais, G.; Mahajan, Y. R.; Micuch, P.; Sebesta, R.; Schweizer, B. W. Helv. Chim. Acta 2003, 86, 1852-1861.
- 93. Davies, H. M. L.; Venkataramani, C. Angew. Chem. Int. Ed. 2002, 41, 2197-2199.
- 94. Sammis, G. M.; Jacobsen, E. N. J. Am. Chem. Soc. 2003, 125, 4442-4443.
- 95. Bower, J. F.; Jumnah, R.; Williams, A. C.; Williams, J. M. J. J. Chem. Soc., Perkin Trans. 1 1997, 1411-1420.
- 96. Duursma, A.; Minnaard, A. J.; Feringa, B. L. J. Am. Chem. Soc. 2003, 125, 3700-3701.
- 97. Elaridi, J.; Thaqi, A.; Prosser, A.; Jackson, W. R.; Robinson, A. J. Tetrahedron: Asymmetry 2005, 16, 1309-1319.
- 98. Tang, W.; Wu, S.; Zhang, X. J. Am. Chem. Soc. 2003, 125, 9570-9571.
- 99. Lefort, L.; Boogers, J. A. F.; de Vries, A. H. M.; de Vries, J. G. Org. Lett. 2004, 6, 1733-1735.
- 100. Stewart, D. E.; Sarkar, A.; Wampler, J. E. J. Mol. Biol. 1990, 214, 253-260.
- 101. Hinderaker, M. P.; Raines, R. T.
Protein Science 2003, 12, 1188-1194. - 102. Weiss, M. S.; Jabs, A.; Hilgenfield, R. Nat. Struct. Biol. 1998, 5, 676.
- 103. Jabs, A.; Weiss, M. S.; Hilgenfield, R. J. Mol. Biol. 1999, 286, 291-304.
- 104. MacArthur, M. W.; Thornton, J. M. J. Mol. Biol. 1991, 218, 397-412.
- 105. Wöhr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.; Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218-9227.
- 106. Haack, T.; Mutter, M. Tetrahedron Lett. 1992, 33, 1589-1592.
- 107. Sampson, W. R.; Patsiouras, H.; Ede, N. J. J. Peptide Sci. 1999, 5, 403-409.
- 108. Wittelsberger, A.; Keller, M.; Scarpellino, L.; Patiny, L.; Acha-Orbea, H.; Mutter, M. Angew. Chem. Int. Ed. 2000, 39, 1111-1115.
- 109. Keller, M.; Miller, A. D. Bioorg. Med. Chem. Lett. 2001, 11, 857-859.
- 110. von Eggelkraut-Gottanka, R.; Machova, Z.; Grouzmann, E.; Beck-Sickinger, A. G.
Chem Bio Chem 2003, 4, 425-433. - 111. White, P.; Keyte, J. W.; Bailey, K.; Bloomberg, G. J. Peptide Sci. 2004, 10, 18-26.
- 112. Magaard, V. W.; Sanchez, R. M.; Bean, J. W.; Moore, M. L. Tetrahedron Lett. 1993, 34, 381-384.
- 113. Bonnett, R.; Clark, V. M.; Giddey, A.; Todd, S. A. J. Chem. Soc. 1959, 2087-2093.
- 114. Aldous, D. J.; Drew, M. G. B.; Hamelin, E. M.-N.; Harwood, L. M.; Jahans, A. B.; Thurairatnam,
S. Synlett 2001, 12, 1836-1840. - 115. Xia, Q.; Ganem, B. Tetrahedron Lett. 2002, 43, 1597-1598.
- 116. Elaridi, J.; Jackson, W. R.; Robinson, A. J. Tetrahedron: Asymmetry 2005, 16, 2025-2029.
- 117. Burk, M. J.; Allen, J. G.; Kiesman, W. F. J. Am. Chem. Soc. 1998, 120, 657-663.
- 118. Teoh, E.; Campi, E. M.; Jackson, W. R.; Robinson, A. J. Chem. Commun. 2002, 978-979.
- 119. Teoh, E.; Campi, E. M.; Jackson, W. R.; Robinson, A. J. New J. Chem. 2003, 27, 387-394.
- 120. Schwab, P.; France, M. B.; Ziller, J. W.; Grubbs, R. H. Angew. Chem., Int. Ed. Engl. 1995, 34, 2039-2041.
- 121. Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1, 953-956.
- 122. Gessler, S.; Randl, S.; Blechert, S. Tetrahedron Lett. 2000, 41, 9973-9976.
- 123. Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J. Am. Chem. Soc. 2000, 122, 8168-8179.
- 124. Grubbs, R. H.; Pine, S. H. Comprehensive Organic Synthesis; Pergamon: New York, 1991; Vol. 5.
- 125. Ivin, K. J.; Moi, J. C. Olefin Metathesis and Metathesis Polymerisation; Academic Press: San Diego, 1997.
- 126. Fürstner, A. Alkene Metathesis in Organic Synthesis; Springer-Verlag: New York, 1998.
- 127. Grubbs, R. H. Handbook of Metathesis; Wiley-VCH: Weinheim, 2003; Vol. 2.
- 128. Trnka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001, 34, 18-29.
- 129. Connon, S. J.; Blechert, S. Angew. Chem. Int. Ed. 2003, 42, 1900-1923.
- 130. Chatterjee, A. K.; Choi, T.-L.; Sanders, D. P.; Grubbs, R. H. J. Am. Chem. Soc. 2003, 125, 11360-11370.
- 131. Jones, R. M.; Bulaj, G. Curr. Opin. Drug Discovery Dev. 2000, 3, 141-154.
- 132. Pons, M.; Albericio, F. R.; Royo,
M. Synlett 2000, 2, 172-181. - 133. Wouters, M. A.; Lau, K. K.; Hog, P. J. Bioessays 2003, 26, 73-79.
- 134. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W. G., C.; Pople, J. A.; Gaussian Inc.: Wallingford Conn., 2004.
- 135. Walker, R.; Yamanaka, T.; Sakakibara, S. Proc. Natl. Acad. Sci. USA 1974, 71, 1901-1905.
- 136. Nutt, R. F.; Verber, D. F.; Saperstein, R. J. Am. Chem. Soc. 1980, 102, 6539-6545.
- 137. Collier, P. N.; Campbell, A. D.; Patel, I.; Raynham, T. M.; Taylor, R. J. K. J. Org. Chem. 2002, 67, 1802-1815.
- 138. Lange, M.; Fischer, P. M. Helv. Chim. Acta 1998, 81, 2053-2061.
- 139. Hase, S.; Morikawa, T.; Sakakibara, S. Experientia 1969, 25, 1239-1240.
- 140. Kambayashi, Y.; Nakajima, S.; Ueda, M.; Inouye, K. FEBS Letters 1989, 248, 28-34.
- 141. Whelan, A.; Elaridi, J.; Harte, M.; Smith, S.; Jackson, W. R.; Robinson, A. J. Tetrahedron Lett. 2004, 45, 9545-9547.
- 142. Whelan, A.; Elaridi, J.; Mulder, R.; Jackson, W. R.; Robinson, A. J. Can. J. Chem. 2005, 83, 875-881.
- 143. Carotenuto, A.; D'Addona, D.; Rivalta, E.; Chelli, M.; Papini, A. M.; Rovero, P.; Ginanneschi, M. Lett. Org. Chem. 2005, 2, 274-279.
- 144. Jost, K.; Sorm, F. Coll. Czech. Chem. Commun. 1971, 36, 234-245.
- 145. Stymiest, J. L.; Mitchell, B. F.; Wong, S.; Vederas, J. C. Org. Lett. 2003, 5, 47-49.
- 146. Stymiest, J. L.; Mitchell, B. F.; Wong, S.; Vederas, J. C. J. Org. Chem. 2005, 70, 7799-7809.
- 147. Cerovsky, V.; Wunsch, E.; Brass, J. Eur. J. Biochem. 1997, 247, 231-237.
- 148. Lange, M.; Cuthbertson, A. S.; Towart, R.; Fischer, P. M. J. Peptide Sci. 1998, 4, 289-293.
- 149. Bhatnagar, P. K.; Agner, E. K.; Alberts, D.; Arbo, B. E.; Callahan, J. F.; Cuthbertson, A. S.; Angelsen, S. J.; Fjerdingstad, H.; Hartmann, M.; Heerding, D.; Hiebl, J.; Huffman, W. F.; Hysben, M.; King, A. G.; Kremminger, P.; Kwon, C.; LoCastro, S.; Lovhaug, D.; Pelus, L. M.; Petteway, S.; Takata, J. S. J. Med. Chem. 1996, 39, 3814-3819.
- 150. Hiebl, J.; Blanka, M.; Guttman, A.; Hollman, H.; Leitner, K.; Mayrhofer, G.; Rovenszky, F.; Winkler, K. Tetrahedron 1998, 54, 2059-2074.
- 151. Williams, R. M.; Yuan, C. J. Org. Chem. 1992, 57, 6519-6527.
- 152. Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. J. Am. Chem. Soc. 1996, 118, 9606-9614.
- 153. Gao, Y.; Lane-Bell, P.; Vederas, J. C. J. Org. Chem. 1998, 63, 2133-2143.
- 154. Williams, R. M.; Lui, J. J. Org. Chem. 1998, 63, 2130-2132.
- 155. Aguilera, B.; Wolf, L. B.; Nieczypor, P.; Rutjes, F. P. J. T.; Overkleeft, H. S.; van Hest, J. C. M.; Schoemaker, H. E.; Wang, B.; Mol, J. C.; Furstner, A.; Overland, M.; van der Marel, G. A.; van Boom, J. H. J. Org. Chem. 2001, 66, 3584-3589.
- 156. Creighton, C. J.; Reitz, A. B. Org. Lett. 2001, 3, 893-895.
- 157. Ghalit, N.; Rijkers, D. T. S.; Kemmink, J.; Versluis, C.; Liskamp, R. M. J. Chem. Commun. 2005, 192-194.
- 158. Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. J. Am. Chem. Soc. 1995, 117, 5855-5856.
- 159. Blackwell, H. E.; Sadowsky, J. D.; Howard, R. J.; Sampson, J. N.; Chao, J. A.; Steinmetz, W. E.; O'Leary, D. J.; Grubbs, R. H. J. Org. Chem. 2001, 66, 5291-5302.
- 160. Clark, T. D.; Ghadiri, M. R. J. Am. Chem. Soc. 1995, 117, 12364-12365.
- 161. Chaleix, V.; Sol, V.; Guilloton, M.; Granet, R.; Krausz, P. Tetrahedron Lett. 2004, 45, 5295-5299.
- 162. Pemerstorfer, J.; Schuster, M.; Blechert, S. Chem. Commun. 1997, 1949-1950.
- 163. Piscopio, A. D.; Miller, J. F.; Koch, K. Tetrahedron Lett. 1997, 38, 7143-7146.
- 164. Piscopio, A. D.; Miller, J. F.; Koch, K. Tetrahedron Lett. 1998, 39, 2667-2670.
- 165. Jarvo, E. R.; Copeland, G. T.; Papaioannou, N.; Bonitatebus, P. J.; Miller, S. J. J. Am. Chem. Soc. 1999, 121, 11638-11643.
- 166. Piscopio, A. D.; Miller, J. F.; Koch, K. Tetrahedron 1999, 55, 8189-8198.
- 167. Schmiedeberg, N.; Kessler, H. Org. Lett. 2002, 4, 59-62.
- 168. Kazmaier, U.; Hebach, C.; Watzke, A.; Maier, S.; Mues, H.; Huch, V. Org. Biomol. Chem. 2005, 3, 136-145.
- 169. Hsieh, H.; Wu, Y.; Chen, S.; Wang, K. Bioorg. Med. Chem. 1999, 7, 1797-1803.
- 170. Suetake, T.; Aizawa, T.; Koganesawa, N.; Osaki, T.; Kobashigawa, Y.; Demura, M.; Kawabata, S.; Kawano, K.; Tsuda, S.; Nitta,
K. PEDS 2002, 15, 763-769. - 171. Adams, D. J.; Alewood, P. F.; Craik, D. J.; Drinkwater, R. D.; Lewis, R. J. Drug Dev. Res. 1999, 46, 219-234.
- 172. Hu, Y.-L.; Huang, F.; Jiang, H.; Fan, C.-X.; Chen, C.-Y.; Chen, J.-S. Wuli Huaxue Xuebao 2005, 21, 474-478.
- 173. Rogers, J. P.; Luginbühl, P.; Shen, G. S.; McCabe, R. T.; Stevens, R. C.; Wemmer, D. E. Biochemistry 1999, 38.
- 174. Maslennikova, I. V.; Shenkareva, Z. O.; Zhmaka, M. N.; Ivanova, V. T.; Methfesselb, C.; Tsetlina, V. I.; Arseniev, A. S. FEBS Letters 1999, 444, 275-280.
- 175. Craik, D. J.; Daly, N. L.; Bond, T.; Waine, C. J. Mol. Biol. 1999, 294, 1327-1336.
- 176. Rosengren, K. J.; Daly, N. L.; Plan, M. R.; Waine, C.; Craik, D. J. J. Biol. Chem. 2003, 278, 8606-8616.
- 177. Hill, C. P.; Yee, J.; Selsted, M. E.; Eisenberg, D. Science 1991, 251, 1481-1485.
- 178. Comet, B.; Bonmatin, J. M.; Hetru, C.; Hoffmann, J. A.; Ptak, M.; Vovelle,
F. Structure 1995, 3, 435-448. - 179. Aumelas, A.; Mangoni, M.; Roumestand, C.; Chiche, L.; Despaux, E.; Grassy, G.; Calas, B.; Chavanieu, A. Eur. J. Biochem. 1996, 237, 575-583.
- 180. Fahrner, R. L.; Dieckmann, T.; Harwig, S. S.; Lehrer, R. I.; Eisenberg, D.; Feigon, J. Chem. Biol. 1996, 3, 543-550.
- 181. Rodighiero, C.; Lencer, W. I. Microbial Pathogenesis and the Intestinal Epithelial Cell 2003, 385-401.
- 182. Chatterjee, A. K.; Sanders, D. P.; Grubbs, R. H. Org. Lett. 2002, 4, 1939-1942.
- 183. Marx, J. N.; Argyle, J. C.; Norman, L. R. J. Am. Chem. Soc. 1974, 96, 2121-2129.
- 184. Klioze, S. S.; Darmory, F. P. J. Org. Chem. 1975, 40, 1588-1592.
- 185. Folkers, K.; Adkins, H. J. Am. Chem. Soc. 1931, 53, 1416-1419.
- 186. Burk, M. J.; Kalberg, C. S.; Pizzaro, A. J. Am. Chem. Soc. 1998, 120, 4345-4353.
- 187. Noyori, R. Asymmetric Catalysis in Organic Synthesis; John Wiley and Sons Inc.: USA, 1994.
- 188. Imamoto, T.; Watanabe, J.; Wada, Y.; Masuda, H.; Yamada, H.; Tsuruta, H.; Matsukawa, S.; Yamaguchi, K. J. Am. Chem. Soc. 1998, 120, 1635-1636.
- 189. Yamanoi, Y.; Imamoto, T. J. Org. Chem. 1999, 64, 2988-2989.
- 190. Gridnev, I. D.; Yamanoi, Y.; Higashi, N.; Tsuruta, H.; Yasutake, M.; Imamoto, T. Adv. Synth. Catal. 2001, 343, 118-136.
- 191. Armstrong, S. K.; Brown, J. M.; Burk, M. J. Tetrahedron Lett. 1993, 34, 879-882.
- 192. Landis, C. R.; Feldgus, S. Angew. Chem. Int. Ed. 2000, 39, 2863-2866.
- 193. Feldgus, S.; Landis, C. R. J. Am. Chem. Soc. 2000, 122, 12714-12727.
- 194. Feldgus, S.; Landis, C. R. Organometallics 2001, 20, 2374-2386.
- 195. Williams, R. M.; Aldous, D. J.; Aldous, S. C. J. Org. Chem. 1990, 55, 4657-4663.
- 196. Legall, P.; Sawhney, K. N.; Conley, J. D.; Kohn, H. Int. J. Peptide Protein Res. 1988, 32, 279-291.
- 197. Schmidt, U.; Lieberknecht, A.; Wild, J. Synthesis 1984, 53-60.
- 198. Burk, M. J.; Gross, M. F.; Harper, T. G. P.; Kalberg, C. S.; Lee, J. R.; Martinez, J. P. Pure Appl. Chem. 1996, 68, 37-44.
- 199. Burk, M. J.; Wang, Y. M.; Lee, J. R. J. Am. Chem. Soc. 1996, 118, 5142-5143.
- 200. Chatterjee, A. K.; Grubbs, R. H. Org. Lett. 1999, 1, 1751-1753.
- 201. Letham, D. S.; Young,
H. Phytochemistry 1971, 10, 23-28. - 202. Schwab, P.; Grubbs, R. H.; Ziller, J. W. J. Am. Chem. Soc. 1998, 118, 100-110.
- 203. Adlhart, C.; Chen, P. J. J. Am. Chem. Soc. 2004, 126, 3496-3510.
- 204. Hong, S. H.; Day, M. W.; Grubbs, R. H. J. Am. Chem. Soc. 2004, 126, 7414-7415.
- 205. Schlummer, B.; Hartwig, J. F. Org. Lett. 2002, 4, 1471-1474.
- 206. Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry of Organic Compounds; John Wiley & Sons, Inc.: New York, 1994.
- 207. Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry of Organic Compounds; John Wiley & Sons, Inc.: New York, 1994.
- 208. Cox, R. J.; Sherwin, W. A.; Lister, K. P. L.; Vederas, J. C. J. Am. Chem. Soc. 1996, 118, 7449-7460.
- 209. Zoller, U.; Ben-Ishai, D. Tetrahedron 1975, 31, 863-866.
- 210. Mauldin, S. C.; Hornback, W. J.; Munroe, J. E. J. Chem. Soc., Perkin Trans. 1 2001, 1554-1558.
- 211. Easton, C. J.; Roselt, P. D.; Tiekink, E. R. T. Tetrahedron 1995, 51, 7809-7822.
- 212. Tanaka, K.; Aim, M.; Watanabe, Y.; Fuji, K. Tetrahedron:
Asymmetry 1996, 7, 1771-1782. - 213. Fürstner, A.; Thiel, O. R.; Lehmann, C. W. Organometallics 2002, 21, 331-335.
- 214. Louie, J.; Grubbs, R. H. Organometallics 2002, 21, 2153-2164.
- 215. Osborn, J. A.; Jardine, F. H.; Young, J. F.; Wilkinson, G. J. Chem. Soc. A 1966, 1711-1736.
- 216. Jardine, J. H.; Osborn, J. A.; Wilkinson, G. J. Chem. Soc. A 1967, 1574-1580.
- 217. Burdett, K. A.; Harris, L. D.; Margl, P.; Maughon, B. R.; Mokhtar-Zadeh, T.; Saucier, P. C.; Wasserman, E. P. Organometallics 2004, 23, 2027-2047.
- 218. Patel, J.; Elaridi, J.; Jackson, W. R.; Robinson, A. J.; Serelis, A. K.; Such, C. Chem. Commun. 2005, 44, 5546-5547.
- 219. Schwab, P.; Grubbs, R. H.; Ziller, J. W. J. Am. Chem. Soc. 1996, 118, 100-110.
- 220. Jourdant, A.; Gonzalez-Zamora, E.; Zhu, J. J. Org. Chem. 2002, 67, 3163-3164.
- 221. Fmoc Solid Phase Peptide Synthesis: A Practical Approach; Chan, W. C.; White, P. D., Eds.; Oxford University Press: England, 2000.
- 222. Illesinghe, J.; Campi, E. M.; Jackson, W. R.; Robinson, A. J. Aust. J. Chem. 2004, 57, 531-536.
- 223. Barrett, A. G. M.; Hennessy, A. J.; Vézouët, R. L.; Procopiou, P. A.; Seale, P. W.; Stefaniak, S.; Upton, R. J.; White, A. J. P.; Williams, D. J. J. Org. Chem. 2004, 69, 1028-1037.
- 224. Schafmiester, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122, 5891-5892.
- 225. Jones, R. M.; Bulaj, G. Curr. Pharm.
Design 2000, 6, 1249-1285. - 226. McIntosh, J. M.; Yoshikami, D.; Mahe, E.; Nielsen, D. B.; Rivier, J. E.; Gray, W. R.; Olivera, B. M. J. Biol. Chem. 1994, 269, 16733-16739.
- 227. Skropeta, D.; Jolliffe, K. A.; Turner, P. J. Org. Chem. 2004, 69, 8804-8809.
- 228. Jolliffe, K. A. Supramolecular Chem. 2005, 17, 81-86.
- 229. Nima, S.; Skropeta, D.; Jolliffe, K. A. Org. Lett. 2005, 7, 5497-5499.
- 230. Paquet, A. Can. J. Chem. 1982, 60, 976-980.
- 231. Kubo, S.; Chino, N.; Kimura, T.; Sakakibara, S. Biopolymers 1996, 38, 733-744.
- 232. Schuster, M.; Blechert, S. Angew. Chem., Int. Ed. Engl. 1997, 36, 2036-2056.
- 233. Moroder, L.; Musiol, H.-J.; Gotz, M.; Renner, C. Biopolymers 2005, 80, 85-97.
- 234. Alewood, P.; Hopping, G.; Armishaw, C. Aust. J. Chem. 2003, 56, 769-774.
- 235. Fazlic, S. Honours Thesis, Monash University, 2004.
- 236. Mayo, K. G.; Nearhoof, E. H.; Kiddie, J. J. Org. Lett. 2002, 4, 1567-1570.
- 237. Yang, C.; Murray, W. V.; Wilson, L. J. Tetrahedron Lett. 2003, 44.
- 238. Grigg, R.; Martin, W.; Morris, J.; Sridharan, V. Tetrahedron Lett. 2003, 44, 4899-4901.
- 239. Efskind, J.; Undheim, K. Tetrahedron Lett. 2003, 44, 2837-2839.
- 240. Balan, D.; Adolfsson, H. Tetrahedron Lett. 2004, 45, 3089-3092.
- 241. Aitken, S. G.; Abell, A. D. Aust. J Chem. 2005, 58, 3-13.
- 242. Appukkuttan, P.; Dehaen, W.; Van der Eycken, E. Org. Lett. 2005, 7, 2723-2726.
- 243. Poulsen, S.; Bornaghi, L. F. Tetrahedron Lett. 2005, 46, 7389-7392.
- 244. Nosse, B.; Schall, A.; Jeong, W. B.; Reiser, O. Adv. Synth. Catal. 2005, 347, 1869-1874.
- 245. Varray, S.; Gauzy, C.; Lamaty, F.; Lazaro, R.; Martinez, J. J. Org. Chem. 2000, 65, 6787-6790.
- 246. Organ, M. G.; Mayer, S.; Lepifre, F.; N′Zemba, B.; Khatri,
J. Molecular Diversity 2003, 7, 211-227. - 247. Personal communication with Professor Paul Alewood, University of Queensland (Australia).
- 248. Rigby, A. C.; Lucas-Meunier, E.; Kalume, D. E.; Czerwiec, E.; Hambe, B.; Dahlqvist, I.; Fossier, P.; Baux, G.; Roepstorff, P.; Baleja, J. D.; Furie, B. C.; Furie, B.; Stenflo, J. Proc Natl Acad Sci USA. 1999, 96, 5758-5763.
- 249. Bulaj, G.; Buczek, O.; Goodsell, I.; Jimenez, E. C.; Kranski, J.; Nielsen, J. S.; Garrett, J. E.; Olivera, B. M. Proc Natl Acad Sci USA. 2003, 100, 14562-14568.
- 250. Buczek, O.; Olivera, B. M.; Bulaj, G. Biochemistry 2004, 43, 1093-1101.
- 251. Dela, C. R.; Whitby, F.; Buczek, O.; Bulaj, G. J. Pept. Res. 2003, 61, 202-212.
- 252. Collaboration with Professor Paul Alewood, University of Queensland (Australia).
- 253. Erdélyi, M.; Gogoll, A. Synthesis 2002, 11, 1592-1596.
- 254. Frost & Sullivan Research Report: Strategic Analysis of the Therapeutic Peptides Market in Europe, October 2004.
- 255. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595-598.
- 256. Fontenot, J. D.; Ball, J. M.; Miller, M. A.; Montelaro, R. C. Pep. Res. 1991, 4, 19-25.
- 257. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
- 258. Davies, S. J.; Ayscough, A. P.; Beckett, R. P.; Bragg, R. A.; Clements, J. M.; Doel, S.; Grew, C.; Launchbury, S. B.; Perkins, G. M.; Pratt, L. M.; Smith, H. K.; Spavold, Z. M.; Thomas, S. W.; Todd, R. S.; Whittaker, M. Bioorg. Med. Chem. Lett. 2003, 13, 2709-2713.
- 259. Berney, D. Helv. Chim. Acta 1982, 65, 1694-1699.
- 260. Saylik, D.; Campi, E. M.; Donohue, A. C.; Jackson, W. R.; Robinson, A. J. Tetrahedron;
Asymmetry 2001, 12, 657-667. - 261. Testa, E.; Cignarella, G.; Pifferi, G.; Furesz, S.; Timbal, M. T.; Schiatti, P.; Maffi, G. Farmaco Ed. Sci. 1964, 19, 895-912.
- 262. Papageorgiou, C.; Borer, X.; French, R. R. Bioorg. Med. Chem. Lett. 1994, 4, 267-272.
- 263. Williams, R. M.; Im, M.-N. Tetrahedron Lett. 1988, 29, 6075-6078.
- 264. Bremner, J. B.; Keller, P. A.; Pyne, S. G.; Robertson, A. D.; Skelton, B. W.; White, A. H.; Witchard, H. M. Aust. J Chem. 2000, 53, 535-540.
- 265. Arvela, R. K.; Leadbeater, N. E.; Sangi, M. S.; Williams, V. A.; Granados, P.; Singer, R. D. J. Org. Chem. 2005, 70, 161-168.
- 266. Spetzler, J. C.; Hoeg-Jensen, T. J. Peptide Sci. 2001, 7, 537-551.
- 267. Heffernan, M. A.; Summers, R. J.; Thorbum, A. W.; Ogru, E.; Gianello, R.; Jiang, W.-J.; Ng, F. M. Endocrinology 2001, 142, 5182-5189.
- 268. Ogru, E.; Wilson, J. C.; Heffernan, M. A.; Jiang, W.-J.; Chalmers, D. K.; Libinaki, R.; Ng, F. M. J. Peptide Res. 2000, 56, 388-397.
- 269. Tam, J. P.; Miao, Z. J. Am. Chem. Soc. 1999, 121, 9013-9022.
-
A.1 The Amino Acids One Three letter letter Amino acid code code Structure Alanine A Ala Allylglycine* — Hag Arginine R Arg Asparagine N Asn Aspartic acid D Asp Crotylglycine* — Crt Cysteine C Cys 5,5-Dimethylproline* — dmP Glutamic acid E Glu Glutamine Q Gln Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Prenylglycine* — Pre Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val *Synthetic amino acids.
Claims (36)
1. A method for the synthesis of an organic compound with a dicarba bridge, comprising:
providing a reactable organic compound having a pair of unblocked complementary metathesisable groups, or two or more reactable organic compounds having between them a pair of unblocked complementary metathesisable groups, and
subjecting the reactable organic compound or compounds to cross-metathesis under microwave radiation conditions to form an organic compound with an unsaturated dicarba bridge.
2. The method of claim 1 , further comprising:
hydrogenating the unsaturated dicarba bridge.
3. The method of claim 1 or claim 2 , wherein the complementary metathesisable groups of the first pair of complementary metathesisable groups are each independently selected from the group consisting of olefins comprising the portion ═CH2, and monosubstituted olefins comprising the group ═CHR5, in which R5 is alkyl or an alkyl substituted by a polar functional group.
4. The method of any one of claims 1 to 3 , wherein the reactable organic compound, or one of the reactable organic compounds, is attached to a solid support during the cross-metathesis of the complementary metathesisable groups.
5. The method of claim 4 , wherein the loading of the reactable organic compound on the solid support is at least 0.2 mmol/g.
6. The method of any one of claims 4 to 5 , wherein each cross-metathesis is performed using a cross-metathesis catalyst, and each cross-metathesis is performed in a solvent combination of a resin-swelling solvent, with a co-ordinating solvent for the catalyst.
7. The method of claim 6 , wherein the co-ordinating solvent is an alcohol.
8. The method of claim 6 or claim 7 , wherein the co-ordinating solvent is used in an amount of about 1-30% by volume, with respect to the total solvent combination.
9. The method of any on eof claims 1 to 8 , wherein the reactable organic compound, or the two or more reactable organic compounds, between them contain a second pair of complementary metathesisable groups which are blocked, and can be unblocked by an unblocking reaction specific to the second pair of complementary metathesisable groups, and the method further comprises unblocking the blocked second pair of complementary metathesisable groups, followed by cross-metathesis of the second pair of cross-methatesisable groups.
10. The method of claim 9 , wherein the blocked second pair of complementary metathesisable groups comprise dialkyl-blocked olefins.
11. The method of claim 9 or claim 10 , wherein the blocked second pair of complementary metathesisable groups are unblocked by cross-methatiesis with a disposable olefin, which is 1,3-butadiene-free.
12. The method of claim 11 , wherein the disposable olefin is a 1,3-butadiene-free olefin or olefin mixture of one or more of the following:
13. The method of any one of claims 9 to 12 , wherein the reactable organic compound comprises a third pair of complementary methathesisable groups, which are blocked and can be unblocked by an unblocking reaction or series of reactions specific to the third pair, and the method comprises subjecting the third pair of complementary metathesisable groups to unblocking reactions specific to those pairs, followed by cross metathesis.
14. The method of claim 13 , wherein the blocking group of the third pair of complementary metathesisable groups comprises an electronic blocking group, and the unblocking reactions comprise conversion of the electronic steric blocking group to a group that is cross-metathesisable.
15. The method of claim 14 , wherein the electronic blocking group comprises a diblocked conjugated diene, and the unblocking reaction comprises subjecting the blocked third pair of complementary metathesisable groups to asymmetric hydrogenation to regioselectively, stereoselectively and chemoselectively hydrogenate one of the diene double bonds to leave a sterically-blocked double bond, followed by cross-metathesis with a disposable olefin to effect removal of the steric blocking groups on the remaining double bond, to yield unblocked complementary metathesisable groups.
16. The method of claim 14 , wherein the electronic blocking group comprises ═CH—CH═CR3R4, in which R3 and R4 are each alkyl, and the unblocking reaction comprises hydrogenation of this group to ═CH—CH2—CHR3R4, followed by cross-metathesis with a disposable olefin to yield the unblocked group ═CHR5, in which R5 is alkyl or an alkyl substituted by a polar functional group.
17. The method of any one of claims 1 to 16 , wherein the reactable organic compound is a peptide.
18. The method of claim 17 , wherein the organic compound with two or more dicarba bridges prepared by the method is a peptidomimetic.
19. The method of claim 17 , wherein the organic compound with two or more dicarba bridges prepared by the method is a pseudopeptide.
20. The method of any one of claims 1 to 19 , wherein the reactable organic compound comprises a peptide comprising a series of amino acids supported on a solid support, wherein two of the amino acids comprise sidechains with a first pair of complementary metathesisable groups.
21. The method of claim 20 , wherein the peptide contains at least one turn-inducing amino acid between the amino acids that comprise the first pair of complementary metathesisable groups.
22. The method of claim 21 , wherein the turn-inducing amino acids are selected from one or more of ψserine, ψthreonine and ψcysteine.
23. The method of claim 22 , further comprising cleaving the protein from the solid support and converting any ψserine, ψthreonine and ψcysteine residues present into serine, threonine and cysteine, respectively.
24. A method for the synthesis of an organic compound with a dicarba bridge, comprising:
synthesising a reactable organic compound to contain a pair of unblocked complementary metathesisable groups, and a turn-inducing group in between the pair of complementary metathesisable groups, and
subjecting the reactable organic compound to cross-metathesis to form a compound with an unsaturated dicarba bridge.
25. The method of claim 24 , further comprising hydrogenating the unsaturated dicarba bridge to form an organic compound with a saturated dicarba bridge.
26. The method of any one of claims 24 to 25 , wherein the reactable organic compound comprises a peptide comprising a series of amino acids supported on a solid support, wherein two of the amino acids comprise sidechains with a first pair of complementary metathesisable groups.
27. The method of claim 26 , wherein the peptide contains at least one turn-inducing amino acid between the amino acids that comprise the first pair of complementary metathesisable groups.
28. The method of claim 27 , wherein the turn-inducing amino acids are selected from one or more of ψserine, ψthreonine and ψcysteine.
29. The method of any one of claims 26 to 28 , wherein the complementary metathesisable groups of the first pair of complementary metathesisable groups are each independently selected from the group consisting of olefins comprising the portion ═CH2, and monosubstituted olefins comprising the group ═CHR5, in which R5 is alkyl or an alkyl substituted by a polar functional group.
30. The method of any one of claims 26 to 29 , wherein the loading of the reactable organic compound on the solid support is at least 0.2 mmol/g.
31. The method of any one of claims 26 to 30 , wherein each cross-metathesis is performed using a cross-metathesis catalyst, and each cross-metathesis is performed in a solvent combination of a resin-swelling solvent, with a co-ordinating solvent for the catalyst.
32. The method of claim 31 , wherein the co-ordinating solvent is an alcohol.
33. The method of claim 31 or claim 32 , wherein the co-ordinating solvent is used in an amount of about 1-30% by volume, with respect to the total solvent combination.
34. An organic compound or a peptide containing a dicarba bridge when produced by the method of any one of claims 1 to 33 .
35. Fmoc-protected prenyl glycine.
36. A method for the synthesis of Fmoc-protected prenyl glycine, the method comprising cross-metathesis of Fmoc-protected allyl glycine with 2-alkyl-2-butylene in the presence of a cross-metathesis catalyst.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/804,693 US20150315243A1 (en) | 2006-02-17 | 2015-07-21 | Methods for the synthesis of dicarba bridges in organic compounds |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2006900799 | 2006-02-17 | ||
AU2006900799A AU2006900799A0 (en) | 2006-02-17 | Methods for the synthesis of dicarba bridges in organic compounds | |
PCT/AU2007/000176 WO2007093013A1 (en) | 2006-02-17 | 2007-02-16 | Methods for the synthesis of dicarba bridges in organic compounds |
US27938309A | 2009-03-13 | 2009-03-13 | |
US14/804,693 US20150315243A1 (en) | 2006-02-17 | 2015-07-21 | Methods for the synthesis of dicarba bridges in organic compounds |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/AU2007/000176 Continuation WO2007093013A1 (en) | 2006-02-17 | 2007-02-16 | Methods for the synthesis of dicarba bridges in organic compounds |
US12/279,383 Continuation US9102708B2 (en) | 2006-02-17 | 2007-02-16 | Methods for the synthesis of dicarba bridges in organic compounds |
Publications (1)
Publication Number | Publication Date |
---|---|
US20150315243A1 true US20150315243A1 (en) | 2015-11-05 |
Family
ID=38371126
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/279,383 Expired - Fee Related US9102708B2 (en) | 2006-02-17 | 2007-02-16 | Methods for the synthesis of dicarba bridges in organic compounds |
US13/453,766 Abandoned US20130023645A1 (en) | 2006-02-17 | 2012-04-23 | Methods for the synthesis of dicarba bridges in organic compounds |
US14/804,693 Abandoned US20150315243A1 (en) | 2006-02-17 | 2015-07-21 | Methods for the synthesis of dicarba bridges in organic compounds |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/279,383 Expired - Fee Related US9102708B2 (en) | 2006-02-17 | 2007-02-16 | Methods for the synthesis of dicarba bridges in organic compounds |
US13/453,766 Abandoned US20130023645A1 (en) | 2006-02-17 | 2012-04-23 | Methods for the synthesis of dicarba bridges in organic compounds |
Country Status (3)
Country | Link |
---|---|
US (3) | US9102708B2 (en) |
EP (1) | EP1984385A4 (en) |
WO (1) | WO2007093013A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7538190B2 (en) | 2006-02-17 | 2009-05-26 | Polychip Pharmaceuticals Pty Ltd | Methods for the synthesis of two or more dicarba bridges in organic compounds |
US7745573B2 (en) | 2006-02-17 | 2010-06-29 | Polychip Pharmaceuticals Pty Ltd. | Conotoxin analogues and methods for synthesis of intramolecular dicarba bridge-containing peptides |
US9102708B2 (en) | 2006-02-17 | 2015-08-11 | Syngene Limited | Methods for the synthesis of dicarba bridges in organic compounds |
EP2576588A4 (en) * | 2010-05-25 | 2014-07-09 | Syngene Ltd | Methods for the synthesis of alkyne-containing dicarba bridges in peptides |
WO2013126703A1 (en) * | 2012-02-22 | 2013-08-29 | New York University | Reversibly crosslinked helical hydrogen bond surrogate macrocycles |
WO2014005197A1 (en) * | 2012-07-06 | 2014-01-09 | Monash University | Amino acid analogues and methods for their synthesis |
US20160185821A1 (en) * | 2014-07-18 | 2016-06-30 | California Institute Of Technology | Z-selective olefin metathesis of peptides |
EP3452459A1 (en) | 2016-05-04 | 2019-03-13 | Haldor Topsøe A/S | New adipate-type compounds and a process of preparing it |
US20230348412A1 (en) | 2020-05-28 | 2023-11-02 | Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. | Method for preparing glp-1 receptor agonist |
TW202206420A (en) | 2020-05-28 | 2022-02-16 | 美商維特衛治療有限責任公司 | Intermediates and methods for preparing a glp-1 receptor agonist |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5811515A (en) * | 1995-06-12 | 1998-09-22 | California Institute Of Technology | Synthesis of conformationally restricted amino acids, peptides, and peptidomimetics by catalytic ring closing metathesis |
US20020040109A1 (en) * | 2000-03-27 | 2002-04-04 | Fogg Deryn Elizabeth | Method for producing saturated polymers and saturated or unsaturated blends |
US20030176749A1 (en) * | 2002-03-15 | 2003-09-18 | Lever John G. | Simplified methods of making 1,3-cyclohexadiene |
WO2005000873A1 (en) * | 2003-06-24 | 2005-01-06 | Isis Innovation Limited | Preparation of glycosylated amino acids, proteins and peptides via olefin metathesis reactions |
US7538190B2 (en) * | 2006-02-17 | 2009-05-26 | Polychip Pharmaceuticals Pty Ltd | Methods for the synthesis of two or more dicarba bridges in organic compounds |
US7745573B2 (en) * | 2006-02-17 | 2010-06-29 | Polychip Pharmaceuticals Pty Ltd. | Conotoxin analogues and methods for synthesis of intramolecular dicarba bridge-containing peptides |
US8748570B2 (en) * | 2010-05-25 | 2014-06-10 | Syngene Limited | Insulin analogues |
US9102708B2 (en) * | 2006-02-17 | 2015-08-11 | Syngene Limited | Methods for the synthesis of dicarba bridges in organic compounds |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5153282A (en) * | 1987-06-08 | 1992-10-06 | Exxon Chemical Patents Inc. | Preparation of polyolefin incorporating functional groups from masked functional group-containing monomers |
US4933402A (en) * | 1988-12-30 | 1990-06-12 | Hercules Incorporated | Phosphorus compounds that delay the metathesis polymerization of cycloolefins |
ZA965853B (en) * | 1995-07-13 | 1998-01-12 | Du Pont Merck Pharma | Asymmetric synthesis of r and s warfarin and its analogs. |
AUPP589598A0 (en) * | 1998-09-14 | 1998-10-08 | University Of Queensland, The | Novel peptides |
US6855805B2 (en) * | 1999-01-22 | 2005-02-15 | University Of Utah Research Foundation | α-conotoxin peptides |
WO2002060923A2 (en) * | 2001-01-29 | 2002-08-08 | Cognetix, Inc. | B-superfamily conotoxins |
US7261876B2 (en) * | 2002-03-01 | 2007-08-28 | Bracco International Bv | Multivalent constructs for therapeutic and diagnostic applications |
JP2007516239A (en) * | 2003-11-20 | 2007-06-21 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Method for removing transition metals, particularly from metathesis products |
US7202332B2 (en) * | 2004-05-27 | 2007-04-10 | New York University | Methods for preparing internally constrained peptides and peptidomimetics |
-
2007
- 2007-02-16 US US12/279,383 patent/US9102708B2/en not_active Expired - Fee Related
- 2007-02-16 WO PCT/AU2007/000176 patent/WO2007093013A1/en active Application Filing
- 2007-02-16 EP EP07701507A patent/EP1984385A4/en not_active Withdrawn
-
2012
- 2012-04-23 US US13/453,766 patent/US20130023645A1/en not_active Abandoned
-
2015
- 2015-07-21 US US14/804,693 patent/US20150315243A1/en not_active Abandoned
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5811515A (en) * | 1995-06-12 | 1998-09-22 | California Institute Of Technology | Synthesis of conformationally restricted amino acids, peptides, and peptidomimetics by catalytic ring closing metathesis |
US20020040109A1 (en) * | 2000-03-27 | 2002-04-04 | Fogg Deryn Elizabeth | Method for producing saturated polymers and saturated or unsaturated blends |
US20030176749A1 (en) * | 2002-03-15 | 2003-09-18 | Lever John G. | Simplified methods of making 1,3-cyclohexadiene |
WO2005000873A1 (en) * | 2003-06-24 | 2005-01-06 | Isis Innovation Limited | Preparation of glycosylated amino acids, proteins and peptides via olefin metathesis reactions |
US7538190B2 (en) * | 2006-02-17 | 2009-05-26 | Polychip Pharmaceuticals Pty Ltd | Methods for the synthesis of two or more dicarba bridges in organic compounds |
US7745573B2 (en) * | 2006-02-17 | 2010-06-29 | Polychip Pharmaceuticals Pty Ltd. | Conotoxin analogues and methods for synthesis of intramolecular dicarba bridge-containing peptides |
US8124726B2 (en) * | 2006-02-17 | 2012-02-28 | Monash University | Contoxin analogues and methods for synthesizing same |
US8362204B2 (en) * | 2006-02-17 | 2013-01-29 | Syngene Limited | Methods for the synthesis of two or more dicarba bridges in organic compounds |
US9102708B2 (en) * | 2006-02-17 | 2015-08-11 | Syngene Limited | Methods for the synthesis of dicarba bridges in organic compounds |
US8748570B2 (en) * | 2010-05-25 | 2014-06-10 | Syngene Limited | Insulin analogues |
Non-Patent Citations (3)
Title |
---|
Connon, Stphen J. and Blechert, Siegfried, "Recent developments in olefin cross-metathesis." Angew. Chem. Int. Ed (2003) 42 p1900-1923 * |
Kappe, C. Oliver and DAllinger, Doris, "The impact of microwave synthesis on drug discovery." Nature Rev. (2006) 5 p51-63 * |
Schmiedeberg, Niko and Kessler, Horst, "Reversible backbone protection enables cominatorial solid phase ring closing metathesis reaction (rcm) in peptides." Org. Lett. (2002) 4(1) p59-62) * |
Also Published As
Publication number | Publication date |
---|---|
US9102708B2 (en) | 2015-08-11 |
EP1984385A1 (en) | 2008-10-29 |
US20100036089A1 (en) | 2010-02-11 |
US20130023645A1 (en) | 2013-01-24 |
WO2007093013A1 (en) | 2007-08-23 |
EP1984385A4 (en) | 2012-03-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8124726B2 (en) | Contoxin analogues and methods for synthesizing same | |
US8362204B2 (en) | Methods for the synthesis of two or more dicarba bridges in organic compounds | |
US20150315243A1 (en) | Methods for the synthesis of dicarba bridges in organic compounds | |
Stymiest et al. | Synthesis of oxytocin analogues with replacement of sulfur by carbon gives potent antagonists with increased stability | |
Jagasia et al. | Peptide cyclization and cyclodimerization by cui-mediated azide− alkyne cycloaddition | |
Yao et al. | Efficient synthesis and stereochemical revision of coibamide A | |
Gleeson et al. | Ring-closing metathesis in peptides | |
US20130345397A1 (en) | Methods for the synthesis of dicarba bridges in peptides | |
Sharma et al. | Tandem thiol switch synthesis of peptide thioesters via N–S acyl shift on thiazolidine | |
JP2008513403A (en) | Peptide cyclization | |
Marti et al. | Solid phase synthesis of β-peptides via Arndt-Eistert homologation of Fmoc-protected amino acid diazoketones | |
Baron et al. | cis-Apa: A practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage | |
Romero-Estudillo et al. | Domino process achieves site-selective peptide modification with high optical purity. Applications to chain diversification and peptide ligation | |
Gisemba et al. | Optimized ring closing metathesis reaction conditions to suppress desallyl side products in the solid-phase synthesis of cyclic peptides involving tyrosine (O-allyl) | |
Lee et al. | Peptide ligation via the suzuki–miyaura cross-coupling reaction | |
Crich et al. | Solid-phase synthesis of peptidyl thioacids employing a 9-fluorenylmethyl thioester-based linker in conjunction with Boc chemistry | |
AU2012203760A1 (en) | Methods for the synthesis of a dicarba bridge in organic compounds | |
Schulz et al. | Preparation of disulfide-bonded polypeptide heterodimers by titration of thio-activated peptides with thiol-containing peptides | |
Chen et al. | Application of tert-Butyl Disulfide-Protected Amino Acids for the Fmoc Solid-Phase Synthesis of Lactam Cyclic Peptides under Mild Metal-Free Conditions | |
Mollica et al. | (Acyloxy) alkoxy moiety as amino acids protecting group for the synthesis of (R, R)-2, 7 diaminosuberic acid via RCM | |
Pedersen et al. | Huisgen cycloaddition in peptidomimetic chemistry | |
Boeglin et al. | Development of a practical solid-phase synthesis approach to 1, 3, 5-triazepan-2, 6-diones | |
Pinnen | Adriano Mollica*, Federica Feliciani, Azzurra Stefanucci 2, Evgeny A. Fadeev 3 and | |
Rodríguez Puentes | Applications of the Ugi reaction in the synthesis of cyclic and N-Alkylated peptides |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |