WO1999055718A2 - Solution phase biopolymer synthesis - Google Patents
Solution phase biopolymer synthesis Download PDFInfo
- Publication number
- WO1999055718A2 WO1999055718A2 PCT/US1999/008939 US9908939W WO9955718A2 WO 1999055718 A2 WO1999055718 A2 WO 1999055718A2 US 9908939 W US9908939 W US 9908939W WO 9955718 A2 WO9955718 A2 WO 9955718A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lpc
- aryl
- tris
- diaza
- alkyl
- Prior art date
Links
- 229920001222 biopolymer Polymers 0.000 title claims abstract description 113
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 101
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 98
- 238000000034 method Methods 0.000 claims abstract description 92
- 150000001875 compounds Chemical class 0.000 claims abstract description 36
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 29
- 239000007791 liquid phase Substances 0.000 claims abstract description 13
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 12
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 11
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 7
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 7
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract 2
- -1 nitro, formyl Chemical group 0.000 claims description 96
- 125000000217 alkyl group Chemical group 0.000 claims description 93
- 125000003118 aryl group Chemical group 0.000 claims description 89
- 125000001072 heteroaryl group Chemical group 0.000 claims description 73
- 125000002577 pseudohalo group Chemical group 0.000 claims description 65
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 50
- 229910052739 hydrogen Inorganic materials 0.000 claims description 49
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 47
- 239000001257 hydrogen Substances 0.000 claims description 46
- 239000000178 monomer Substances 0.000 claims description 45
- 125000002947 alkylene group Chemical group 0.000 claims description 43
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 42
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 40
- 239000000243 solution Substances 0.000 claims description 36
- 125000000623 heterocyclic group Chemical group 0.000 claims description 30
- 125000004122 cyclic group Chemical group 0.000 claims description 27
- 239000007983 Tris buffer Substances 0.000 claims description 23
- 125000004475 heteroaralkyl group Chemical group 0.000 claims description 23
- 125000004415 heterocyclylalkyl group Chemical group 0.000 claims description 23
- 108091093037 Peptide nucleic acid Proteins 0.000 claims description 22
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 22
- QMKYBPDZANOJGF-UHFFFAOYSA-N benzene-1,3,5-tricarboxylic acid Chemical compound OC(=O)C1=CC(C(O)=O)=CC(C(O)=O)=C1 QMKYBPDZANOJGF-UHFFFAOYSA-N 0.000 claims description 21
- 150000008300 phosphoramidites Chemical class 0.000 claims description 20
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 18
- 125000001140 1,4-phenylene group Chemical group [H]C1=C([H])C([*:2])=C([H])C([H])=C1[*:1] 0.000 claims description 17
- 239000000539 dimer Substances 0.000 claims description 16
- 125000001188 haloalkyl group Chemical group 0.000 claims description 15
- 125000005843 halogen group Chemical group 0.000 claims description 15
- 125000003545 alkoxy group Chemical group 0.000 claims description 14
- 125000000732 arylene group Chemical group 0.000 claims description 14
- 229910052760 oxygen Inorganic materials 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 13
- 239000013638 trimer Substances 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 125000004450 alkenylene group Chemical group 0.000 claims description 12
- 125000004419 alkynylene group Chemical group 0.000 claims description 12
- 125000005647 linker group Chemical group 0.000 claims description 12
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 12
- 239000012071 phase Substances 0.000 claims description 12
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 claims description 12
- 125000004103 aminoalkyl group Chemical group 0.000 claims description 11
- 229910052736 halogen Inorganic materials 0.000 claims description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 11
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].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.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 claims description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 11
- 125000001424 substituent group Chemical group 0.000 claims description 11
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 10
- 150000002825 nitriles Chemical group 0.000 claims description 10
- 125000003342 alkenyl group Chemical group 0.000 claims description 9
- 125000004414 alkyl thio group Chemical group 0.000 claims description 9
- 150000001408 amides Chemical class 0.000 claims description 9
- 125000005110 aryl thio group Chemical group 0.000 claims description 9
- 125000001118 alkylidene group Chemical group 0.000 claims description 8
- 125000000304 alkynyl group Chemical group 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 125000004104 aryloxy group Chemical group 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 229910052717 sulfur Inorganic materials 0.000 claims description 8
- 125000004453 alkoxycarbonyl group Chemical group 0.000 claims description 7
- 125000004457 alkyl amino carbonyl group Chemical group 0.000 claims description 7
- 125000004947 alkyl aryl amino group Chemical group 0.000 claims description 7
- 125000005125 aryl alkyl amino carbonyl group Chemical group 0.000 claims description 7
- 125000002102 aryl alkyloxo group Chemical group 0.000 claims description 7
- 125000005100 aryl amino carbonyl group Chemical group 0.000 claims description 7
- 125000005161 aryl oxy carbonyl group Chemical group 0.000 claims description 7
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims description 7
- 125000004663 dialkyl amino group Chemical group 0.000 claims description 7
- 125000004473 dialkylaminocarbonyl group Chemical group 0.000 claims description 7
- 239000002777 nucleoside Substances 0.000 claims description 7
- 229920001774 Perfluoroether Polymers 0.000 claims description 6
- 125000003302 alkenyloxy group Chemical group 0.000 claims description 6
- 125000000278 alkyl amino alkyl group Chemical group 0.000 claims description 6
- 125000003282 alkyl amino group Chemical group 0.000 claims description 6
- 125000004471 alkyl aminosulfonyl group Chemical group 0.000 claims description 6
- 125000003806 alkyl carbonyl amino group Chemical group 0.000 claims description 6
- 125000004448 alkyl carbonyl group Chemical group 0.000 claims description 6
- 125000004644 alkyl sulfinyl group Chemical group 0.000 claims description 6
- 125000004390 alkyl sulfonyl group Chemical group 0.000 claims description 6
- 125000005133 alkynyloxy group Chemical group 0.000 claims description 6
- 125000004397 aminosulfonyl group Chemical group NS(=O)(=O)* 0.000 claims description 6
- 125000005128 aryl amino alkyl group Chemical group 0.000 claims description 6
- 125000001769 aryl amino group Chemical group 0.000 claims description 6
- 125000005141 aryl amino sulfonyl group Chemical group 0.000 claims description 6
- 125000004658 aryl carbonyl amino group Chemical group 0.000 claims description 6
- 125000005129 aryl carbonyl group Chemical group 0.000 claims description 6
- 125000005135 aryl sulfinyl group Chemical group 0.000 claims description 6
- 125000004391 aryl sulfonyl group Chemical group 0.000 claims description 6
- 125000001316 cycloalkyl alkyl group Chemical group 0.000 claims description 6
- 125000004985 dialkyl amino alkyl group Chemical group 0.000 claims description 6
- 125000004472 dialkylaminosulfonyl group Chemical group 0.000 claims description 6
- 125000004986 diarylamino group Chemical group 0.000 claims description 6
- 125000004446 heteroarylalkyl group Chemical group 0.000 claims description 6
- 125000005223 heteroarylcarbonyl group Chemical group 0.000 claims description 6
- 125000006684 polyhaloalkyl group Chemical group 0.000 claims description 6
- 125000005031 thiocyano group Chemical group S(C#N)* 0.000 claims description 6
- 125000005078 alkoxycarbonylalkyl group Chemical group 0.000 claims description 5
- 125000004466 alkoxycarbonylamino group Chemical group 0.000 claims description 5
- 125000005162 aryl oxy carbonyl amino group Chemical group 0.000 claims description 5
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 5
- 125000003835 nucleoside group Chemical group 0.000 claims description 5
- 125000005549 heteroarylene group Chemical group 0.000 claims description 4
- 150000002772 monosaccharides Chemical class 0.000 claims description 4
- 229910052710 silicon Inorganic materials 0.000 claims description 4
- 239000010703 silicon Substances 0.000 claims description 4
- OFKUAGPTUWMPSX-UHFFFAOYSA-N n-(2-aminoethyl)-3-[3-[3-(2-aminoethylamino)-3-oxopropoxy]-2,2-bis[[3-(2-aminoethylamino)-3-oxopropoxy]methyl]propoxy]propanamide Chemical compound NCCNC(=O)CCOCC(COCCC(=O)NCCN)(COCCC(=O)NCCN)COCCC(=O)NCCN OFKUAGPTUWMPSX-UHFFFAOYSA-N 0.000 claims description 3
- LMDLEWFKFNESMM-UHFFFAOYSA-N 4-[2-[bis[2-(3-carboxypropanoylamino)ethyl]amino]ethylamino]-4-oxobutanoic acid Chemical compound OC(=O)CCC(=O)NCCN(CCNC(=O)CCC(O)=O)CCNC(=O)CCC(O)=O LMDLEWFKFNESMM-UHFFFAOYSA-N 0.000 claims description 2
- BJVRDWSCILKKCD-UHFFFAOYSA-N 3-[2-[3-[3-[3-[2-[(2-carboxyacetyl)amino]ethylamino]-3-oxopropoxy]-2,2-bis[[3-[2-[(2-carboxyacetyl)amino]ethylamino]-3-oxopropoxy]methyl]propoxy]propanoylamino]ethylamino]-3-oxopropanoic acid Chemical compound OC(=O)CC(=O)NCCNC(=O)CCOCC(COCCC(=O)NCCNC(=O)CC(O)=O)(COCCC(=O)NCCNC(=O)CC(O)=O)COCCC(=O)NCCNC(=O)CC(O)=O BJVRDWSCILKKCD-UHFFFAOYSA-N 0.000 claims 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 abstract description 17
- 238000002360 preparation method Methods 0.000 abstract description 12
- 239000000969 carrier Substances 0.000 abstract description 11
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 9
- 125000000524 functional group Chemical group 0.000 abstract description 5
- 125000000896 monocarboxylic acid group Chemical group 0.000 abstract 1
- 150000004820 halides Chemical class 0.000 description 79
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 73
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 57
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 44
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 42
- 238000006243 chemical reaction Methods 0.000 description 38
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 32
- 150000002148 esters Chemical class 0.000 description 25
- 239000000047 product Substances 0.000 description 25
- 238000005227 gel permeation chromatography Methods 0.000 description 22
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 22
- 238000000746 purification Methods 0.000 description 22
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 22
- 239000002904 solvent Substances 0.000 description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- 229910019142 PO4 Inorganic materials 0.000 description 18
- 235000021317 phosphate Nutrition 0.000 description 18
- 239000003480 eluent Substances 0.000 description 17
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- 125000002843 carboxylic acid group Chemical group 0.000 description 15
- 238000004587 chromatography analysis Methods 0.000 description 15
- 150000001266 acyl halides Chemical class 0.000 description 14
- 125000004432 carbon atom Chemical group C* 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 14
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 14
- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical class C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 14
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 14
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical class OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 13
- 150000001262 acyl bromides Chemical class 0.000 description 13
- 150000001263 acyl chlorides Chemical class 0.000 description 13
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical compound [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 description 13
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical class [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 13
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 13
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 13
- MPQXHAGKBWFSNV-UHFFFAOYSA-N oxidophosphanium Chemical class [PH3]=O MPQXHAGKBWFSNV-UHFFFAOYSA-N 0.000 description 13
- 150000003003 phosphines Chemical class 0.000 description 13
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 13
- 150000003871 sulfonates Chemical class 0.000 description 13
- 150000003457 sulfones Chemical class 0.000 description 13
- 150000003462 sulfoxides Chemical class 0.000 description 13
- 150000003463 sulfur Chemical class 0.000 description 13
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 150000001412 amines Chemical class 0.000 description 12
- 230000004048 modification Effects 0.000 description 12
- 238000012986 modification Methods 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- 125000004149 thio group Chemical group *S* 0.000 description 12
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 11
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 11
- 238000005160 1H NMR spectroscopy Methods 0.000 description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 10
- 238000002515 oligonucleotide synthesis Methods 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- UBTJZUKVKGZHAD-UPRLRBBYSA-N 1-[(2r,4s,5r)-5-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-4-hydroxyoxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC(OC)=CC=1)(C=1C=CC=CC=1)OC[C@@H]1[C@@H](O)C[C@H](N2C(NC(=O)C(C)=C2)=O)O1 UBTJZUKVKGZHAD-UPRLRBBYSA-N 0.000 description 8
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 150000002367 halogens Chemical class 0.000 description 8
- LYGJENNIWJXYER-UHFFFAOYSA-N nitromethane Chemical compound C[N+]([O-])=O LYGJENNIWJXYER-UHFFFAOYSA-N 0.000 description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 8
- 239000011541 reaction mixture Substances 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 238000010549 co-Evaporation Methods 0.000 description 7
- 239000000543 intermediate Substances 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 238000010532 solid phase synthesis reaction Methods 0.000 description 7
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 6
- KJUGUADJHNHALS-UHFFFAOYSA-N 1H-tetrazole Chemical compound C=1N=NNN=1 KJUGUADJHNHALS-UHFFFAOYSA-N 0.000 description 6
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 6
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000010898 silica gel chromatography Methods 0.000 description 6
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 5
- 125000004429 atom Chemical group 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 239000007859 condensation product Substances 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 5
- 125000002950 monocyclic group Chemical group 0.000 description 5
- 238000006386 neutralization reaction Methods 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- 0 **c1cc(**)cc([*-])c1 Chemical compound **c1cc(**)cc([*-])c1 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 4
- 239000005977 Ethylene Substances 0.000 description 4
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- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- CRDYSYOERSZTHZ-UHFFFAOYSA-M selenocyanate Chemical compound [Se-]C#N CRDYSYOERSZTHZ-UHFFFAOYSA-M 0.000 description 1
- 150000004756 silanes Chemical group 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000000475 sulfinyl group Chemical group [*:2]S([*:1])=O 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 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
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 125000004953 trihalomethyl group Chemical group 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C211/00—Compounds containing amino groups bound to a carbon skeleton
- C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
- C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
- C07C211/14—Amines containing amino groups bound to at least two aminoalkyl groups, e.g. diethylenetriamines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
- C07C233/77—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
- C07C233/78—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C25/00—Compounds containing at least one halogen atom bound to a six-membered aromatic ring
- C07C25/18—Polycyclic aromatic halogenated hydrocarbons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C255/00—Carboxylic acid nitriles
- C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
- C07C255/16—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms containing cyano groups and singly-bound oxygen atoms bound to the same carbon atom of an acyclic carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C43/00—Ethers; Compounds having groups, groups or groups
- C07C43/02—Ethers
- C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
- C07C43/225—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
- C07C69/708—Ethers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
-
- 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
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
- C07K14/003—Peptide-nucleic acids (PNAs)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/11—Compounds covalently bound to a solid support
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S585/00—Chemistry of hydrocarbon compounds
- Y10S585/929—Special chemical considerations
- Y10S585/93—Process including synthesis of nonhydrocarbon intermediate
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S585/00—Chemistry of hydrocarbon compounds
- Y10S585/929—Special chemical considerations
- Y10S585/93—Process including synthesis of nonhydrocarbon intermediate
- Y10S585/932—Carboxyl-containing, e.g. acid
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S585/00—Chemistry of hydrocarbon compounds
- Y10S585/929—Special chemical considerations
- Y10S585/93—Process including synthesis of nonhydrocarbon intermediate
- Y10S585/933—N-containing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S585/00—Chemistry of hydrocarbon compounds
- Y10S585/929—Special chemical considerations
- Y10S585/93—Process including synthesis of nonhydrocarbon intermediate
- Y10S585/934—Chalcogen-containing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S585/00—Chemistry of hydrocarbon compounds
- Y10S585/929—Special chemical considerations
- Y10S585/93—Process including synthesis of nonhydrocarbon intermediate
- Y10S585/935—Halogen-containing
Definitions
- LPCs Liquid phase carriers
- methods of use of LPCs in biopolymer synthesis are provided.
- multifunctional LPCs and methods of use of multifunctional LPCs in oligonucleotide synthesis are provided.
- oligonucleotides are needed only in small amounts (nano- to milligrams), for use in, e.g., polymerase chain reactions (see, e.g., K.B. Mullis Angew. Chem. 1994, 106, 1 271 -1 276 and K.B. Mullis et al. Methods Enzvmol. 1987, 1 55, 335-3508) and as diagnostic tools (see, e.g. , S. Beck et aL Anal. Chem. 1990, 62, 2258-2270; C.
- Milligan et aL J. Med. Chem. 1993, 36, 1 923-1 937) may be used as therapeutic agents.
- Current methods of biopolymer synthesis such as solid phase synthesis of oligonucleotides and peptides, result in preparation of small amounts of a desired biopolymer (see, e.g . , N. D. Sinha et al. Tetrahedron Lett. 1983, 24, 5843-5846; H . K ⁇ ster et al. Tetrahedron 1984, 40, 1 03-1 1 2; and N.D. Sinha et al. Nucleic Acids Res. 1984, __2, 4539-4557) .
- a further drawback of solution phase techniques is the lack of solubility of the substrate biopolymer in organic solvents when the number of monomeric units exceeds 3-5. Such lack of solubility also contributes to lower yields seen in solution phase synthesis of biopolymers. Thus, there is a need for carriers that mediate the solubility of the growing biopolymeric chain.
- LPCs Liquid phase carriers
- biopolymers that can be prepared using the LPCs and methods described herein include, but are not limited to, oligonucleotides, peptides, peptide nucleic acids (PNAs) and oligosaccharides.
- PNAs peptide nucleic acids
- a desired biopolymer may be prepared by the methods provided herein, or modification thereof using techniques known to those of skill in the art.
- the LPCs provided herein are multifunctional compounds that allow for the simultaneous construction of three or more biopolymers, which may be of differing structure, but more typically are identical in structure.
- the LPCs have the formula Sp(X 1 ) n .
- Sp refers to a group that serves as the scaffold upon which synthesis of a plurality of oligomers is effected.
- X is the reactive group for effecting synthesis of a selected biopolymer, and "n" is preferably 3 or more.
- Sp is a polyvalent group, such as an atomic group, a cyclic group or an aromatic group (heterocycles, carbocycles, aryls, heteroaryls, such that the resulting structure is symmetrically disposed around the center of the cyclic group), that has more than two points of attachment, n is the number of points of attachment in Sp, and is preferably 3-6, and X 1 is a reactive group for performing synthesis of a biopolymer. Sp is preferably a symmetrical group such that all X 1 groups are equivalent. It will be appreciated by those of skill in the art that such symmetry allows for uniformity in the synthesis of n biopolymers, where n is preferably 3 or more, using the LPCs provided herein.
- the LPCs provided herein are intended for use for synthesis of three or more biopolymers.
- the LPCs have one of formulae (I) :
- Sp is (R 1 ) p -A, E or a cyclic group (i.e. heterocycles, carbocycles, aryls, heteroaryls, such that the resulting structure is symmetrically disposed around the center of the cyclic group) with the linked "Z t " moieties, where t is 0 or 1 .
- the cyclic and aromatic rings have 6 members; it is understood that fewer or higher-number membered rings may also be used, as long as the resulting structure possesses the requisite symmetry, and the number of linkages for synthesis of the biopolymers is greater than two.
- A is carbon or silicon; E is nitrogen or P(O); R 1 and R 3 are each independently hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylalkyl; p is 0 or 1 ; Z, where t is 0 or 1 , is a divalent hydrocarbon, containing one or more or mixtures of phenylene or alkylene groups, and contains a sufficient number of carbons (0, where t is 0, up to as many as 24 or more) to prevent or to reduce interactions among the chains in each biopolymer during synthesis.
- Z typically containing from 1 up to 30 carbons or more, is any combination alkylene and arylene units, preferably methylene and phenylene units, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2 units, preferably selected from 1 ,2-, 1 ,3- or 1 ,4-, preferably 1 ,4-, phenylene and alkylene units, more preferably methylene units, which units may be combined in any order;
- X 1 is any reactive group that is used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups
- Patent No. 5, 1 98,540 is preferably halide, OH, SH, NH 2 , COR 5 or COOR 4 ; n is preferably 3 or 4; R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; R 5 is halide, heteroaryl or pseudohalide; Y 1 is CH 2 , NH, S or O; and Y 2 is selected from CH and N.
- Z is a multivalent group, particularly a trivalent or higher valency hydrocarbon, with three or more points of attachment: The points of attachment are to A, E, or the cyclic nucleus, and the others are to two or more X 1 groups. In these embodiments, Z is chosen such that the symmetry of the LPC is maintained and all of the X 1 groups are equivalent.
- the resulting LPCs are chosen from compounds such as, but not limited to, the following:
- the LPCs contain two or more of the compounds of formula (I) that are linked together so that the symmetry of the resulting LPC is maintained (i.e., all X 1 groups are equivalent) .
- the LPCs have formulae:
- R 20 is alkylene, alkenylene, alkynylene, arylene or heteroarylene of any desired length, preferably from 0 to 24, more preferably from 1 to about 1 2; k is 2 or 3; and j is 0 or 1 .
- any of the groups R ⁇ R 3 , R 4 , R 5 , X 1 , Y ⁇ Y 2 and Z may be unsubstituted or substituted with one or more substituents each independently selected from Q, which, as defined herein, is halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylidene, arylalkylidene, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, alkoxy- carbonylalkyl, aryloxycarbonyl, aryloxycarbonyl
- the LPCs are chosen as above, with the proviso that Z contains more than two methylene groups. In other embodiments, the LPCs are chosen with the proviso that Z is not 1 ,4- arylene. In more preferred embodiments, the LPCs are chosen with the proviso that Z contains more than two methylene groups and is not 1 ,4- arylene. In other more preferred embodiments, the LPCs are chosen with the proviso that Z contains at least one, preferably at least two, more preferably at least three or more phenylene or alkylene units.
- the LPCs and methods permit synthesis of relatively large amounts, e ⁇ therapeutically useful amounts or preparative amounts (on the order of at least 0.1 grams, preferably a gram or more in volumes at which solid phase methods and other liquid phase methods produce substantially lower amounts) of biopolymers, including, but not limited to, oligonucleotides, peptides, peptide nucleic acids (PNAs) and oligosaccharides, may be produced by the methods. It is possible to prepare not only naturally occurring biopolymers, but also analogs of the biopolymers.
- Such analogs include, for example, analogs of oligonucleotides such as oligo-triesters or oligophosphonates.
- Use of the LPCs provided herein in the methods afford biopolymers of high purity and in significantly higher yields than methods known in the art.
- the methods involve the steps of (a) reacting an LPC of formula Sp(X ) n , provided herein, with a first monomer N 1 ; (b) separating and purifying the product of step (a) to afford a compound of formula Sp(X 1 -N 1 ) n ; (c) reacting the product of step (b) with a second monomer N 2 , a dimer N 2 -N 3 or a trimer N 2 -N 3 -N 4 ; and (d) repeating steps (b) and (c) using the desired monomers, dimers or trimers to produce the desired LPC-bound biopolymer of formula Sp(X 1 -N 1 -N 2 -...-N m ) n , where m is 3 to 1 00, preferably 3-50, more preferably 3-25, most preferably 3- 10.
- the methods further involve the step of (e) cleaving the biopolymer from the LPC.
- the methods involve the steps of: (a) reacting an LPC of formula Sp(X 1 ) n with a first monomer N 1 ; (b) separating and purifying the product of step (a) to afford a compound of formula Sp(X 1 -N 1 ) n ;
- step (c) reacting the product of step (b) with a second monomer N 2 , a dimer N 2 -N 3 or a trimer N 2 -N 3 -N 4 ;
- step (d) repeating steps (b) and (c) to produce an LPC-bound biopolymer, preferably an oligonucleotide, of formula Sp(X 1 -N 1 -N 2 -...- N m ) n , where m is 3 to 100, wherein:
- Sp is a polyvalent group that has two or more points of attachment, n corresponds to the number of points of attachment in Sp and X 1 is a reactive group for biopolymer, preferably oligonucleotide, synthesis;
- N ⁇ N 2 , N 3 ...N m are biopolymer monomers, preferably nucleotides or nucleosides; the dimers and trimers comprise the monomers; and the protocol used in steps (c) and (d) to synthesize the biopolymer, preferably the oligonucleotide, is the phosphoramidite protocol.
- Monomers (N) used in preparing the biopolymer will depend on the biopolymer desired .
- monomers which are used in the methods include nucleotides, nucleosides, natural and unnatural amino acids, PNA monomers and monosaccharides.
- synthesis of the biopolymeric chain may be performed by any protocol known to those of skill in the art.
- a preferred protocol for oligonucleotide synthesis is the phosphoramidite protocol (see, e.g., S. L. Beaucage et al. Tetrahedron 1992, 48, 2223-231 1 and S. L. Beaucage et al. Tetrahedron Lett. 1981 , 22, 1 859-1 862) .
- DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS see, e.g., S. L. Beaucage et al. Tetrahedron 1992, 48, 2223-231 1 and S. L. Beaucage et al. Tetrahedron Let
- peptide nucleic acid refers to nucleic acid analogs in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds.
- MALDI-TOF refers to matrix assisted laser desorption ionization-time of flight mass spectrometry.
- a liquid phase carrier is a symmetrical compound that possesses functionality compatible with biopolymer synthesis.
- functionality includes, but is not limited to, alcohol, thiol, amino, carboxylic acid, acyl halide, ester, acyl heteroaryl and acyl pseudohalide groups.
- the symmetry of the LPC is such that all of the functional groups are equivalent.
- Multifunctional LPCs are LPCs that possess three or more functional groups for use in biopolymer synthesis.
- a biopolymer is any compound found in nature, or derivatives thereof, made up of monomeric units.
- Biopolymers include, but are not limited to, oligonucleotides, peptides, peptide nucleic acids (PNAs) and oligosaccharides.
- the monomeric units include, but are not limited to, nucleotides, nucleosides, amino acids, PNA monomers, monosaccharides, and derivatives thereof.
- substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
- TLC thin layer chromatography
- HPLC high performance liquid chromatography
- MS mass spectrometry
- alkyl, alkenyl and alkynyl carbon chains if not specified contain from 1 to 20 carbons, preferably 1 to 1 6 carbons, and are straight or branched.
- Alkenyl carbon chains of from 2 to 20 carbons preferably contain 1 to 8 double bonds, and the alkenyl carbon chains of 1 to 1 6 carbons preferably contain 1 to 5 double bonds.
- Alkynyl carbon chains of from 2 to 20 carbons preferably contain 1 to 8 triple bonds, and the alkynyl carbon chains of 2 to 1 6 carbons preferably contain 1 to 5 triple bonds.
- the alkyl, alkenyl and alkynyl groups may be optionally substituted, with one or more groups, preferably alkyl group substituents that may be the same or different.
- lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having less than about 6 carbons.
- an "alkyl group substituent" includes halo, haloalkyl, preferably halo lower alkyl, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy alkoxycarbonyl, oxo and cycloalkyl.
- aryl refers to cyclic groups containing from 3 to 1 9 carbon atoms.
- Aryl groups include, but are not limited to groups, such as phenyl, substituted phenyl, naphthyl, substituted naphthyl, in which the substituent is lower alkyl, halogen, or lower alkoxy.
- an "aryl group substituent” includes alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl optionally substituted with 1 or more, preferably 1 to 3, substituents selected from halo, halo alkyl and alkyl, arylalkyl, heteroarylalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, halo, hydroxy, haloalkyl and polyhaloalkyl, preferably halo lower alkyl, especially trifluoromethyl, formyl, alkylcarbonyl, arylcarbonyl that is optionally substituted with 1 or more, preferably 1 to 3, substituents selected from halo, halo alkyl and alkyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
- cycloalkyl refers to a saturated mono- or multi- cyclic ring system, preferably of 3 to 1 0 carbon atoms, more preferably 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to mono- or multicyclic ring systems that respectively include at least one double bond and at least one triple bond. Cycloalkenyl and cycloalkynyl groups may preferably contain 3 to 10 carbon atoms, with cycloalkenyl groups more preferably containing 4 to 7 carbon atoms and cycloalkynyl groups more preferably containing 8 to 1 0 carbon atoms.
- ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion, and may be optionally substituted with one or more alkyl group substituents.
- heteroaryl refers to a monocyclic or multicyclic ring system, preferably of about 5 to about 1 5 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
- heteroaryl may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
- exemplary heteroaryl groups include, for example, furyl, imidazinyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl and isoquinolinyl, with pyridyl and quinolinyl being preferred.
- heterocyclic refers to a monocyclic or multicyclic ring system, preferably of 3 to 10 members, more preferably 4 to 7 members, even more preferably 5 to 6 members, where one or more, preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
- the heterocycle may be optionally substituted with one or more, preferably 1 to 3 aryl group substituents.
- Preferred substituents of the heterocyclic group include hydroxy, amino, alkoxy containing 1 to 4 carbon atoms, halo lower alkyl, including trihalomethyl, such as trifluoromethyl, and halogen.
- the term heterocycle may include reference to heteroaryl.
- alkyl refers to saturated carbon chains that contain one or more carbons; the chains may be straight or branched or include cyclic portions or be cyclic.
- alicyclic refers to aryl groups that are cyclic.
- halogen or halide refers to F, Cl, Br or I.
- pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X , in which X is a halogen, such as Cl or Br) .
- Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethyl and azide.
- haloalkyl refers to a lower alkyl radical in which one or more of the hydrogen atoms are replaced by halogen including, but not limited to, chloromethyl, trifluoromethyl, 1 -chloro-2-fluoroethyl and the like.
- haloalkoxy refers to RO- in which R is a haloalkyl group.
- sulfinyl refers to -S(O)-.
- sulfonyl refers to -S(O) 2 -.
- aminocarbonyl refers to -C(O)NH 2 .
- alkylaminocarbonyl refers to -C(O)NHR in which R is hydrogen or alkyl, preferably lower alkyl.
- dialkyl- aminocarbonyl refers to -C(O)NR R in which R and R are independently selected from hydrogen or alkyl, preferably lower alkyl;
- carboxamide refers to groups of formula -NR ' COR.
- diarylaminocarbonyl refers to -C(O)NRR' in which R and R' are independently selected from aryl, preferably lower aryl, more preferably phenyl.
- arylalkylaminocarbonyl refers to -C(O)NRR' in which one of R and R' is aryl, preferably lower aryl, more preferably phenyl, and the other of R and R' is alkyl, preferably lower alkyl.
- arylaminocarbonyl refers to -C(O)NHR in which
- R is aryl, preferably lower aryl, more preferably phenyl.
- alkoxycarbonyl refers to -C(O)OR in which R is alkyl, preferably lower alkyl.
- aryloxycarbonyl refers to -C(O)OR in which R is aryl, preferably lower aryl, more preferably phenyl.
- alkoxy and RS- refer to RO- and RS-, in which R is alkyl, preferably lower alkyl.
- aryloxy and “arylthio” refer to RO- and RS-, in which R is aryl, preferably lower aryl, more preferably phenyl.
- alkylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms, more preferably 1 to 1 2 carbons, even more preferably lower alkylene. The alkylene group is optionally substituted with one or more "alkyl group substituents.
- alkylene groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene ( — (CH 2 ) 3 -), cyclohexylene (-C 6 H 10 -), methylenedioxy (-O-CH 2 - O-) and ethylenedioxy (-O-(CH 2 ) 2 -O-) .
- lower alkylene refers to alkylene groups having 1 to 6 carbons.
- alkylene groups are lower alkylene, with alkylene of 1 to 3 carbon atoms being particularly preferred.
- alkenylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one double bond, more preferably 1 to 1 2 carbons, even more preferably lower alkenylene.
- the alkenylene group is optionally substituted with one or more "alkyl group substituents. " There may be optionally inserted along the alkenylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
- lower alkenylene refers to alkenylene groups having 2 to 6 carbons.
- Preferred alkenylene groups are lower alkenylene, with alkenylene of 3 to 4 carbon atoms being particularly preferred.
- alkynylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one triple bond, more preferably 1 to 1 2 carbons, even more preferably lower alkynylene.
- the alkynylene group is optionally substituted with one or more "alkyl group substituents. " There may be optionally inserted along the alkynylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
- Exemplary alkynylene groups include - C ⁇ C-C ⁇ C- , -C ⁇ C- and -C ⁇ C-CH 2 -.
- the term "lower alkynylene” refers to alkynylene groups having 2 to 6 carbons.
- Preferred alkynylene groups are lower alkynylene, with alkynylene of 3 to 4 carbon atoms being particularly preferred.
- arylene refers to a monocyclic or polycyclic, preferably monocyclic, bivalent aromatic group, preferably having from 3 to about 20 carbon atoms and at least one aromatic ring, more preferably 3 to 1 2 carbons, even more preferably lower arylene.
- the arylene group is optionally substituted with one or more "alkyl group substituents. " There may be optionally inserted around the arylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
- Exemplary arylene groups include 1 ,2-, 1 ,3- and 1 ,4- phenylene.
- the term "lower arylene” refers to arylene groups having 5 or 6 carbons. Preferred arylene groups are lower arylene.
- heteroarylene refers to a bivalent monocyclic or multicyclic ring system, preferably of about 5 to about 1 5 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
- the heteroarylene group may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
- arylalkylidene refers to an alkylidene group in which either R' or R" is and aryl group.
- amido refers to a bivalent group, either -C(O)NH- or -HNC(O)-.
- Thioamido refers to a bivalent group, either -C(S)CH- or - HNC(S)-.
- Oxyamido refers to a bivalent group, either -OC(O)NH- or - HNC(O)O-.
- Thiaamido refers to a bivalent group, either -SC(O)NH- or - HNC(O)S-.
- Dithiaamido refers to a bivalent group, either -SC(S)NH- or -HNC(S)S-.
- Ureido refers to the bivalent group -HNCONH-.
- Thioureido refers to the bivalent group -HNCSNH-.
- amino acid refers to ⁇ -amino acids which are racemic, or of either the D- or L-configuration.
- any particular group such as phenyl or pyridyl
- Preferred substituents where not specified are halo, halo lower alkyl, and lower alkyl.
- preparative or therapeutically useful amounts are amounts sufficient for use of the resulting products as therapeutics in which mgs to gms are administered. Typically, such amounts are at least 0.1 gms per synthesis, more preferably about gms to kilograms.
- Use of the LPCs provided herein results in improved yields and thereby permits development of preparative scale syntheses.
- LPCs Liquid phase carriers for use in solution phase synthesis of biopolymers are provided.
- the LPCs provided herein are multifunctional LPCs that preferably possess three or more functional moieties for biopolymer synthesis.
- the biopolymers prepared on a given LPC may be of differing structure, but more typically are identical in structure.
- the LPCs have the formula Sp(X 1 ) n , wherein Sp is a polyvalent group that has more than two points of attachment, n corresponds to the number of points of attachment in Sp and X 1 is a reactive group that is compatible from the point of view of biopolymer chemistry. Sp is preferably a symmetrical group such that all X 1 groups are equivalent. It will be appreciated by those of skill in the art that such symmetry allows for uniformity in the synthesis of n biopolymers using these LPCs.
- multifunctional liquid phase carriers in which the groups X 1 can form a silyl ether, trityl ether or ester bond with the 3'- or 5'-hydroxyl groups of a nucleoside, nucleotide or oligonucleotide are used.
- the reactive group X 1 which are customary in nucleotide chemistry are described in Liebigs Ann. Chem. 1978, 839- 853 and in Nucleic Acids Research, Symposium Series No. 7, 1980, 39- 59. Typical examples are, inter alia, the following:
- Acid halides in particular acid chlorides and acid bromides; 2.
- Carboxylic acid groups which can react with 5'-OH groups, for example in the presence of condensing agents; they can also be converted into activated trityl chloride derivatives according to the following equation:
- carrier molecules of the general formula Sp(OH) n are used directly in the provided methods if they are to react with reactive carboxyl groups.
- they are usually employed in the form of activated compounds, which can be obtained, for example, in the following manner: a) by reaction with dicarboxylic acid anhydrides, for example succinic or adipic anhydride, according to the equation -Sp-OH + (OC- R-CO) 2 O - - >
- the carrier molecule Sp(X 1 ) n and its derivatives obtained in the course of biopolymer synthesis are soluble in the particular reaction medium used and can be chromatographed .
- the LPCs have formulae (I) :
- A is carbon; E is nitrogen; R 1 and R 3 are each independently hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylalkyl; p is 0 or 1 ; Z is a divalent hydrocarbon as described above, and is preferably any combination of 0-1 2, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2 units in which each unit is preferably selected from 1 ,4-phenylene and methylene, which units may be combined in any order; t is 0 or 1 ; X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sul
- Patent No. 5, 1 98,540 is preferably halide, OH, SH, NH 2 , COR 5 or COOR 4 ; n is 3 or 4; R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; R 5 is halide, heteroaryl or pseudohalide; Y 1 is CH 2 , NH, S or O; and Y 2 is selected from CH and N.
- any of the groups R ⁇ R 3 , R 4 , R 5 , X 1 , Y ⁇ Y 2 and Z may be unsubstituted or substituted with one or more substituents each independently selected from Q, which, as defined herein, is halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylidene, arylalkylidene, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, alkoxyca- rbonylalkyl, aryloxycarbonyl, aryloxy
- Q is preferably selected from groups that are electron donating .
- groups include alkyl, alkoxy, dialkylamino, diarylamino, alkylarylamino, alkylthio and arylthio, with alkoxy, preferably methoxy, being preferred .
- the LPCs are chosen as above, with the proviso that Z contains more than two methylene groups. In other embodiments, the LPCs are chosen with the proviso that Z is not 1 ,4- arylene. In other preferred embodiments, the LPCs are chosen with the proviso that Z contains more than two methylene groups and is not 1 ,4- arylene. In other more preferred embodiments, the LPCs are chosen with the proviso that Z contains at least one, preferably at least two, more preferably at least three or more phenylene or alkylene units. 1 . Acyclic Multifunctional LPCs
- the LPCs are acyclic, multifunctional compounds in which all of the functional groups are equivalent. These LPCs generally have a center of symmetry at a single atom.
- the LPCs have formulae (Ma) or (lib) :
- R 1 is hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylalkyl; p and t are each indpendently 0 or 1 ; Z is any combination of phenylene and methylene units, selected from 0- 1 2, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order; X 1 is any reactive group that is used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono
- the LPCs are of formula (Ha) wherein p is 0 or 1 , preferably 0, and n is 3 or 4, preferably 4.
- the LPCs of formula (lla) have the formula C-(Z- X 1 ) 4 , where Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or
- Patent No. 5, 1 98,540 is preferably OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs of formula (I Is) have the formula C-(Z-X 1 ) 4 , where Z is C 1 -12 , preferably C 2 . 12 , more preferably C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 12 or C 8 12 alkylene and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs of formula (Ma) have the formula C-(Z-X 1 ) 4 , where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is OH, SH or NH 2 , preferably OH.
- the LPCs of formula (Ma) have the formula C-(Z-X 1 ) 4 , where Z is methylene or ethylene, preferably methylene, and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is OH, SH or NH 2 , preferably OH.
- the presently most preferred LPC of formula (Ma) is pentaerythritol.
- the LPCs are of formula (Mb) where Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2- 1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (halide or pseudohalide) and phosphorous
- Patent No. 5, 1 98,540 is preferably OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (Mb), where Z is C,. ⁇ , preferably C 2 . 12 , more preferably C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (Mb), where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is OH, SH or NH 2 , preferably NH 2 .
- the LPCs have formula (Mb), where Z is methylene or ethylene, preferably ethylene, and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is OH, SH or NH 2 , preferably NH 2 .
- the presently most preferred LPC of formula (Mb) is tri(2-aminoethyl)amine.
- the LPCs of formulae (I Is) and (Mb) are chosen as above, with the proviso that Z contains more than two, preferably more than three, more preferably more than four methylene groups. In other embodiments, the LPCs are chosen with the proviso that Z is not 1 ,4-arylene. In other preferred embodiments, the LPCs are chosen with the proviso that Z contains more than two methylene groups and is not 1 ,4-arylene. In other more preferred embodiments, the LPCs are chosen with the proviso that Z contains at least one, preferably at least two, more preferably at least three or more phenylene or alkylene units.
- the LPCs are cyclic, multifunctional compounds in which all of the functional groups are equivalent. These LPCs generally have a center of symmetry at the center of a core cycloalkyl, heterocyclyl, aryl or heteroaryl ring. a. Cyclic Multifunctional LPCs of Formulae (He) or (lid) In more preferred embodiments, the LPCs have formulae (lie) or (lid) :
- Z is any combination alkylene and phenylene units, of 0-1 2 units, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2 units, preferably selected from 1 ,4-phenylene and methylene, which units may be combined in any order;
- t is 0 or 1 ;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (
- Patent No. 5, 1 98,540 is preferably halide, OH, SH, NH 2 , COR 5 or COOR 4 ; n is 3 or 4; R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs are of formula (lie) where Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2- 1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order; t is 0 or 1 ;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (halide or
- Patent No. 5, 1 98,540 is preferably halide, OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (lie), where Z is C,. ⁇ 2 , preferably C 2 . 12 , more preferably C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (lie), where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide. In more preferred embodiments, X 1 is COR 5 or COOR 4 , preferably COOR 4 .
- the LPCs have formula (lie), where Z contains 0, 1 or 2 methylene units, preferably 0; t is preferably 0; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is COR 5 or COOR 4 , preferably COOR 4 .
- the LPCs are of formula (lid) where Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2- 1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order; t is 0 or 1 ;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (halide or
- Patent No. 5, 1 98,540 is preferably halide, OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (lid), where Z is C-,. 12/ preferably C 2 . 12 , more preferably C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from halide, preferably chloride, OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (lid), where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 .
- X 1 is selected from halide, preferably chloride, OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is halide or COR 5 or COOR 4 , preferably chloride or COOR 4 .
- the LPCs have formula (lid), where Z contains 0, 1 or 2 methylene groups, preferably 0 (i.e., Z is absent); t is preferably 0; and X 1 is selected from halide, preferalby chloride, OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl, aryl or pseudohalide.
- X 1 is halide, COR 5 or COOR 4 , preferably chloride or COOR 4 .
- the presently most preferred LPCs of formula (lid) are 1 ,3,5- benzenetricarboxylic acid and 1 ,3,5-tri(chloromethyl)benzene.
- the LPCs of formulae (lie) and (lid) are chosen as above, with the proviso that Z contains more than two methylene groups. In other embodiments, the LPCs are chosen with the proviso that Z is not 1 ,4-arylene. In other preferred embodiments, the LPCs are chosen with the proviso that Z contains more than two methylene groups and is not 1 ,4-arylene. In other more preferred embodiments, the LPCs are chosen with the proviso that Z contains at least one, preferably at least two, more preferably three or more phenylene or methylene units. b. Cyclic Multifunctional LPCs of Formulae (le) and (If)
- the LPCs have formulae (le) or (If) :
- Z is any combination of alklyene or arylene units as described above, of 0-1 2 units, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2 units preferably selected from 1 ,4-phenylene and methylene, which units may be combined in any order;
- t is 0 or 1 ;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or ary
- the LPCs are of formula (le) where Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2- 1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order; t is 0 or 1 ;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (halide or
- Patent No. 5, 1 98,540 is preferably OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (le), where Z is C ⁇ . 12 , preferably C 2 . 12 , more preferably C 3 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 12 or C 8 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (le), where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide. In more preferred embodiments, X 1 is COR 5 or COOR 4 , preferably COOR 4 .
- the LPCs have formula (le), where Z contains 0, 1 or 2 methylene units, preferably 0; t is preferably 0; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is COR 5 or COOR 4 , preferably COOR 4 , most preferably COOH.
- the LPCs are of formula (If) where
- Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order;
- t is 0 or 1 ;
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (halide or pseudohalide) and phosphorous derivatives such as
- Patent No. 5, 1 98,540 is preferably OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (If), where Z is C ⁇ 2> preferably C 2 . 12 , more preferably C 3 . 12 , particularly C 4 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have formula (If), where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- X 1 is COR 5 or COOR 4 , preferably COOR 4 .
- the LPCs have formula (If), where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide,
- Z contains 0, 1 or 2 methylene groups, preferably 0 (i.e., Z is absent); t is preferably 0; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl, aryl or pseudohalide.
- X 1 is COR 5 or COOR 4 , preferably COOR 4 , most preferably COOH.
- the LPCs of formulae (le) and (If) are chosen as above, with the proviso that Z contains more than two methylene groups. In other embodiments, the LPCs are chosen with the proviso that Z is not 1 ,4-arylene. In other preferred embodiments, the LPCs are chosen with the proviso that Z contains more than two methylene groups and is not 1,4-arylene. In other more preferred embodiments, the LPCs are chosen with the proviso that Z contains at least one, preferably at least two, more preferably three or more phenylene or methylene units.
- the LPCs are composed of two of the groups of formula (I).
- the LPCs of this embodiment are symmetrical compounds such that all of the X 1 groups are equivalent.
- the LPCs have formulae:
- Patent No. 5, 1 98,540 is preferably OH, SH, NH 2 , COR 5 or COOR 4 ; n is 3 or 4; R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; R 5 is halide, heteroaryl or pseudohalide; Y 1 is CH 2 , NH, S or O; and Y 2 is selected from CH and N.
- Y 1 is CH 2 and Y 2 is CH.
- Any of the groups R ⁇ R 3 , R 4 , R 5 , X 1 , Y ⁇ Y 2 and Z may be unsubstituted or substituted with one or more substituents each independently selected from Q.
- the LPCs are of the above formulae where Z is any combination of 0-1 2, preferably 1 -1 2, more preferably 2-1 2, particularly 3-1 2, more particularly 4-1 2, most preferably 6-1 2, 7-1 2 or 8-1 2, units selected from 1 ,4-phenylene and methylene, which units may be combined in any order; t is 0 or 1 ; R 20 is C ⁇ ., 2 , preferably ⁇ .
- X 1 is any reactive group which can be used in biopolymer synthesis, including, but not limited to, acyl halides and acyl pseudohalides, particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride derivatives thereof, activated esters, OH, SH and other sulfur derivatives such as sulfoxides, sulfones and sulfonates, NH 2 and mono-substituted amino groups, halides, pseudohalides, trityl derivatives, silyl derivatives such as Si(alkyl or aryl) 2 (halide or pseudohalide) and phosphorous derivatives such as phosphines, phosphine oxides, phosphonates and phosphates (see, e.g., U.S.
- acyl halides and acyl pseudohalides particularly acyl chlorides and acyl bromides, carboxylic acid groups and activated trityl chloride
- Patent No. 5, 1 98,540 is preferably halide, OH, SH, NH 2 , COR 5 or COOR 4 ;
- R 4 is selected from hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl; and
- R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have the above formulae, where Z is C-,. 12 , preferably C 2 . 12 , more preferably C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 . 12 or C 8 . 12 alkylene; t is 1 ; R 20 is lower alkylene; and X 1 is selected from halide, OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide.
- the LPCs have the above formulae, where Z is C 3 . 12 , particularly C 4 . 12 , most preferably C 6 . 12 , C 7 12 or C 8 . 12 alkylene; t is 1 ; R 20 is lower alkylene; and X 1 is selected from OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl or pseudohalide. In more preferred embodiments, X 1 is COR 5 or COOR 4 , preferably COOR 4 .
- the LPCs have formula (lid), where Z contains 0, 1 or 2 methylene groups, preferably 0 (i.e., Z is absent) or 1 ; t is 0 or 1 ; R 20 is lower alkylene; and X 1 is selected from halide, preferably chloride, OH, SH, NH 2 , COR 5 and COOR 4 , where R 4 is hydrogen, alkyl, aryl or aralkyl and R 5 is halide, heteroaryl, aryl or pseudohalide.
- X 1 is halide, COR 5 or COOR 4 , preferably chloride or COOR 4 , most preferably chloride or COOH.
- Most preferred compounds of the above formulae include:
- LPCs for Oligonucleotide Synthesis LPCs for use in oligonucleotide synthesis will preferably possess a carboxylic acid group or a derivative thereof at the terminus of X 1 . This type of functionality may be used to form an ester linkage to, for example, the 3'-OH group of a first nucleotide or nucleoside monomer.
- X 1 is OH or SH
- the modification involves reaction with, for example, acrylonitrile, followed by alcoholysis of the nitrile and reaction of the resulting ester with an ⁇ , ⁇ -diamine, to form an amino derivative, for example, of the general formula Sp(O,S-(CH 2 ) 2 -C(O)-NH-(CH 2 ) x -NH 2 ) n where x is 0-6, preferably 1 -4, more preferably 2.
- the presently most preferred LPC of this formula is tetrakis(8-amino-6-aza-2-oxa-5- oxooctyDmethane.
- the amine may then be further derivatized by reaction with, for example, an ⁇ , ;-alkanedioic acid anhydride to provide a compound of formula Sp(O,S-(CH 2 ) 2 -C(O)-NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x - COOH) n , where x is 0-6, preferably 1 -4, more preferably 2.
- the presently most preferred LPC of this formula is tetrakisd 1 -carboxy-6,9- diaza-5, 1 0-dioxo-2-oxaundecyl)methane.
- the LPC may be derivatized by reaction with, for example, an ⁇ , ⁇ -alkanedioic acid anhydride to provide a compound of formula Sp(NH-C(O)-(CH 2 ) x -COOH) n , where x is 0-6, preferably 1 -4, more preferably 2.
- the presently most preferred LPC of this formula is tris(3-aza-6-carboxy-4-oxohexyl)amine.
- the LPC may be derivatized by reaction with, for example, an ⁇ ,to/-alkylenediamine, to form an amino derivative, for example, of the general formula Sp(C(O)- NH-(CH 2 ) x -NH 2 ) n where x is 0-6, preferably 1 -4, more preferably 2.
- the presently most preferred LPC of this formula is 1 ,3,5- benzenetricarboxylic acid tris-N-(2-aminoethyl)amide.
- the amine may then be further derivatized by reaction with, for example, an a, ⁇ - alkanedioic acid anhydride to provide a compound of formula Sp(C(O)- NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n , where x is 0-6, preferably 1 -4, more preferably 2.
- LPC of this formula is 1 ,3,5- benzenetricarboxylic acid tris-N-(3-aza-6-carboxy-4-oxohexyl)amide.
- the carboxyl derivatives of formulae Sp(O,S-(CH 2 ) 2 -C(O)-NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n , Sp(NH- C(O)-(CH 2 ) x -COOH) n and Sp(C(O)-NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n may be further derivatized by reaction with a first monomer unit for synthesis of a biopolymer.
- a first monomer unit in the synthesis of oligonucleotides may be 5'-O-dimethoxytrityl-2'- deoxythymidine or 2'-deoxythymidine.
- Preferred LPCs of this embodiment include tetrakis ⁇ 6,9-diaza-1 3-[5'-O-(4,4'- dimethoxytriphenylmethyl)-2'-deoxythymidine-3'-O-yl]-2-oxa-5, 1 0, 1 3- trioxotridecyl ⁇ methane ((DMT-dT) 4 -PE-LPC), 1 ,3,5-tris ⁇ 2,5-diaza-9-[5'-O- (4,4'-dimethoxytriphenyl-methyl)-2'-deoxythymidine-3'-O-yl]-1 ,6,9- trioxononyl ⁇ -benzene ((DMT-dT) 3 -Aryl-LPC),
- LPCs for Peptide Synthesis will preferably possess a haloalkyl, hydroxyl, thio or carboxyl group at the terminus of X 1 .
- the hydroxyl or thio groups may be used to form ester or thioester linkages to the carboxyl group of a first amino acid monomer.
- Preferred haloalkyl groups are benzylic halides that can form benzylic esters with the carboxyl terminus of a first monomer.
- the modification involves reaction with, for example, acrylonitrile, followed by alcoholysis of the nitrile and reaction of the resulting ester with an ⁇ , ⁇ »-diol, dithiol or oxy- thiol, to form a hydroxy or thio derivative, for example, of the general formula Sp(O,S-(CH 2 ) 2 -C(O)-O,S-(CH 2 ) x -OH,SH) n where x is 0-6, preferably 1 -4, more preferably 2.
- the LPC may be derivatized by reaction with, for example, an ⁇ ,w-alkanedioic acid anhydride to provide a compound of formula Sp(NH-C(O)-(CH 2 ) x -COOH) n , where x is 0-6, preferably 1 -4, more preferably 2.
- Reaction of the resulting acid with an dithiol or oxy-thiol forms a hydroxy or thio derivative, for example, of the general formula Sp(NH-C(O)-(CH 2 ) x -CO-O,S-(CH 2 ) x - OH,SH) n , where x is 0-6, preferably 1 -4, more preferably 2.
- the LPC may be derivatized by reaction with, for example, an a, ⁇ -d o ⁇ , dithiol or oxy-thiol, to form a hydroxy or thio derivative, for example, of the general formula Sp(C(O)-O,S-(CH 2 ) x -OH,SH) n , where x is 0-6, preferably 1 -4, more preferably 2.
- the hydroxy or thio derivatives of formulae Sp(O,S-(CH 2 ) 2 -C(O)-O,S-(CH 2 ) x -OH,SH) n , Sp(NH- C(O)-(CH 2 ) x -CO-O,S-(CH 2 ) x -OH,SH) n or Sp(C(O)-O,S-(CH 2 ) x -OH,SH) n may be further derivatized by reaction with a first monomer unit for synthesis of a biopolymer.
- LPCs for Peptide Nucleic Acid (PNA) Synthesis LPCs for use in PNA synthesis will preferably possess a haloalkyl, hydroxyl, thio or carboxyl group at the terminus of X 1 .
- the hydroxyl or thio groups may be used to form ester or thioester linkages to the carboxyl group of a first PNA monomer.
- Preferred haloalkyl groups are benzylic halides that can form benzylic esters with the carboxyl terminus of a first monomer.
- the carboxyl group of X 1 may be used to form an amide linkage to the amino group of a first PNA monomer. LPCs possessing these groups at the terminus of X 1 are described above. d. LPCs for Oligosaccharide Synthesis
- LPCs for use in oligosaccharide synthesis will preferably possess a hydroxyl, thio or carboxyl group at the terminus of X 1 .
- the hydroxyl or thio groups may be used to form glycosyl linkages to the a first saccharide monomer.
- the carboxyl group may be used to form an ester linkage to a hydroxyl (e.g. , the 5- or 6-hydroxyl) group of a first saccharide monomer.
- LPCs possessing these groups at the terminus of X 1 are described above. e. LPCs Coupled to Linkers Alternatively, or in conjunction with the modifications described above, the LPC may be coupled with any linker known to those of skill in the art for use in biopolymer synthesis.
- linkers have traditionally been used in solid phase synthesis of biopolymers.
- the linkers include photocleavable, traceless, safety-catch, "cleanable” or other linkers well known to those of skill in the art.
- the linkers include, but are not limited to, trityl ethers, imines, hydrazones, oxime esters and ethers, hydroxymethylphenylacteates, hydroxymethylphenoxyacetates, 2- nitrophenylpropionates, hydroxyacetates, acylsulfonamides, acyls, nitrobenzyls, nitrobenzoyls, phenacyls, benzyl ethers and amines, tertiary ethers and amines, 9-fluorenylmethyl ethers, silanes, oxazolidinones, Pro- Lys, hydroazides, thiols, acrylates, dithianes, /?-benzoylpropionate
- liquid phase carriers may be prepared by the methods described in detail herein, by minor modification of the methods or by other methods known to those of skill in the art. Certain of the LPCs described herein are commercially available from, for example, Aldrich Chemical Co., Milwaukee, Wl.
- LPCs of formula (la) is exemplified by the syntheses of tetrakis ⁇ 6,9-diaza-13-[5'-O-(4,4'-dimethoxytriphenylmethyl)-2'- deoxythymidine-3'-O-yl]-2-oxa-5,10,13-trioxotridecyl ⁇ methane ((DMT- dT) 4 -PE-LPC) and tetrakis[13-(2'-deoxythymidin-3'-O-yl)-6,9-diaza-2-oxa- 5,10,13-trioxotridecyl]-methane (dT 4 -PE-LPC), shown below (see also, Examples 1 and 3).
- Other LPCs of formula (la) may be prepared by minor modification of this method.
- the four primary amino functions were then acylated by the reactive p-nitrophenylester of 5'-O-dimethoxytrityl-deoxythymidine- 3'-O-succinate in pyridine, leading to the highly symmetrical compound tetrakis- ⁇ 6,9-diaza-1 3-[5'-O-(4,4'-dimethoxytriphenylmethyl)-2'- deoxythymidine-3'-O-yl]-2-oxa-5, 1 0, 1 3-trioxotridecyl ⁇ methane ((DMT- dT) 4 -PE-LPC) .
- cationic ion exchanger resins in their protonated form may be used for combined dedimethoxytritylation and purification by stepwise variation of the polarity of the solvent system (solid phase extraction) .
- Nonpolar solvents diichloromethane/ethanol mixtures
- the 5'-O-deprotected LPC is eluted next with polar solvent systems such as ethanol or THF/ethanol mixtures.
- the yield of dT 4 -PE-LPC was 93%.
- purification may be performed with different types of chromatography media: a) by solid phase extraction using LiChroprep ® DIOL, or b) by GPC using Sephadex LH20 and additional extraction of remaining dimethoxytritylcarbinol with diethyl ether, or c) neutralization with n-trioctylamine, purification by reversed phase chromatography using NUCLEOPREP ® 300-30 C1 8 (eluant: acetonitrile/THF/water 56: 1 4:30 v/v), or d) by reversed phase chromatography using NUCLEOPREP ® 300-30 C1 8 in combination with gel permeation chromatography using Sephadex LH20 eluant: THF/water 70:30 v/v.
- LPCs of formula (lb) are exemplified by the synthesis of tris- ⁇ 3-aza-4,7-dioxo-7-[5'-O-(4,4'-dimethoxytriphenylmethyl)-2'- deoxythymidine-3'-O-yl]-heptyl ⁇ -amine ((DMT-dT) 3 -Amine-LPC) and tris- [3-aza-7-(2'-deoxythymidine-3'-O-yl)-4,7-dioxoheptyl]-amine (dT 3 -Amine- LPC), shown below (see also, Examples 5 and 6) .
- Other LPCs of formula (lb) may be prepared by minor modification of this method.
- This LPC may be prepared by minor modification of the method described above for synthesis of LPCs of formula (la). Synthesis starting with tris(2-aminoethyl)amine provided (DMT-dT) 3 -Amine-LPC in good yield (74%). Dedimethoxytritylation under conditions described for LPCs of formula (la) gave dT 3 -Amine-LPC. 3. Preparation of LPCs of Formulae (Ic) and (Id)
- LPCs of formulae (Ic) and (Id) are exemplified by the synthesis of 1 ,3,5-tris- ⁇ 2,5-diaza-9-[5'-O-(4,4'- dimethoxytriphenylmethyl)-2'-deoxythymidine-3'-O-yl]-1,6,9- trioxononylj-benzene ((DMT-dT) 3 -Aryl-LPC) and 1 ,3,5-tris-[9-(2'- deoxythymidin-3'-O-yl)-2,5-diaza-1 ,6,9-trioxononyl]-benzene (dT 3 -Aryl- LPC), shown below (see also, Examples 2 and 4).
- Other LPCs of formulae (Ic) and (Id) may be prepared by minor modification of this method.
- Biopolymers including, but not limited to, oligonucleotides, peptides, PNAs and oligosaccharides, may be produced by the methods. It is possible to prepare not only naturally occurring biopolymers, but also analogs of the biopolymers. Such analogs include, for example, analogs of oligonucleotides such as oligo-triesters or oligophosphonates.
- Use of the LPCs provided herein in the methods provided afford biopolymers of high purity and in significantly higher yields than methods known in the art.
- the methods involve the steps of (a) reacting an LPC of formula Sp(X 1 ) n , provided herein, with a first monomer N 1 ; (b) separating and purifying the product of step (a) to afford a compound of formula Sp(X 1 -N 1 ) n ; (c) reacting the product of step (b) with a second monomer N 2 , a dimer N 2 -N 3 or trimer N 2 -N 3 -N 4 ; (d) repeating steps (b) and (c) using the desired monomers, dimers or trimers to produce the desired LPC-bound biopolymer of formula Sp(X 1 -N 1 -N 2 -...-N ) n , where m is 3 to 100, preferably 3-50, more preferably 3-30, most preferably 3-1 5 or 3-1 0; and (e) cleaving the biopolymer from the LPC.
- m is preferably 3-30, more preferably 3-1 5. In embodiments where the biopolymer is an oligosaccharide, m is preferably 3-1 5, more preferably 3-1 0.
- the separation and purification steps may be accomplished by any method known to those of skill in the art.
- Preferred methods include gel permeation chromatography (GPC) and chromatography on reverse phase (RP) silica gel.
- GPC gel permeation chromatography
- RP reverse phase silica gel.
- combinations of GPC and RP-silica gel chromatography are used.
- Cleavage of the biopolymer from the LPC is achieved through standard means and depends on the nature of the linkage between the LPC and the biopolymer.
- ester linkages may be cleaved with a base, such as hydroxide, alkoxides and amines. In certain embodiments, ammonium hydroxide is preferred.
- the linkages may be silyl or trityl ethers, or glycosyl bonds.
- treatment with acid such as acetic acid, will cleave the biopolymer from the LPC.
- the cleavage conditions will depend on the nature of the linker. For example, light of a specific wavelength can be used to release a biopolymer from a photocleavable linker. 1 .
- Monomers (N), dimers and trimers used in preparing the biopolymer will depend on the biopolymer desired.
- monomers which are used in the methods include nucleotides, nucleosides, natural and unnatural amino acids, PNA monomers and monosaccharides. Dimers and trimers are composed of the monomers.
- the monomers are used or obtained in the course of synthesis in protected form; typical examples of such protective groups are well known to those of skill in the art (see, e.g.. Tetrahedron 1981 , 363-369; Liebigs Ann. Chem. 1978, 839-853; Nucleic Acids Research,
- synthesis of the biopolymeric chain may be carried out using any protocols known to those of skill in the art.
- protocols known to those of skill in the art.
- the phosphate triester, phosphite triester, H-phosphonate, phosphoramidite, phosphorothioate, phosphorodithioate, activated ester or amide, or alcoholate protocols may be used.
- a preferred protocol is the ⁇ - cyanoethyl N,N-dialkylamino/N-morpholinomonochloro phosphoamidite method.
- protection of the monomers may be necessary. Such protection is well known to those of skill in the art.
- Isolation of the oligonucleotide may be accomplished by a variety of methods, including the use of affinity techniques. See, e.g., N. D. Sinha et ah Tetrahedron Lett. 1983, 24, 5843-5846; H. K ⁇ ster et aL
- protocols for use herein include, but are not limited to, Fmoc protocols, reagents such as HOBt, HATU, HBTU, activated ester protocols, and phosphoryl azide protocols. See, U.S. Patent Nos. 5,726,243, 5,705,333, 4,749,742, 5,644,029, 5,637,71 9, 5,262,331 , 4,794, 1 50, 5,221 ,736 and 5,641 ,862.
- protocols for use herein include any known protocols. Such protocols are well known to those of skill in the art.
- Preferred protocols include, but are not limited to, template directed polymerization and the use of reagents such as HOBt or HBTU. See, Brown et aL Science 1994, 265, 777-780; Norton et aL Bioorg. & Med. Chem. 1995, 3, 437-445; Schmidt et aL Nucl. Acids Res. 1997, 25, 4792-4796 and U.S. Patent Nos. 5,700,922, 5,736,336, 5,71 9,262, 5,71 4,331 , 5,705,333 and 5,539,083.
- protocols for use herein include, but are not limited to, the use of glucoconjugates, glycosidases, glycosyltransferases, fucosyltransferases, glucosaminyltransferases and galactosyltransferases. See, U.S. Patent Nos. 5,246,840, 5,61 6,698, 5,374,655, 5,288,637, 5,532, 147 and 5,583,042.
- LPCs for use in the methods are any of those provided herein.
- the LPCs have the formula Sp(X 1 ) n , wherein Sp is a polyvalent group that has two or more, preferably three or more, more preferably 3-6, points of attachment, n corresponds to the number of points of attachment in Sp and X 1 is a reactive group that is compatible from the point of view of biopolymer chemistry.
- Sp is preferably a symmetrical group such that all X 1 groups are equivalent. It will be appreciated by those of skill in the art that such symmetry allows for uniformity in the synthesis of n biopolymers using these LPCs.
- the LPCs used in the methods are of formulae (I) or derivatives thereof as described above, more preferably of formulae (la), (lb) and (Id), or derivatives thereof. In more preferred embodiments, the LPCs used in the methods are of formulae (lla), (lib) or (lid), or derivatives thereof.
- the LPCs are suitable for oligonucleotide synthesis.
- Such LPCs include the carboxyl derivatives of formulae (lla), (Mb) or (lid) that have the general formulae Sp(O,S-(CH 2 ) 2 -C(O)-NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n , Sp(NH- C(O)-(CH 2 ) x -COOH) n and Sp(C(O)-NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n .
- the LPCs used in the methods are the carboxyl derivatives of formulae Sp(O,S-(CH 2 ) 2 -C(O)-NH- (CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n , Sp(NH-C(O)-(CH 2 ) x -COOH) n and Sp(C(0)- NH-(CH 2 ) x -NH-C(O)-(CH 2 ) x -COOH) n that are further derivatized by reaction with a first monomer unit for synthesis of a biopolymer, preferably an oligonucleotide.
- a first monomer unit in the synthesis of oligonucleotides may be 5'-O-dimethoxytrityl-2'- deoxythymidine or 2'-deoxythymidine.
- Preferred LPCs of this embodiment include tetrakis ⁇ 6,9-diaza-1 3-[5'-O-(4,4'- dimethoxytriphenylmethyl)-2'-deoxythymidine-3'-O-yl]-2-oxa-5, 1 0, 1 3- trioxotridecyl ⁇ methane ((DMT-dT) 4 -PE-LPC), 1 ,3,5-tris ⁇ 2,5-diaza-9-[5'-O- (4,4'-dimethoxytriphenyl-methyl)-2'-deoxythymidine-3'-O-yl]- 1 ,6,9- trioxononylj-benzene ((DMT-dT) 3 -Aryl-LPC),
- the method is used to prepare oligonucleotides. This is achieved through chain elongation in the 3' ⁇ 5' direction with monomeric, dimeric or trimeric phosphoramidites.
- the procedure represents a way to a purification oriented and effective technique for the synthesis of oligonucleotides and their analogs in preparative scale.
- the purification mainly achieved by gel permeation chromatography (GPC) on Sephadex LH20, is facilitated through the coupling of the intermediates to the LPC.
- GPC gel permeation chromatography
- the considerable difference in size between reaction products and all other reagents including the excess phosphoramidites allows separation by gel filtration.
- the condensation product as the largest component, is eluted first near the void volume.
- the LPC mediates the solubility in organic solvents during the different reaction steps and the chromatography process.
- the reaction products can be analyzed by MALDI-TOF mass spectrometry.
- MALDI-TOF mass spectrometry a direct reaction control on the step of the fully base and phosphate protected intermediates is possible.
- the scale of synthesis is mainly determined by the dimension of the Sephadex LH20 column and can be varied in a wide range, including up to grams or kilograms.
- the method involves the synthesis of the decanucleotide d(GACGGCCAGT) using monomeric phosphoramidites and an LPC based on a 1 ,3,5-benzene tricarboxylic acid derivative (see, Example 7) .
- the condensation was monitored by thin layer chromatography (TLC) .
- TLC thin layer chromatography
- the resulting mixture of product and excess reagents including several equivalents of phosphoramidite was carefully reduced to a volume up to 1 0 ml and separated on a Sephadex LH20 column with THF/methanol 80:20 (v/v) as eluent.
- the condensation product eluted near the void volume, followed by the excess phosphoramidite components and finally the solvent, pyridine.
- the fractions containing the condensation product were combined and oxidized with t-butylhydroperoxide (t-BuOOH) at 0 °C.
- the t-butylhydroperoxide (boiling point 81 °C) can easily be removed by co-evaporation with the solvents used in the oxidation (THF and methanol) .
- the solution was reduced to dryness and the residue dissolved in dichloromethane/nitromethane/methanol 80: 1 9: 1 (v/v) .
- the resulting colorless solution was mixed with trifluoroacetic acid (TFA) in the same solvent system, so that a final acid concentration not higher than 2% (by volume) was obtained.
- TFA trifluoroacetic acid
- Table 1 shows the yields, achieved in the nine cycles for condensation and oxidation (81 - 99%), dedimethoxytritylation (81 -99%) and the whole cycle (81 -99%) .
- the total yield over all cycles amount to 33% for d(5'-O-DMT-
- the average yield for dedimethoxytritylation is 87%.
- Preferred embodiments of the method avoid the use of water in chromatography.
- Other polar solvents for GPC on Sephadex LH20, such as pyridine or N,N-dimethylformamide, are favored for this purpose.
- Table 1 Yields achieved in the oligonucleotide synthesis in solution using dT 3 - Aryl-LPC for the preparation of decamer d(GACGGCCAGT)
- dimer phosphoramidites were used in the synthesis of the pentanucleotide d(GCCCT) (see, Example 8) .
- Such building blocks are advantageous in oligonucleotide synthesis in solution because the number of coupling cycles and purification steps is reduced.
- Pentaerythritol (6.84 g, 50 mmol) was dissolved in 20 ml dioxane and 2 ml water and mixed up with 40 % aqueous potassium hydroxide solution (w/v, 1 ml) . After cooling to 0 °C in an ice bath, acrylonitrile (1 6.2 g, 300 mmol) was added and the mixture stirred for 48 h at room temperature. Solvents were evaporated, the remaining oil was diluted in 100 ml dichloromethane and washed with 1 0 % aqueous sodium chloride solution (w/v, 50 ml) .
- 1,3,5-Benzenetricarboxylic acid trimethyl ester (1.04 g 4.0 mmol) was treated with ethylenediamine (66.7 ml, 1 mol), following the procedure described in Example 1.C. The reaction was complete after 24 h. After lyophilization with dioxane the product was obtained as a white solid; yield: 99 % (1,35 g, 4.0 mmol).
- 1 H-NMR 400 MHz, d 6 -DMSO) ⁇ 1.55 (br, 6 H, -NH 2 ), 2.72 (m, 6 H, -CH 2 NH 2 ), 3.32 (m, 6 H, -NHCH 2 -),
- Dimethoxytrityl-compounds were washed of with n-hexane/THF/ethanol 50:50:5 (v/v, 100 ml), THF/ethanol 95:5 (v/v, 100 ml) and THF/Ethanol 50:50 (v/v, 150 ml).
- dT 4 -PE-LPC was eluted with pure ethanol, solvent was removed and thoroughly co-evaporation with toluene gave the product as a colorless solid, yield: 93 % (263 mg, 139 //mol).
- Dimethoxytrityl-compounds were washed of with n-hexane/THF/ethanol 60:35:5 (v/v, 200 ml), THF/n-hexane/ethanol 60:35:5 (v/v, 200 ml) and THF/ethanol 95:5 (v/v, 200 ml) .
- dT 3 -Aryl-LPC was eluted with THF/ethanol 65:35 and THF/ethanol 50:50 (v/v, 300 ml each) .
- the compound d(5'-O- DMT-G ib p.A bz p.C bz p_G' b p_G lb p_C bz p_C bz p_A bz p_G lb p_T) 3 -Aryl-LPC ( 1 3 mg, 1 //mol) was treated with ammonium hydroxide (32%, 3 ml) for 20 h at 55 °C.
- ammonium hydroxide 32%, 3 ml
- the product was analyzed by ion exchange HPLC using the Pharmacia SMART system (column: Mono Q 1 .6/5, eluents were 25 mM tris-HCI, 1 mM EDTA, pH 8.0 (A) and: 25 mM Tris-HCI, 1 mM EDTA, 1 M NaCl, pH 8.0 (B), flow: 1 00 //l/min: start: 20% B, 4 min isocratic, 20 to 60% B in 26 min) and compared to d(GACCGGCAGT), achieved from solid phase synthesis on CPG with included capping step.
- Pharmacia SMART system column: Mono Q 1 .6/5, eluents were 25 mM tris-HCI, 1 mM EDTA, pH 8.0 (A) and: 25 mM Tris-HCI, 1 mM EDTA, 1 M NaCl, pH 8.0 (B), flow: 1 00 //l/min: start: 20% B, 4 min iso
- the 5'-hydroxyl component dT 3 -Aryl-LPC (Example 4) ( 1 31 mg, 100 //mol, in 5 ml pyridine) was treated with d(5'-O-DMT-C tl ⁇ C tl ) phosphoramidite (total: 1 .79 g, 1 .36 mmol, 4.5 equivalents) in the above described procedures.
- d(5'-O-DMT-C tl ⁇ C tl ) phosphoramidite (total: 1 .79 g, 1 .36 mmol, 4.5 equivalents) in the above described procedures.
- 5-(4-nitrophenyl)-tetrazole was used ( 1 .4 mmol, as suspension in acetonitrile) .
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Abstract
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Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU36643/99A AU3664399A (en) | 1998-04-27 | 1999-04-26 | Solution phase biopolymer synthesis |
EP99918819A EP1073668A2 (en) | 1998-04-27 | 1999-04-26 | Solution phase biopolymer synthesis |
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US09/067,337 | 1998-04-27 | ||
US09/067,337 US7094943B2 (en) | 1998-04-27 | 1998-04-27 | Solution phase biopolymer synthesis |
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WO1999055718A2 true WO1999055718A2 (en) | 1999-11-04 |
WO1999055718A3 WO1999055718A3 (en) | 1999-12-16 |
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PCT/US1999/008939 WO1999055718A2 (en) | 1998-04-27 | 1999-04-26 | Solution phase biopolymer synthesis |
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US (2) | US7094943B2 (en) |
EP (1) | EP1073668A2 (en) |
AU (1) | AU3664399A (en) |
WO (1) | WO1999055718A2 (en) |
Cited By (3)
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US7038103B2 (en) | 1998-04-27 | 2006-05-02 | Hubert Koster | Solution phase biopolymer synthesis |
JP2010275254A (en) * | 2009-05-29 | 2010-12-09 | Tokyo Univ Of Agriculture & Technology | Hydrophobic group-linked nucleoside, hydrophobic group-linked nucleoside solution and method of synthesizing hydrophobic group-linked oligonucleotide |
WO2013179412A1 (en) * | 2012-05-30 | 2013-12-05 | 北海道システム・サイエンス株式会社 | Oligonucleotide synthesis method using highly dispersible liquid-phase support |
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US20060264608A1 (en) * | 2001-08-03 | 2006-11-23 | Wender Paul A | Bi-directional synthesis of oligoguanidine transport agents |
JP4741458B2 (en) | 2003-01-16 | 2011-08-03 | カプロテック・バイオアナリティクス・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 analyzing capture compounds, collections thereof, and proteomes and composite compositions |
EP2394175B1 (en) * | 2009-02-09 | 2016-02-03 | caprotec bioanalytics GmbH | Devices, systems and methods for separating magnetic particles |
EP2971166B1 (en) | 2013-03-14 | 2021-05-19 | Children's Medical Center Corporation | Psap peptide for treating cd36 expressing cancers |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7038103B2 (en) | 1998-04-27 | 2006-05-02 | Hubert Koster | Solution phase biopolymer synthesis |
JP2010275254A (en) * | 2009-05-29 | 2010-12-09 | Tokyo Univ Of Agriculture & Technology | Hydrophobic group-linked nucleoside, hydrophobic group-linked nucleoside solution and method of synthesizing hydrophobic group-linked oligonucleotide |
WO2013179412A1 (en) * | 2012-05-30 | 2013-12-05 | 北海道システム・サイエンス株式会社 | Oligonucleotide synthesis method using highly dispersible liquid-phase support |
US9284344B2 (en) | 2012-05-30 | 2016-03-15 | Hokkaido System Science Co., Ltd. | Oligonucleotide synthesis method using highly dispersible liquid-phase support |
Also Published As
Publication number | Publication date |
---|---|
EP1073668A2 (en) | 2001-02-07 |
US7038103B2 (en) | 2006-05-02 |
US7094943B2 (en) | 2006-08-22 |
WO1999055718A3 (en) | 1999-12-16 |
US20020007048A1 (en) | 2002-01-17 |
US20020016451A1 (en) | 2002-02-07 |
AU3664399A (en) | 1999-11-16 |
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