CA1290481C - Graft copolymers of crosslinked polymers and polyoxyethylene, processes for their production, and their usage - Google Patents
Graft copolymers of crosslinked polymers and polyoxyethylene, processes for their production, and their usageInfo
- Publication number
- CA1290481C CA1290481C CA000498925A CA498925A CA1290481C CA 1290481 C CA1290481 C CA 1290481C CA 000498925 A CA000498925 A CA 000498925A CA 498925 A CA498925 A CA 498925A CA 1290481 C CA1290481 C CA 1290481C
- Authority
- CA
- Canada
- Prior art keywords
- graft copolymer
- meq
- reaction
- polymer
- polyoxyethylene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
Abstract
ABSTRACT OF THE DISCLOSURE
The invention relates to graft copolymers of crosslinked polymers and linear polyoxyethylene, processes for their production, and their use as a substrate for peptide or nucleotide synthesis or for the covalent fixation of peptides, active protein compounds, and enzymes for affinity chromotography, diagnostic agents, and biotechnological methods.
The invention relates to graft copolymers of crosslinked polymers and linear polyoxyethylene, processes for their production, and their use as a substrate for peptide or nucleotide synthesis or for the covalent fixation of peptides, active protein compounds, and enzymes for affinity chromotography, diagnostic agents, and biotechnological methods.
Description
``` ~2904~3~
BACKGROUND AND SUMMARY OF THE I~IENTION
. The present invention relates to graft copolymers of crosslinked polymers and linear polyoxyethylene, processes for their production, and their use.
S Graft copolymers of crosslinked, insoluble polymers and polyoxyethylene are of importance as substrates for peptide synthesis and for the immobilization of low-molecular and high-molecular ~ active agents for affinity chromatography, diagnostic agents, and biotechnological methods. Heretofore, such graft copolymers have been prepared ~rom crosslinked, chloromethylated polystyrene and shorter polyethylene glycols in accordance with the Williamson ether synthesis:
~~~~ ~vv~
+ ~ 0C112CIIZ)n-O~
Cll Cl C112- (0CH2C~12)n-0~1 . . .
.
1 2~
(c~. Makromol. Chem. Rapid Commun. 3 : 217 [1982];
~, 621 [1981]). One disadvantage of this process resides in that the polystyrene is frequently inadequately charged with polyoxyethylene. The yields drop very drastically, primarily with an increasing molecular weight of polyoxyethylene, and only relatively short oligoethylene glycol chains with molecular weights of up to 1320 could be bound to the polystyrene. Another drawback in the ether synthesis is the formation of cyclic ethers by the reaction of both terminal hydroxy groups of polyoxyethylene with the chloromethylated polystyrene whereby the terminal hydroxy groups, required for the carrier function, are once again decreased.
lS The graft copolymers produced in this way exhibit, in their usage, inadequate reactivity, a charging with polyoxyethylene that is too low, and an insufficient stability of the bond during immobilization. Therefore, linear, homogeneously soluble polymers, such as polyoxyethylene, have frequently been employed for peptide synthesis. These soluble polymers, though, can be separated only with extreme difficulty.
It is thus an object oE the present invention to 9~1~8~
provide graft copolymers exhibiting higher reactivity, higher charging, and higher stability of the bond during immobili~ation than conventional polymers, as well as a process for producing these graft copolymers, which avoids the disadvantages of the above-descr ibed prior art procec:s.
This obj ect is obtained by the graf t copolymers of the present invention, exhibiting, on a crosslinked polymer, several polyoxyethylene residues with an average molecular weight of 500 - 50,000, and having 0.02 - 15 meq free hydroxy groups per gram of copolymer. Preferably, the amount of hydroxy groups is 0.05 - 15 meq/g, most preferably 0.05 - 10 meq/g.
With the use of crosslinked polystyrenes, this range is preferably 0.02 - 2 meq/g, especially preferably 0.05 - 0.7 meq/g. When using polyvinyl - ~ alcohols as the crosslinked polymers, this range is 1 -15 meq/g, pref erably 1 - 10 meq/g.
T h e a v e r a g e m o l e c u l a r w e i g h t o f t h e polyoxyethylene chains is preferably from 800 - 10,000, especially from 900 to 6,000 with the optimum range being from 2,000 to 3,000.
The crosslinked polymer is pref erably a polyvinyl alcohol, polyhydroxystyrene, a polymer produced f rom chloromethylated polystyrene and ethylene glycol or .
~ ' .
,"'''~ ~
. ` ~
'...... ~ ''' ~
oli~oethylene glycol, or a polyacrylate or polymethacrylate functionalized by hydroxy groups. The degree of crosslinking of these polymers herein is generally 0.05 - 10~6, preferably 0.1 - 8%, especially S preferably 0.2 - 5%. The most suitable degree of crosslinking is 1 - 2%, especially when using polystyrenes crosslinked with divinylbenzene.
Binding of the polyoxyethylene chains to the crosslinked polymers takes place preferably by way of - lû hydroxy or amino groups of the cr osslinked polymer .
These can be present per se in the polymer, such as, for example, in the polyvinyl alcohol and polyhydroxystyrene, or they can be introduced subsequently by functionalizing. The amount of hydroxy 15 groups (degree of functionalization) is generally in a range from 0.02 to 25 meq/g of crosslinked polymer, preferably 0.05 - 15 meq/g. Most suitably, a polystyrene is utilized having a degr ee of functionalization of 0.05 - 0.7 meq/g, or a polyvinyl 20 alcohol is utilized with a degree of functionalization of 1 - 15 meq/g.
The process for preparing the graft copolymers of the present invention is characterized by reacting crosslinked polymers with ethylene oxide.
By suitably choosing the reaction temperature, . . .
the reaction period, the monomer concentration, and the solvent, the reaction can be controlled so that any desired average molecular weight can be obtained for the polyoxyethylene chain. Preferably, the reaction temperature is in the range from 20 to 100 C, especially preferably in a range from 60 to 80 C.
The reaction time is preferably 30 minutes to 150 hours.
The reaction medium employed i~ one of the aprotic, organic solvents inert to the reaction; ethers are especially suitable, such as, ~or example, dioxane, tetrahydrofuran, or diglycol ethers, as well as toluene, benzene, xylene, dimethylformamide~ or dimethyl sulfoxide.
The reaction is optionally conducted in the presence of alkaline or acidic catalysts. Suitable alkaline catalysts are, for example, alkali metals, such as lithium, sodium, or potassium; metallic hydrides, such as sodium hydride, calcium hydride;
alkali metal amides, such as sodium amide; Grignard compounds or alcoholates. Preferably, potassium is employed. Suitable acidic catalysts are, for example, hydrogen chloride, sulfuric acid, or p-toluenesulfonic acid.
Advantageously, in a first stage, oligoethylene ... ': ': :
' ' ' -'', ' : ' glycol chains of the formula H-(OCH2CH2)n-OH, wherein n stands for 2-20, are bound to the crosslinked polymer.
This reaction is carried out under conditions customary ~or etherification or Williamson synthesis. An aqueous sodium hydroxide solution can also serve as the base f~r the Williamson synthesis.
In a second stage, the oligoethylene chain is then extended with ethylene oxide. This two-stage process is suitable, in particular, for the production of polystyrene-polyoxyethylene graft copolymers.
The graft copolymers of the present invention can be utilized as substrates for peptide synthesis, nucleotide synthesis, afEinity chromatography, for the immobilization of enzymes in biotechnological reactions and as active agents in diagnostic media.
On account of the hydroxy groups present in the graft copolymers of the present invention, peptides can be built up stepwise by means of conventional methods of peptide synthesis (Peptides, vol. 2, Academic Press, 1979). Surprisingly, such immobilized polyoxyethylenes with an average molecular weight of l,000 - 2,000 show, in peptide coupling reactions, a higher reaction velocity than non-immobilized polyoxyethylenes in solution. This high reactivlty thus also permits immobilization of proteins, enzymes, and other active ~ 1 ~ 9~4 compounds.
The degree of polymerization and/or the average molecular weight of the grafted copolymers can be affected by the parameters of temperature, time, and monomer concentration. For example, it has been found in connection with PSPOE (polystyrene-polyoxyethylene) that high degrees of polymerization cannot be attained at low reaction temperatures (56-58 C), in spite of high amounts of monomer added and a long reaction period. An average molecular weight is obtained for polyoxyethylene (POE) grafted onto a modified polystyrene substrate of 2,000 (PSPOE-2000).
Reaction temperatures that are too high, or polymerization velocities that are too high, lead to destruction of the polystyrene substrate matrix. A
reaction temperature of 70-73 C proved to be favorable. Different degrees of polymerization can be obtained by varying the amounts of monomer added and the reaction time. The curve for PSPOE-5600 in Figure l illustrates the course of the reaction wi~h relatively low amounts of monomer added, while the curve for PSPOE-6900 illustrates the course of the reaction at higher amounts of added monomer. Data for the graft copolymers PSPOE are listed in Table l, with the course of the reaction being shown in Figure l.
, .~ . .
~. . ..
' .. . . .
. '' :. -. . .
, ~.29~4~31 DETAIL~ RI~TION OF THE INVENTXQN
The following Examples are given as being exemplary of the present invention and accordingly should not be considered as limiting the scope of the present invention.
Examp~e 1 Production of a Substrat~ from Crosslinked Chloro-methylated Polystyrene (PS) and Tetraethylene Glycol ~PSTEG) A solution of 350 ml of dioxane, 23 ml (133 mmol) of tetraethylene glycol (TEG), and 13.5 ml of a 20~
aqueous NaOH solution is combined with 10 g (12.5 meq Cl) of chloromethylated polystyrene crosslinked with 1%
,.-,.
divinylbenzene (DVB). The mixture is heated to boiling. The reaction is finished after 120 hours; the substrate is suctioned off and washed 10 times with respectively 100 ml of dioxane, dioxane/H2O 1:1;
ethanol, CH2C12, dioxane, methanol, and dried over P4Olo under vacuum. The elementary analysis is as ~ollows:
C70H735.2 :
Calculated: C 84.1 H 7.4 O 8.3 Found: C 83.8 H 8.1 O 8.1 The hydroxy number was determined to be 0.99 meq/g of copolymer.
E~ample 2 Production of Polystyrene-Polyethylene Glycol L~verage S Mol~ular Weiaht_of Polyethylene GlycoL_Chain = _2 ~00 psPo~-20QO)l 9.4 g ~about 9.4 meq OH) of the PSTEG substrate obtained in accordance with Example 1 is suspended with 370 mg (9.4 mmol) of potassium in 600 ml of dry dioxane and stirred vigorously under a N2 atmosphere overnight at 60-70 C. During the procedure, the solution assumes an orange coloring. The reaction temperature is regulated to be 56-58 C and, within 53.5 hours, 267 g t6.6 mol) of ethylene oxide is introduced into the reaction solution. The reaction system is then sealed, and the reaction mixture is agitated for another 52.5 hours. Under pressure, another 37 g t0.85 mol) of ethylene oxide is introduced within 2 hours into the closed system. After a total reaction period of 320 Z0 hours, the reaction is terminated. Excess ethylene oxide is driven out with N2, the reaction solution is cooled, acidified with dilute aqueous HCl to a pH 3-4, and the product is worked up.
. .
~ ,, ."''. .' .. ...
~m~
At 60-70 C, 10 g (about 10 mmol OH) of the PSTEG
substrate is suspended under nitrogen with 500 mg (12.7 5 meq) of potassium and agitated overnight. During this step, the reaction solution assumes a yellow-orange discoloration. At 70 C, ethylene oxide is initially introduced into the reaction solution within 15 hours in an amount of 25 g (0.57 mol). During this step, the 10 reaction temperature increases to 72-73 C. In the subsequent 2.5 hours, 15 g (0.34 mol), then within 5 hours, 71 9 (1.6 mol) of ethylene oxide are introduced into the reaction mixture. The reaction system is then sealed, and 14 g of ethylene oxide is forced under 15 pressure into the gas space of the reaction system within 30 minutes. The mixture is agitated for another 14 hours, the temperature of the reaction solution dropping to 68 C. A further addition of ethylene oxide (86 g in 5 hours) leads initially to a 20 temperature increase to 70 C, but thereafter the reaction temperature drops up to termination of the reaction to 65 C after a total reaction period of 30.5 hours. Excess ethylene oxide is driven out with N2, and the reaction solution is cooled off, acidified to : -: .... ~ ... . ...
~ ~9~48~
pH 3-4 with dilute aqueous HCl, and worked up.
Example 4 Pre~arat~on of Polvstvrene-Polv~xyethylene Lp~poE-6~Qo) Under N2, 10 g (about 10 mmol O~) of PSTEG
substrate is suspended with 500 mg (12.7 mmol) of potassium in 6,000 ml of dioxane and stirred overnight at 65-70 C. The reation solution assumes a slightly - yellow coloring. The temperature of the reaction mixture is initially 70 C. Then, within 12 hours, 185 g (4.2 mol) o ethylene oxide is introduced into the reaction solution, adding 42 g t0.95 mol) in the first two hours. During this step the reaction temperature rises to 73 C. The reactor is sealed, and the mixture is agitated for another 12.5 hours, the temperature - 15 deopping to 68 C up to the end of the reaction period.
Excess ethylene oxide is driven out with N2, and the reaction mixture is cooled, acidified to a pH 3-4 with dilute a~ueous CHl, and worked up.
WQrkina llp of the PSPOE Co~olymers q~he polymer is separated from the reaction solution through a porous plate (G3-mesh size) and washed respectively 8 times with dioxane, dioxane/H2o " -.. :
. . .
.
, ': ' .
l:l, water, ethanol, dioxane, and methylene chloride.
After the last washing step, the mixture is combined with methylene chloride/diethyl ether l:l and suctioned o~f. ~uring this step, the copolymer shrinks somewhat~
S The product is washed once with ether, once with methylene chloride, and then three times with ether.
In order to dissolve out any still present, soluble PO~, the product is extracted for 24 hours with THF in a Soxhlet apparatus, then washed three times with ether, and dried over P4Olo under vacuum.
~xam~le 5 S~nthe~i~_Q~ gr~in~L_~ecape~de of the Insulin B
fi~n~ Q~ PSPOE Graft So~lymers 44 g of the PSPOE polymer produced according to 15 ~xample 4 is esterified with BOC-glycine according to the methods of llquid-phase synthesis (The Peptides, 2 : 285 et eq~, Academic Press, New York 1979) so that 3~65 mmol o BOC-glycine is bound. Then the decapeptide is built up stepwise with the amino acid 20 derivativeS listed in the table according to the methods of substrate-bound peptide synthesis.
A suspension in DMF is prepared ~rom 6 g of the decapeptide polymer obtairled according to the above . ., 904~
directions, and irradiated under N2 with a mercury vapor lamp at 330 nm and 25 C for 22 hours.
Thereafter, the product is filtered of from the polymer, and the solution of the peptide is evaporated 5 to dryness. The blocked decapeptide in solution is purified on a silica gel column with chloroform/-methanol/ethyl acetate/glacial acetic acid (65:25:9:1) as the eluent. The peptide fractions are collected and subsequently purified with methanol as the eluent over 10 a "Sephadex"~LH 20 column, thus obtaining 157 mg of blocked peptide revealing an amino acid analysis of 1.10 Glu, 1.06 Arg, 1.01 Gly, 2.01 Phe, 1.03 Tyr, 0.96 Thr, 0.95 Pro, 1.02 Lys and 1.00 Ala. The blocking groups can be split off with HF, thus yielding the free 15 peptide.
..~, .: .
: ~ f ' .
Example 6 Immob;lizat;on o~ Bovine Serum Albumin on ~-Polystyrene - r~ uccin;mido-oxycarbonyl)-ethy~carbonyl]aminopoly-(oxyethylene~
Derivatization of the PSPOE substrate (mol.wt.pOE
= 5,100, 163 ~mol OH/g) takes place analogously to Makromol. Chem. 1~2 : 1379-1384 (1981) and, respectively, Angew. Chem. ~ : 863-874 (1975)~ The G~r k 13 : , `` 1291~)4~31 capacity of I amounts to 75.6% o the originally present capacity.
2.5 g ~37 ~mol) of bovine serum albumin is dissolved in 10 ml of water and adjusted to a pH of 8 5 wi~h 13 ml of a l-molar Na~CO3 solution. The albumin solution is centrifuged, then combined with 160 mg of substrate I and stirred for 22 hours under darkness at room temperature. After the reaction is finished, the mixture is suctioned off and the surface carefully 10 washed with water. Once no more albumin can be detected in the filtrate, washing is repeated at least 15 more times, and the product dried under vacuum over P401 o .
Charging of albumin: 0.5 ~mol/g = 33 mg of 15 albumin/g of substrate.
, - .
.
1~ .
~L29048~
,, , , , 11 ,~ , , ~ I
~ , , ,~ , 11 a) I I a) 1 1l ~ I I ~1 1 11 n5 1 1 ~t I 11 a I l ~0~ 1 . . . . . . . . . . Il ~ I I ~ 1 11 o O I CO~
h I I 1 11 I I ~ 1 11 1 ~1 0 ~ I 11 O I I Ql rl :1 1 O ~ ~ D 1~ N t~
P. I I ~ h O I ~`1 ~ 11 I O O ~ 1 11 .q ~ I I 1 11 ~ I I I m ll ~ N
'-1 1 1 1 1 0 ~5 1 1 1 ~ 11 O ~ I I I 1 11 Cl U I I I N
S: I I I O t~
I I I ~I -- O -- O 0 11 N I .R N
,Crl I I ~ I O O a~ o ~: m 1l P~ E t ~t p ~ Pt C~
"~ m I I ~ I U _ U ~ U~ ~ _ u _ c~
o c; I I ~n I o m o ~ o ~ o ~ o m o ~ o ~ o ,¢ o ~ o .¢ ll ' m-- m-- m-- m-- m-- m--m-- m-- m-- m ~ ll _ I 1 11 ~a~ I I 1 11 , U, I I g , 11 u I I a) I N N N ~ 1 N N
40'~ I O O O O O O 0 11 ,1 1 1 u~ I m m m m m m m m m m 11 o) I I 1 11 I I . I
I I o 1 11 (~ I I Z I -1 N ~ 0 I~ 00 cn O 1 1 E t I I . ~
, . ~ .
1~9048~
I o 1 11 ~ o ~ 1 11 I a~ 1 11 I
I I ~l l ~ O O ll I ~ P~ I o ~ o o ~ ~ ~ ~ ~ 11 oo o co o ~ 11 X ~ ~ ~ o u~ In ~1~ ~ 11 1l o o ~ 1 11 I In a~ l ll I I ~ I ~ o ~r o 11 .q II ~ ~ I o In O ~ o o a~ ~D ~ 11 ~ ~ o ~ o ~ 11 X I ~ Ul o U~ ~ ~ ~ ~ 11 , ~ ~ , 11 ~, , , 11 o, , , 11 ~ ~ I 1 11 o . , , 11 I , o , 11 o ~ ~ 11 I, o , 11 II ~ a) 1 11 ~,,, ~, ~ o o 11 S~ II ~ ~ I o ~ o,J a~ ~ Lr~ ~ ~ 11 V II O ~ I ~ O ~ ~1 o~,, U~ X, ~ o ~ ~ ~ ~ 1l U~ ~ , , o 11 ~, , , ~ 11 o I I I ~ ~
~ '" ~ I , I o ~ o ~ a~ , ,. 11 o I I ~ ~ ~
o ~ ~ Il O h ~ ~ ~ ~ 4~ ul 11 .1 ~ I I I ~ ~ ~ O ~ a) o I I I h ~ 1 X-- ~ O ~d ~ ~ ~ 11 I I I U ~O O O ~ ~
~ fd ~ ~I td O tn a) o rd O ~d O ~d 11 a) I I I ~ ~~ ~ ~ o ~ ~ ~ ~ u ~ ~ ¢, ~ 11 R I I I O ~1 X ~ O ~;~ ~1 11 E~ I L I . . ... _................ Il , "
~. ..-
BACKGROUND AND SUMMARY OF THE I~IENTION
. The present invention relates to graft copolymers of crosslinked polymers and linear polyoxyethylene, processes for their production, and their use.
S Graft copolymers of crosslinked, insoluble polymers and polyoxyethylene are of importance as substrates for peptide synthesis and for the immobilization of low-molecular and high-molecular ~ active agents for affinity chromatography, diagnostic agents, and biotechnological methods. Heretofore, such graft copolymers have been prepared ~rom crosslinked, chloromethylated polystyrene and shorter polyethylene glycols in accordance with the Williamson ether synthesis:
~~~~ ~vv~
+ ~ 0C112CIIZ)n-O~
Cll Cl C112- (0CH2C~12)n-0~1 . . .
.
1 2~
(c~. Makromol. Chem. Rapid Commun. 3 : 217 [1982];
~, 621 [1981]). One disadvantage of this process resides in that the polystyrene is frequently inadequately charged with polyoxyethylene. The yields drop very drastically, primarily with an increasing molecular weight of polyoxyethylene, and only relatively short oligoethylene glycol chains with molecular weights of up to 1320 could be bound to the polystyrene. Another drawback in the ether synthesis is the formation of cyclic ethers by the reaction of both terminal hydroxy groups of polyoxyethylene with the chloromethylated polystyrene whereby the terminal hydroxy groups, required for the carrier function, are once again decreased.
lS The graft copolymers produced in this way exhibit, in their usage, inadequate reactivity, a charging with polyoxyethylene that is too low, and an insufficient stability of the bond during immobilization. Therefore, linear, homogeneously soluble polymers, such as polyoxyethylene, have frequently been employed for peptide synthesis. These soluble polymers, though, can be separated only with extreme difficulty.
It is thus an object oE the present invention to 9~1~8~
provide graft copolymers exhibiting higher reactivity, higher charging, and higher stability of the bond during immobili~ation than conventional polymers, as well as a process for producing these graft copolymers, which avoids the disadvantages of the above-descr ibed prior art procec:s.
This obj ect is obtained by the graf t copolymers of the present invention, exhibiting, on a crosslinked polymer, several polyoxyethylene residues with an average molecular weight of 500 - 50,000, and having 0.02 - 15 meq free hydroxy groups per gram of copolymer. Preferably, the amount of hydroxy groups is 0.05 - 15 meq/g, most preferably 0.05 - 10 meq/g.
With the use of crosslinked polystyrenes, this range is preferably 0.02 - 2 meq/g, especially preferably 0.05 - 0.7 meq/g. When using polyvinyl - ~ alcohols as the crosslinked polymers, this range is 1 -15 meq/g, pref erably 1 - 10 meq/g.
T h e a v e r a g e m o l e c u l a r w e i g h t o f t h e polyoxyethylene chains is preferably from 800 - 10,000, especially from 900 to 6,000 with the optimum range being from 2,000 to 3,000.
The crosslinked polymer is pref erably a polyvinyl alcohol, polyhydroxystyrene, a polymer produced f rom chloromethylated polystyrene and ethylene glycol or .
~ ' .
,"'''~ ~
. ` ~
'...... ~ ''' ~
oli~oethylene glycol, or a polyacrylate or polymethacrylate functionalized by hydroxy groups. The degree of crosslinking of these polymers herein is generally 0.05 - 10~6, preferably 0.1 - 8%, especially S preferably 0.2 - 5%. The most suitable degree of crosslinking is 1 - 2%, especially when using polystyrenes crosslinked with divinylbenzene.
Binding of the polyoxyethylene chains to the crosslinked polymers takes place preferably by way of - lû hydroxy or amino groups of the cr osslinked polymer .
These can be present per se in the polymer, such as, for example, in the polyvinyl alcohol and polyhydroxystyrene, or they can be introduced subsequently by functionalizing. The amount of hydroxy 15 groups (degree of functionalization) is generally in a range from 0.02 to 25 meq/g of crosslinked polymer, preferably 0.05 - 15 meq/g. Most suitably, a polystyrene is utilized having a degr ee of functionalization of 0.05 - 0.7 meq/g, or a polyvinyl 20 alcohol is utilized with a degree of functionalization of 1 - 15 meq/g.
The process for preparing the graft copolymers of the present invention is characterized by reacting crosslinked polymers with ethylene oxide.
By suitably choosing the reaction temperature, . . .
the reaction period, the monomer concentration, and the solvent, the reaction can be controlled so that any desired average molecular weight can be obtained for the polyoxyethylene chain. Preferably, the reaction temperature is in the range from 20 to 100 C, especially preferably in a range from 60 to 80 C.
The reaction time is preferably 30 minutes to 150 hours.
The reaction medium employed i~ one of the aprotic, organic solvents inert to the reaction; ethers are especially suitable, such as, ~or example, dioxane, tetrahydrofuran, or diglycol ethers, as well as toluene, benzene, xylene, dimethylformamide~ or dimethyl sulfoxide.
The reaction is optionally conducted in the presence of alkaline or acidic catalysts. Suitable alkaline catalysts are, for example, alkali metals, such as lithium, sodium, or potassium; metallic hydrides, such as sodium hydride, calcium hydride;
alkali metal amides, such as sodium amide; Grignard compounds or alcoholates. Preferably, potassium is employed. Suitable acidic catalysts are, for example, hydrogen chloride, sulfuric acid, or p-toluenesulfonic acid.
Advantageously, in a first stage, oligoethylene ... ': ': :
' ' ' -'', ' : ' glycol chains of the formula H-(OCH2CH2)n-OH, wherein n stands for 2-20, are bound to the crosslinked polymer.
This reaction is carried out under conditions customary ~or etherification or Williamson synthesis. An aqueous sodium hydroxide solution can also serve as the base f~r the Williamson synthesis.
In a second stage, the oligoethylene chain is then extended with ethylene oxide. This two-stage process is suitable, in particular, for the production of polystyrene-polyoxyethylene graft copolymers.
The graft copolymers of the present invention can be utilized as substrates for peptide synthesis, nucleotide synthesis, afEinity chromatography, for the immobilization of enzymes in biotechnological reactions and as active agents in diagnostic media.
On account of the hydroxy groups present in the graft copolymers of the present invention, peptides can be built up stepwise by means of conventional methods of peptide synthesis (Peptides, vol. 2, Academic Press, 1979). Surprisingly, such immobilized polyoxyethylenes with an average molecular weight of l,000 - 2,000 show, in peptide coupling reactions, a higher reaction velocity than non-immobilized polyoxyethylenes in solution. This high reactivlty thus also permits immobilization of proteins, enzymes, and other active ~ 1 ~ 9~4 compounds.
The degree of polymerization and/or the average molecular weight of the grafted copolymers can be affected by the parameters of temperature, time, and monomer concentration. For example, it has been found in connection with PSPOE (polystyrene-polyoxyethylene) that high degrees of polymerization cannot be attained at low reaction temperatures (56-58 C), in spite of high amounts of monomer added and a long reaction period. An average molecular weight is obtained for polyoxyethylene (POE) grafted onto a modified polystyrene substrate of 2,000 (PSPOE-2000).
Reaction temperatures that are too high, or polymerization velocities that are too high, lead to destruction of the polystyrene substrate matrix. A
reaction temperature of 70-73 C proved to be favorable. Different degrees of polymerization can be obtained by varying the amounts of monomer added and the reaction time. The curve for PSPOE-5600 in Figure l illustrates the course of the reaction wi~h relatively low amounts of monomer added, while the curve for PSPOE-6900 illustrates the course of the reaction at higher amounts of added monomer. Data for the graft copolymers PSPOE are listed in Table l, with the course of the reaction being shown in Figure l.
, .~ . .
~. . ..
' .. . . .
. '' :. -. . .
, ~.29~4~31 DETAIL~ RI~TION OF THE INVENTXQN
The following Examples are given as being exemplary of the present invention and accordingly should not be considered as limiting the scope of the present invention.
Examp~e 1 Production of a Substrat~ from Crosslinked Chloro-methylated Polystyrene (PS) and Tetraethylene Glycol ~PSTEG) A solution of 350 ml of dioxane, 23 ml (133 mmol) of tetraethylene glycol (TEG), and 13.5 ml of a 20~
aqueous NaOH solution is combined with 10 g (12.5 meq Cl) of chloromethylated polystyrene crosslinked with 1%
,.-,.
divinylbenzene (DVB). The mixture is heated to boiling. The reaction is finished after 120 hours; the substrate is suctioned off and washed 10 times with respectively 100 ml of dioxane, dioxane/H2O 1:1;
ethanol, CH2C12, dioxane, methanol, and dried over P4Olo under vacuum. The elementary analysis is as ~ollows:
C70H735.2 :
Calculated: C 84.1 H 7.4 O 8.3 Found: C 83.8 H 8.1 O 8.1 The hydroxy number was determined to be 0.99 meq/g of copolymer.
E~ample 2 Production of Polystyrene-Polyethylene Glycol L~verage S Mol~ular Weiaht_of Polyethylene GlycoL_Chain = _2 ~00 psPo~-20QO)l 9.4 g ~about 9.4 meq OH) of the PSTEG substrate obtained in accordance with Example 1 is suspended with 370 mg (9.4 mmol) of potassium in 600 ml of dry dioxane and stirred vigorously under a N2 atmosphere overnight at 60-70 C. During the procedure, the solution assumes an orange coloring. The reaction temperature is regulated to be 56-58 C and, within 53.5 hours, 267 g t6.6 mol) of ethylene oxide is introduced into the reaction solution. The reaction system is then sealed, and the reaction mixture is agitated for another 52.5 hours. Under pressure, another 37 g t0.85 mol) of ethylene oxide is introduced within 2 hours into the closed system. After a total reaction period of 320 Z0 hours, the reaction is terminated. Excess ethylene oxide is driven out with N2, the reaction solution is cooled, acidified with dilute aqueous HCl to a pH 3-4, and the product is worked up.
. .
~ ,, ."''. .' .. ...
~m~
At 60-70 C, 10 g (about 10 mmol OH) of the PSTEG
substrate is suspended under nitrogen with 500 mg (12.7 5 meq) of potassium and agitated overnight. During this step, the reaction solution assumes a yellow-orange discoloration. At 70 C, ethylene oxide is initially introduced into the reaction solution within 15 hours in an amount of 25 g (0.57 mol). During this step, the 10 reaction temperature increases to 72-73 C. In the subsequent 2.5 hours, 15 g (0.34 mol), then within 5 hours, 71 9 (1.6 mol) of ethylene oxide are introduced into the reaction mixture. The reaction system is then sealed, and 14 g of ethylene oxide is forced under 15 pressure into the gas space of the reaction system within 30 minutes. The mixture is agitated for another 14 hours, the temperature of the reaction solution dropping to 68 C. A further addition of ethylene oxide (86 g in 5 hours) leads initially to a 20 temperature increase to 70 C, but thereafter the reaction temperature drops up to termination of the reaction to 65 C after a total reaction period of 30.5 hours. Excess ethylene oxide is driven out with N2, and the reaction solution is cooled off, acidified to : -: .... ~ ... . ...
~ ~9~48~
pH 3-4 with dilute aqueous HCl, and worked up.
Example 4 Pre~arat~on of Polvstvrene-Polv~xyethylene Lp~poE-6~Qo) Under N2, 10 g (about 10 mmol O~) of PSTEG
substrate is suspended with 500 mg (12.7 mmol) of potassium in 6,000 ml of dioxane and stirred overnight at 65-70 C. The reation solution assumes a slightly - yellow coloring. The temperature of the reaction mixture is initially 70 C. Then, within 12 hours, 185 g (4.2 mol) o ethylene oxide is introduced into the reaction solution, adding 42 g t0.95 mol) in the first two hours. During this step the reaction temperature rises to 73 C. The reactor is sealed, and the mixture is agitated for another 12.5 hours, the temperature - 15 deopping to 68 C up to the end of the reaction period.
Excess ethylene oxide is driven out with N2, and the reaction mixture is cooled, acidified to a pH 3-4 with dilute a~ueous CHl, and worked up.
WQrkina llp of the PSPOE Co~olymers q~he polymer is separated from the reaction solution through a porous plate (G3-mesh size) and washed respectively 8 times with dioxane, dioxane/H2o " -.. :
. . .
.
, ': ' .
l:l, water, ethanol, dioxane, and methylene chloride.
After the last washing step, the mixture is combined with methylene chloride/diethyl ether l:l and suctioned o~f. ~uring this step, the copolymer shrinks somewhat~
S The product is washed once with ether, once with methylene chloride, and then three times with ether.
In order to dissolve out any still present, soluble PO~, the product is extracted for 24 hours with THF in a Soxhlet apparatus, then washed three times with ether, and dried over P4Olo under vacuum.
~xam~le 5 S~nthe~i~_Q~ gr~in~L_~ecape~de of the Insulin B
fi~n~ Q~ PSPOE Graft So~lymers 44 g of the PSPOE polymer produced according to 15 ~xample 4 is esterified with BOC-glycine according to the methods of llquid-phase synthesis (The Peptides, 2 : 285 et eq~, Academic Press, New York 1979) so that 3~65 mmol o BOC-glycine is bound. Then the decapeptide is built up stepwise with the amino acid 20 derivativeS listed in the table according to the methods of substrate-bound peptide synthesis.
A suspension in DMF is prepared ~rom 6 g of the decapeptide polymer obtairled according to the above . ., 904~
directions, and irradiated under N2 with a mercury vapor lamp at 330 nm and 25 C for 22 hours.
Thereafter, the product is filtered of from the polymer, and the solution of the peptide is evaporated 5 to dryness. The blocked decapeptide in solution is purified on a silica gel column with chloroform/-methanol/ethyl acetate/glacial acetic acid (65:25:9:1) as the eluent. The peptide fractions are collected and subsequently purified with methanol as the eluent over 10 a "Sephadex"~LH 20 column, thus obtaining 157 mg of blocked peptide revealing an amino acid analysis of 1.10 Glu, 1.06 Arg, 1.01 Gly, 2.01 Phe, 1.03 Tyr, 0.96 Thr, 0.95 Pro, 1.02 Lys and 1.00 Ala. The blocking groups can be split off with HF, thus yielding the free 15 peptide.
..~, .: .
: ~ f ' .
Example 6 Immob;lizat;on o~ Bovine Serum Albumin on ~-Polystyrene - r~ uccin;mido-oxycarbonyl)-ethy~carbonyl]aminopoly-(oxyethylene~
Derivatization of the PSPOE substrate (mol.wt.pOE
= 5,100, 163 ~mol OH/g) takes place analogously to Makromol. Chem. 1~2 : 1379-1384 (1981) and, respectively, Angew. Chem. ~ : 863-874 (1975)~ The G~r k 13 : , `` 1291~)4~31 capacity of I amounts to 75.6% o the originally present capacity.
2.5 g ~37 ~mol) of bovine serum albumin is dissolved in 10 ml of water and adjusted to a pH of 8 5 wi~h 13 ml of a l-molar Na~CO3 solution. The albumin solution is centrifuged, then combined with 160 mg of substrate I and stirred for 22 hours under darkness at room temperature. After the reaction is finished, the mixture is suctioned off and the surface carefully 10 washed with water. Once no more albumin can be detected in the filtrate, washing is repeated at least 15 more times, and the product dried under vacuum over P401 o .
Charging of albumin: 0.5 ~mol/g = 33 mg of 15 albumin/g of substrate.
, - .
.
1~ .
~L29048~
,, , , , 11 ,~ , , ~ I
~ , , ,~ , 11 a) I I a) 1 1l ~ I I ~1 1 11 n5 1 1 ~t I 11 a I l ~0~ 1 . . . . . . . . . . Il ~ I I ~ 1 11 o O I CO~
h I I 1 11 I I ~ 1 11 1 ~1 0 ~ I 11 O I I Ql rl :1 1 O ~ ~ D 1~ N t~
P. I I ~ h O I ~`1 ~ 11 I O O ~ 1 11 .q ~ I I 1 11 ~ I I I m ll ~ N
'-1 1 1 1 1 0 ~5 1 1 1 ~ 11 O ~ I I I 1 11 Cl U I I I N
S: I I I O t~
I I I ~I -- O -- O 0 11 N I .R N
,Crl I I ~ I O O a~ o ~: m 1l P~ E t ~t p ~ Pt C~
"~ m I I ~ I U _ U ~ U~ ~ _ u _ c~
o c; I I ~n I o m o ~ o ~ o ~ o m o ~ o ~ o ,¢ o ~ o .¢ ll ' m-- m-- m-- m-- m-- m--m-- m-- m-- m ~ ll _ I 1 11 ~a~ I I 1 11 , U, I I g , 11 u I I a) I N N N ~ 1 N N
40'~ I O O O O O O 0 11 ,1 1 1 u~ I m m m m m m m m m m 11 o) I I 1 11 I I . I
I I o 1 11 (~ I I Z I -1 N ~ 0 I~ 00 cn O 1 1 E t I I . ~
, . ~ .
1~9048~
I o 1 11 ~ o ~ 1 11 I a~ 1 11 I
I I ~l l ~ O O ll I ~ P~ I o ~ o o ~ ~ ~ ~ ~ 11 oo o co o ~ 11 X ~ ~ ~ o u~ In ~1~ ~ 11 1l o o ~ 1 11 I In a~ l ll I I ~ I ~ o ~r o 11 .q II ~ ~ I o In O ~ o o a~ ~D ~ 11 ~ ~ o ~ o ~ 11 X I ~ Ul o U~ ~ ~ ~ ~ 11 , ~ ~ , 11 ~, , , 11 o, , , 11 ~ ~ I 1 11 o . , , 11 I , o , 11 o ~ ~ 11 I, o , 11 II ~ a) 1 11 ~,,, ~, ~ o o 11 S~ II ~ ~ I o ~ o,J a~ ~ Lr~ ~ ~ 11 V II O ~ I ~ O ~ ~1 o~,, U~ X, ~ o ~ ~ ~ ~ 1l U~ ~ , , o 11 ~, , , ~ 11 o I I I ~ ~
~ '" ~ I , I o ~ o ~ a~ , ,. 11 o I I ~ ~ ~
o ~ ~ Il O h ~ ~ ~ ~ 4~ ul 11 .1 ~ I I I ~ ~ ~ O ~ a) o I I I h ~ 1 X-- ~ O ~d ~ ~ ~ 11 I I I U ~O O O ~ ~
~ fd ~ ~I td O tn a) o rd O ~d O ~d 11 a) I I I ~ ~~ ~ ~ o ~ ~ ~ ~ u ~ ~ ¢, ~ 11 R I I I O ~1 X ~ O ~;~ ~1 11 E~ I L I . . ... _................ Il , "
~. ..-
Claims (5)
1. A graft copolymer comprising a crosslinked polymer containing polyoxyethylene residues having an average molecular weight of 500 - 50,000, containing 0.02 - 15 meq free hydroxy groups per g of copolymer, a degree of crosslinking of 0.05 to 10% and wherein the crosslinked polymer is selected from the group consisting of a polyvinyl alcohol, polyhydroxystyrene, and a polymer produced from chloromethylated polystryene and ethylene glycol or oligoethylene glycols, or a polyacrylate or polymethacrylate functionalized by hydroxy groups.
2. The graft copolymer of claim 1 wherein the amount of hydroxy groups is 0.05 - 10 meq/g.
3. The graft copolymer of claim 1 wherein the crosslinked polymer is crosslinked polystyrenes containing hydroxy groups in the amount of 0.05 - 0.7 meq/g.
4. The graft copolymer of claim 1 wherein the crosslinked polymer is polyvinyl alcohols containing hydroxy groups in the amount of 1 - 10 meq/g.
5. The graft copolymer of claim 1 wherein the average molecular weight of the polyoxyethylene residue is 800 - 10,000.
5. The graft copolymer of claim 1 wherein the average molecular weight of the polyoxyethylene residue is 2,000 to 3,000.
7. The graft copolymer of claim 1 wherein the degree of crosslinking of the polymer is 0.05 - 10%.
8. A process for the preparation of the graft copolymers of claim 1 which comprises reacting the crosslinked polymers with ethylene oxide.
9. The process according to claim 8 wherein the reaction is conducted at a temperature in the range of from 20°C to 100°C.
10. The process according to claim 9 wherein the reaction period is 30 minutes to 150 hours.
11. The process according to claim 8 wherein in a first stage, oligoethylene glycol chains of the formula H-(OCH2CH2)n-OH
wherein n stands for 2-20 is bonded to the crosslinked polymer, and the oligoethylene glycol chain is extended with ethylene oxide in a second stage.
12. The process according to claim 11 wherein chloro-methylated polystyrene is used as the polymer in the first stage.
13. The use of a graft copolymer according to any one of claims 1 to 7 as a substrate for peptide or nucleotide synthesis, or for the covalent fixation of peptides, active protein compounds, and enzymes for affinity chromatography, diagnostic media, and biotechnological methods.
5. The graft copolymer of claim 1 wherein the average molecular weight of the polyoxyethylene residue is 2,000 to 3,000.
7. The graft copolymer of claim 1 wherein the degree of crosslinking of the polymer is 0.05 - 10%.
8. A process for the preparation of the graft copolymers of claim 1 which comprises reacting the crosslinked polymers with ethylene oxide.
9. The process according to claim 8 wherein the reaction is conducted at a temperature in the range of from 20°C to 100°C.
10. The process according to claim 9 wherein the reaction period is 30 minutes to 150 hours.
11. The process according to claim 8 wherein in a first stage, oligoethylene glycol chains of the formula H-(OCH2CH2)n-OH
wherein n stands for 2-20 is bonded to the crosslinked polymer, and the oligoethylene glycol chain is extended with ethylene oxide in a second stage.
12. The process according to claim 11 wherein chloro-methylated polystyrene is used as the polymer in the first stage.
13. The use of a graft copolymer according to any one of claims 1 to 7 as a substrate for peptide or nucleotide synthesis, or for the covalent fixation of peptides, active protein compounds, and enzymes for affinity chromatography, diagnostic media, and biotechnological methods.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19853500180 DE3500180A1 (en) | 1985-01-04 | 1985-01-04 | Graft copolymers from crosslinked polymers and polyoxyethylene, process for their preparation and their use |
| DEP3500180.1 | 1985-01-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1290481C true CA1290481C (en) | 1991-10-08 |
Family
ID=6259282
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000498925A Expired - Lifetime CA1290481C (en) | 1985-01-04 | 1986-01-03 | Graft copolymers of crosslinked polymers and polyoxyethylene, processes for their production, and their usage |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4908405A (en) |
| EP (1) | EP0187391B1 (en) |
| JP (1) | JPH0811768B2 (en) |
| AT (1) | ATE88195T1 (en) |
| CA (1) | CA1290481C (en) |
| DE (2) | DE3500180A1 (en) |
Families Citing this family (109)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0263184B1 (en) * | 1986-03-28 | 1992-10-28 | Toray Industries, Inc. | Immobilized physiologically active material |
| FR2615194B1 (en) * | 1987-05-11 | 1991-06-14 | Rhone Poulenc Chimie | COMPRISING POLYMER PARTICLES IMPLANTED ON THE SURFACE OF AMPHIPHILIC MOLECULES CARRYING IONOGENIC OR REAGENT GROUPS, THEIR PREPARATION PROCESS AND THEIR APPLICATION IN BIOLOGY |
| DE3816458A1 (en) * | 1988-05-13 | 1989-12-21 | Josowicz Mira | ULTRAMICROELECTRODE, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
| CA1316858C (en) * | 1988-06-10 | 1993-04-27 | Keith E. Taylor | Method and device for attachment of biologically useful materials to a solid phase |
| FR2634023B1 (en) * | 1988-07-08 | 1994-03-25 | Bio Merieux | REAGENT IN THE FORM OF A SOLID SUPPORT FOR COVALENTLY FIXING AN AMINE BIOLOGICAL LIGAND, ITS PREPARATION AND ITS USE |
| US5258454A (en) * | 1988-09-01 | 1993-11-02 | Riso National Laboratory | Peptide synthesis method and solid support for use in the method |
| SE465221B (en) * | 1989-12-29 | 1991-08-12 | Berol Nobel Ab | COVALENTLY BIND PROTEIN TO HYDROPHILA SURFACE |
| US5545698A (en) * | 1990-08-31 | 1996-08-13 | University Of Minnesota | Polyethylene glycol derivatives for solid-phase applications |
| EP0546055B1 (en) * | 1990-08-31 | 1996-07-10 | Regents Of The University Of Minnesota | Polyethylene glycol derivatives for solid-phase applications |
| US5235028A (en) * | 1990-08-31 | 1993-08-10 | University Of Minnesota | Polyethylene glycol derivatives for solid-phase applications |
| DE4134967C1 (en) * | 1991-10-23 | 1992-12-10 | Th. Goldschmidt Ag, 4300 Essen, De | |
| EP0547720A3 (en) * | 1991-12-19 | 1993-09-01 | Eniricerche S.P.A. | Polymer containing chelating groups, process for preparing it and its use in water purification |
| US5278303A (en) * | 1992-06-12 | 1994-01-11 | University Of Toronto Innovations Foundation | Polymer-supported solution synthesis of oligosaccharides |
| US6039876A (en) * | 1992-07-24 | 2000-03-21 | Yang; Yan-Bo | Hydrophilic polystrene divinylbenzene based matrixes for chromatography |
| DE4301693A1 (en) * | 1993-01-22 | 1994-07-28 | Cytech Biomedical Inc | Amplification methods and methods for the detection of solid phase polynucleotide sequences |
| US5391667A (en) * | 1993-03-04 | 1995-02-21 | Isis Pharmaceuticals | Copolymers of N-vinyl-lactams suitable for oligomer solid phase synthesis |
| DK72493D0 (en) * | 1993-06-18 | 1993-06-18 | Risoe Forskningscenter | SOLID SUPPORTS FOR USE IN PEPTIDE SYNTHESIS AND ASSAYS |
| FR2714061B1 (en) * | 1993-12-16 | 1996-03-08 | Genset Sa | Process for the preparation of polynucleotides on solid support and apparatus allowing its implementation. |
| US5616698A (en) * | 1994-01-10 | 1997-04-01 | University Of Toronto Innovations Foundation | Polymer-supported solution synthesis of oligosaccharides |
| US5503933A (en) * | 1994-02-25 | 1996-04-02 | Purdue Research Foundation | Covalently bonded coatings |
| US5502246A (en) * | 1994-03-22 | 1996-03-26 | Eli Lilly And Company | Solid-phase synthesis utilizing photochemical carbon-sulfur bond cleavage of thioethers |
| US5466758A (en) * | 1994-04-13 | 1995-11-14 | Yoon-Sik; Lee | Process for preparing polystyrenes having β hydroxy group and polyglycol-grafted polystyrenes thereof |
| US5552471A (en) * | 1994-08-17 | 1996-09-03 | The Perkin-Elmer Corporation | Solid support reagents for the synthesis of 3'-Nitrogen containing polynucleotides |
| US5961923A (en) * | 1995-04-25 | 1999-10-05 | Irori | Matrices with memories and uses thereof |
| US5751629A (en) * | 1995-04-25 | 1998-05-12 | Irori | Remotely programmable matrices with memories |
| US6025129A (en) * | 1995-04-25 | 2000-02-15 | Irori | Remotely programmable matrices with memories and uses thereof |
| US6284459B1 (en) | 1995-04-25 | 2001-09-04 | Discovery Partners International | Solid support matrices with memories and combinatorial libraries therefrom |
| US6017496A (en) * | 1995-06-07 | 2000-01-25 | Irori | Matrices with memories and uses thereof |
| US6331273B1 (en) | 1995-04-25 | 2001-12-18 | Discovery Partners International | Remotely programmable matrices with memories |
| US6416714B1 (en) | 1995-04-25 | 2002-07-09 | Discovery Partners International, Inc. | Remotely programmable matrices with memories |
| US6100026A (en) * | 1995-04-25 | 2000-08-08 | Irori | Matrices with memories and uses thereof |
| US5741462A (en) * | 1995-04-25 | 1998-04-21 | Irori | Remotely programmable matrices with memories |
| US5874214A (en) * | 1995-04-25 | 1999-02-23 | Irori | Remotely programmable matrices with memories |
| US6329139B1 (en) | 1995-04-25 | 2001-12-11 | Discovery Partners International | Automated sorting system for matrices with memory |
| US5656707A (en) * | 1995-06-16 | 1997-08-12 | Regents Of The University Of Minnesota | Highly cross-linked polymeric supports |
| US5858534A (en) * | 1995-09-05 | 1999-01-12 | Solid Phase Sciences Corp. | Method of making and using derivatized paramagnetic polymer beads |
| DE19781535T1 (en) * | 1996-01-23 | 1999-01-28 | Argonaut Technologies Inc | Highly functionalized polystyrene carriers grafted with polyethylene glycol |
| US5736626A (en) | 1996-01-29 | 1998-04-07 | The Perkin-Elmer Corporation | Solid support reagents for the direct synthesis of 3'-labeled polynucleotides |
| US6247995B1 (en) | 1996-02-06 | 2001-06-19 | Bruce Bryan | Bioluminescent novelty items |
| US5876995A (en) | 1996-02-06 | 1999-03-02 | Bryan; Bruce | Bioluminescent novelty items |
| US20040147022A1 (en) * | 1996-06-06 | 2004-07-29 | Baker Brenda F. | 2'-methoxy substituted oligomeric compounds and compositions for use in gene modulations |
| US20040203024A1 (en) * | 1996-06-06 | 2004-10-14 | Baker Brenda F. | Modified oligonucleotides for use in RNA interference |
| US20040161777A1 (en) * | 1996-06-06 | 2004-08-19 | Baker Brenda F. | Modified oligonucleotides for use in RNA interference |
| US20040171028A1 (en) * | 1996-06-06 | 2004-09-02 | Baker Brenda F. | Phosphorous-linked oligomeric compounds and their use in gene modulation |
| US20040266706A1 (en) * | 2002-11-05 | 2004-12-30 | Muthiah Manoharan | Cross-linked oligomeric compounds and their use in gene modulation |
| US20050119470A1 (en) * | 1996-06-06 | 2005-06-02 | Muthiah Manoharan | Conjugated oligomeric compounds and their use in gene modulation |
| US5898031A (en) | 1996-06-06 | 1999-04-27 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides for cleaving RNA |
| US20050053976A1 (en) * | 1996-06-06 | 2005-03-10 | Baker Brenda F. | Chimeric oligomeric compounds and their use in gene modulation |
| US7812149B2 (en) * | 1996-06-06 | 2010-10-12 | Isis Pharmaceuticals, Inc. | 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations |
| US9096636B2 (en) * | 1996-06-06 | 2015-08-04 | Isis Pharmaceuticals, Inc. | Chimeric oligomeric compounds and their use in gene modulation |
| US20050042647A1 (en) * | 1996-06-06 | 2005-02-24 | Baker Brenda F. | Phosphorous-linked oligomeric compounds and their use in gene modulation |
| US6416960B1 (en) | 1996-08-08 | 2002-07-09 | Prolume, Ltd. | Detection and visualization of neoplastic tissues and other tissues |
| US6124372A (en) * | 1996-08-29 | 2000-09-26 | Xerox Corporation | High performance polymer compositions having photosensitivity-imparting substituents and thermal sensitivity-imparting substituents |
| US5958995A (en) | 1996-08-29 | 1999-09-28 | Xerox Corporation | Blends containing photosensitive high performance aromatic ether curable polymers |
| US5994425A (en) * | 1996-08-29 | 1999-11-30 | Xerox Corporation | Curable compositions containing photosensitive high performance aromatic ether polymers |
| US5849809A (en) | 1996-08-29 | 1998-12-15 | Xerox Corporation | Hydroxyalkylated high performance curable polymers |
| US5761809A (en) * | 1996-08-29 | 1998-06-09 | Xerox Corporation | Process for substituting haloalkylated polymers with unsaturated ester, ether, and alkylcarboxymethylene groups |
| AU5426398A (en) | 1996-10-28 | 1998-05-22 | Versicor Inc | Methods for solid-phase synthesis of hydroxylamine compounds and derivatives, and combinatorial libraries thereof |
| US6281245B1 (en) | 1996-10-28 | 2001-08-28 | Versicor, Inc. | Methods for solid-phase synthesis of hydroxylamine compounds and derivatives, and combinatorial libraries thereof |
| NZ335453A (en) | 1996-12-12 | 2001-07-27 | Prolume Ltd | Microelectronic device with microlocations including photodetector for detecting bioluminescence |
| US6033918A (en) * | 1997-11-10 | 2000-03-07 | Bayer Corporation | Method and device for the detection of analyte in a fluid sample |
| US6114466A (en) * | 1998-02-06 | 2000-09-05 | Renal Tech International Llc | Material for purification of physiological liquids of organism |
| US6136424A (en) * | 1998-02-06 | 2000-10-24 | Renal Tech International, Llc | Method of and material for purification of physiological liquids of organism, and method of producing the material |
| EP1925320A3 (en) | 1998-03-27 | 2008-09-03 | Prolume, Ltd. | Luciferases, fluorescent proteins, nucleic acids encoding the luciferases and fluorescent proteins and the use thereof in diagnostics |
| ATE388224T1 (en) | 1998-03-27 | 2008-03-15 | Prolume Ltd | LUCIFERASE, GFP FLUORESCENT PROTEINS, CODING NUCLEIC ACID AND THEIR USE IN DIAGNOSTICS |
| GB9814876D0 (en) * | 1998-07-10 | 1998-09-09 | Zeneca Ltd | Polymer |
| WO2000012575A1 (en) | 1998-08-28 | 2000-03-09 | Jerini Bio Tools Gmbh | Method for producing polymeric solid phase supporting materials |
| WO2000027896A1 (en) * | 1998-11-05 | 2000-05-18 | Beadtech Inc. | Modified polystyrene spherical resin and process for decontaminating waste water by using the same |
| US6139920A (en) * | 1998-12-21 | 2000-10-31 | Xerox Corporation | Photoresist compositions |
| SE9902133D0 (en) * | 1999-06-06 | 1999-06-06 | Amersham Pharm Biotech Ab | A method for surface modification, a novel support matrix and the use of the matrix |
| US7192729B2 (en) | 1999-07-06 | 2007-03-20 | General Atomics | Methods for assaying homocysteine |
| GB9918437D0 (en) * | 1999-08-05 | 1999-10-06 | Anglo American Platinum Corp | Separation of platinium group metals |
| RU2002113076A (en) | 1999-11-18 | 2004-03-10 | Корвас Интернэшнл, Инк. (Us) | Nucleic acids encoding endotheliases, endotheliases and their use |
| US7700341B2 (en) | 2000-02-03 | 2010-04-20 | Dendreon Corporation | Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon |
| AU2001227679A1 (en) * | 2000-02-25 | 2001-09-03 | General Atomics | Mutant nucleic binding enzymes and use thereof in diagnostic, detection and purification methods |
| US7109315B2 (en) * | 2000-03-15 | 2006-09-19 | Bruce J. Bryan | Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items |
| US6610504B1 (en) | 2000-04-10 | 2003-08-26 | General Atomics | Methods of determining SAM-dependent methyltransferase activity using a mutant SAH hydrolase |
| WO2001096357A2 (en) * | 2000-06-13 | 2001-12-20 | Proligo Llc | Universal solid supports for solid phase oligosynthesis and methods for their preparation and use |
| US8975328B2 (en) * | 2000-06-30 | 2015-03-10 | Institute Curie | Non-thermosensitive medium for analyzing species in a channel and for minimizing adsorption and/or electroosomosic phenomena |
| FR2811083B1 (en) | 2000-06-30 | 2002-11-22 | Inst Curie | NON-THERMOSENSITIVE LIQUID MEDIUM FOR THE ANALYSIS OF SPECIES WITHIN A CHANNEL |
| FR2810905B1 (en) * | 2000-06-30 | 2003-05-16 | Inst Curie | ADDITIVE FOR MINIMIZING ADSORPTION AND / OR ELECTROOSMOSIS PHENOMENES |
| DK1352003T3 (en) * | 2000-11-20 | 2006-05-29 | Matrix Innovation Inc | Process for the preparation of amphiphilic solid support for peptide synthesis, bioorganic and organic chemistry |
| AU2002305052A1 (en) | 2001-03-13 | 2002-09-24 | Corvas International, Inc. | Nucleic acid molecules encoding a transmembrane serine protease 7, the encoded polypeptides and methods based thereon |
| CA2441378A1 (en) | 2001-03-22 | 2002-10-03 | Dendreon San Diego Llc | Nucleic acid molecules encoding serine protease cvsp14, the encoded polypeptides and methods based thereon |
| CA2442089A1 (en) | 2001-03-27 | 2002-10-03 | Dendreon San Diego Llc | Nucleic acid molecules encoding a transmembran serine protease 9, the encoded polypeptides and methods based thereon |
| EP1539942A4 (en) | 2001-05-14 | 2005-10-19 | Dendreon Corp | Nucleic acid molecules encoding a transmembrane serine protease 10, the encoded polypeptides and methods based thereon |
| US6642334B2 (en) | 2001-07-09 | 2003-11-04 | Carlsberg A/S | Beaded polyethylene glycol-based resins |
| AU2002357004A1 (en) * | 2001-11-20 | 2003-06-10 | Dendreon San Diego Llc | Nucleic acid molecules encoding serine protease 17, the encoded polypeptides and methods based thereon |
| JP3890556B2 (en) * | 2001-11-28 | 2007-03-07 | 富士フイルム株式会社 | Nucleic acid separation and purification method and nucleic acid separation and purification unit |
| EP1572937B1 (en) * | 2002-04-09 | 2012-02-08 | The Scripps Research Institute | Motif-grafted hybrid polypeptides and uses thereof |
| GB0209539D0 (en) * | 2002-04-26 | 2002-06-05 | Avecia Ltd | Monomer Polymer and process |
| US20040001801A1 (en) * | 2002-05-23 | 2004-01-01 | Corvas International, Inc. | Conjugates activated by cell surface proteases and therapeutic uses thereof |
| CA2504929C (en) * | 2002-11-05 | 2014-07-22 | Charles Allerson | Compositions comprising alternating 2'-modified nucleosides for use in gene modulation |
| EP1440995B1 (en) * | 2003-01-23 | 2019-08-07 | Rapp Polymere GmbH | Hydroxyl-group containing polymers |
| US6861473B2 (en) * | 2003-02-28 | 2005-03-01 | Baxter International Inc. | Macromolecular ketoaldehydes |
| US20060241072A1 (en) * | 2003-06-20 | 2006-10-26 | Isis Pharmaceuticals, Inc. | Oligomeric compounds for use in gene modulation |
| EP1687343B1 (en) * | 2003-08-04 | 2014-07-23 | Matrix Innovation Inc. | New polyether based monomers and highly cross-linked amphiphile resins |
| US20060035242A1 (en) * | 2004-08-13 | 2006-02-16 | Michelitsch Melissa D | Prion-specific peptide reagents |
| US8569474B2 (en) * | 2004-03-09 | 2013-10-29 | Isis Pharmaceuticals, Inc. | Double stranded constructs comprising one or more short strands hybridized to a longer strand |
| US7384760B2 (en) * | 2004-04-30 | 2008-06-10 | General Atomics | Methods for assaying inhibitors of S-adenosylhomocysteine (SAH) hydrolase and S-adenosylmethionine (SAM)-dependent methyltransferase |
| US8394947B2 (en) * | 2004-06-03 | 2013-03-12 | Isis Pharmaceuticals, Inc. | Positionally modified siRNA constructs |
| AU2005252663B2 (en) * | 2004-06-03 | 2011-07-07 | Isis Pharmaceuticals, Inc. | Double strand compositions comprising differentially modified strands for use in gene modulation |
| WO2006029023A2 (en) * | 2004-09-02 | 2006-03-16 | Isis Pharmaceuticals, Inc. | Polymeric beads for oligonucleotide synthesis |
| US7884086B2 (en) * | 2004-09-08 | 2011-02-08 | Isis Pharmaceuticals, Inc. | Conjugates for use in hepatocyte free uptake assays |
| WO2006076683A2 (en) * | 2005-01-13 | 2006-07-20 | Novartis Vaccines And Diagnostics Inc. | Isolation and detection of pathogenic prions |
| WO2006076687A2 (en) * | 2005-01-13 | 2006-07-20 | Novartis Vaccines And Diagnostics Inc. | Elisa assays using prion-specific peptide reagents |
| JP5443682B2 (en) * | 2007-11-05 | 2014-03-19 | 日東電工株式会社 | Porous resin particles having hydroxy group or primary amino group and method for producing the same |
| CN102083450A (en) * | 2008-04-30 | 2011-06-01 | 诺华有限公司 | Assay for pathogenic conformers |
| EP2159228A1 (en) | 2008-08-28 | 2010-03-03 | Polymer Laboratories Limited | Amphiphilic solid support |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE571756A (en) * | 1957-04-02 | |||
| US3173892A (en) * | 1962-05-02 | 1965-03-16 | Rohm & Haas | Process of preparing reaction products of a halomethylated cross-linked copolymer, and resulting product |
| GB1002343A (en) * | 1962-09-11 | 1965-08-25 | Canadian Ind | Process for hydroxyalkylating polymers containing carboxyl groups |
| GB1213030A (en) * | 1968-08-16 | 1970-11-18 | Daicel Ltd | Process for producing a graft copolymer |
| SE394820B (en) * | 1970-01-23 | 1977-07-11 | Exploaterings Ab Tbf | MAKE BULLY COOLANTLY BINDING TO A WATEROLO POLYMER COOLANTLY ACTIVATELY, AS AMINO ACIDS, PEPTIDES, PROTEINS, NUCLEOTIDES AND NUCLEIC ACIDS BY REACTION WITH AN SUCH POLY |
| US4085168A (en) * | 1971-02-22 | 1978-04-18 | Cpc International Inc. | Chemically joined, phase separated self-cured hydrophilic thermoplastic graft copolymers and their preparation |
| AU466178B2 (en) * | 1972-04-10 | 1975-10-23 | Dow Corning Co. | Implantable extendable member |
| US3941849A (en) * | 1972-07-07 | 1976-03-02 | The General Tire & Rubber Company | Polyethers and method for making the same |
| DE2239401A1 (en) * | 1972-08-10 | 1974-02-21 | Basf Ag | BLOCK OR GRAFT COPOLYMERS MADE FROM POLYALKYLENE OXIDES AND VINYLAROMATE OR DIEN POLYMERIZED |
| US4083834A (en) * | 1973-02-12 | 1978-04-11 | Japan Synthetic Rubber Co., Ltd. | Process for producing polymer having functional groups at its chain terminals |
| JPS5247754A (en) * | 1975-10-14 | 1977-04-15 | Kenichiro Minami | Automatic camera for subscribers message meters |
| US4140667A (en) * | 1977-03-31 | 1979-02-20 | Olin Corporation | Graft copolymers from vinyl monomers and unsaturated polyols containing alkenyl aryl constituents and polyurethanes prepared therefrom |
| DE3380726D1 (en) * | 1982-06-24 | 1989-11-23 | Japan Chem Res | Long-acting composition |
| JPS60232243A (en) * | 1984-04-28 | 1985-11-18 | Res Dev Corp Of Japan | Adsorbent comprising crosslinked high-molecular polymer |
-
1985
- 1985-01-04 DE DE19853500180 patent/DE3500180A1/en not_active Withdrawn
- 1985-12-27 JP JP60299717A patent/JPH0811768B2/en not_active Expired - Lifetime
- 1985-12-31 DE DE8585116662T patent/DE3587269D1/en not_active Expired - Lifetime
- 1985-12-31 EP EP85116662A patent/EP0187391B1/en not_active Expired - Lifetime
- 1985-12-31 AT AT85116662T patent/ATE88195T1/en active
-
1986
- 1986-01-02 US US06/815,634 patent/US4908405A/en not_active Expired - Lifetime
- 1986-01-03 CA CA000498925A patent/CA1290481C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0811768B2 (en) | 1996-02-07 |
| EP0187391B1 (en) | 1993-04-14 |
| EP0187391A3 (en) | 1989-01-04 |
| ATE88195T1 (en) | 1993-04-15 |
| JPS61171712A (en) | 1986-08-02 |
| EP0187391A2 (en) | 1986-07-16 |
| DE3587269D1 (en) | 1993-05-19 |
| DE3500180A1 (en) | 1986-07-10 |
| US4908405A (en) | 1990-03-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1290481C (en) | Graft copolymers of crosslinked polymers and polyoxyethylene, processes for their production, and their usage | |
| EP0801082B1 (en) | Method for making graft resins for solid-phase peptide synthesis | |
| US5482996A (en) | Protein-containing polymers and a method of synthesis of protein-containing polymers in organic solvents | |
| JPH08510274A (en) | Membrane formed by acrylonitrile-based polymer | |
| GB1571182A (en) | Carrier-bound biologically active substances and process for their manufacture | |
| EP0713885A4 (en) | Modified conjugated diene copolymer, process for producing the same, and composition thereof | |
| KR20010015856A (en) | Heterofunctionalized star-shaped poly(ethylene glycols) for protein modification | |
| JPS5858026B2 (en) | Packing material for chromatography and its manufacturing method | |
| IE841841L (en) | Copolymers and shaped articles therefrom | |
| US5545698A (en) | Polyethylene glycol derivatives for solid-phase applications | |
| Garret‐Flaudy et al. | Use of the avidin (imino) biotin system as a general approach to affinity precipitation | |
| WO1993021257A1 (en) | Polyetheramidoamine hydrogels as heparinizable materials | |
| US3795664A (en) | Process for preparing peptides or proteins | |
| EP0425268A2 (en) | Phosphazene polymer carrier for biologically active substance | |
| US3981775A (en) | Enzyme insolubilization | |
| Zühlke et al. | Synthesis and application of new microcarriers for animal cell culture. Part I: design of polystyrene based microcarriers | |
| US4350791A (en) | Vinylpyrrolidone polymers, their preparation, their use in the preparation of plasma substitutes, and the substitutes thus obtained | |
| EP0420015B1 (en) | Method of preparing water soluble silated polyvinyl alcohol | |
| US4079021A (en) | Polymeric carrier for a controlled synthesis of peptides | |
| Renil et al. | Synthesis, characterization and application of tetraethylene glycol diacrylate crosslinked polystyrene support for gel phase peptide synthesis | |
| EP0587357B1 (en) | New chain-terminated N-vinyl lactam polymers and graft-copolymers and methods for making same | |
| US7282237B2 (en) | Method for surface modification, a novel support matrix and the use of the matrix | |
| WO2014058755A1 (en) | Synthesis support resin | |
| JP4131450B2 (en) | Sugar-containing thermosensitive copolymer and method for producing the same | |
| Miyagawa et al. | Immobilization of glycoconjugate polymers on cellulose membrane for affinity separation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |