CS272491B1 - Copolymers of allyl derivatives of oligo and / or polysaccharides with β vinyl monomers and methods for their preparation - Google Patents

Abstract

The solution relates to new substances - copolymers of allyl derivatives of oligo and/or polysaccharides and vinyl monomers, which can be prepared by copolymerization of a solution from a mixture of an allyl derivative of oligo and/or polysaccharide, a vinyl monomer and a crosslinker in the presence of a polymerization initiator and, if applicable, a suspension stabilizer, at a temperature of max. 80 °C. The solution will find application in biotechnology, especially in the isolation and purification of carbohydrate-binding proteins of the lectin type, oligo and polysaccharide hydrolases, as well as antibodies to carbohydrate antigens, as well as in medicine in the identification of these proteins using gel electrophoresis or affinity chromatography. In addition, they can be used as carriers of biologically active substances, enzymes and other proteins.

Description

* 1 CS 272 491 Bl

The present invention relates to copolymers of oligo and / or polysaccharide allyl derivatives 8 by vinyl monorene as well as processes for their preparation. The subject copolymers are particularly suitable for the affinity protein-affinity sorbents for the appropriate oligo or polysaccharide incorporated in the copolymer (lectins, antibodies, oligo or polysaccharide hydrolase). In the field of new polymeric materials. bases of copolymers of polysaccharides with monolayers containing double bonds, the development has so far been avoided to form water-soluble or partially water-soluble copolymers, i.e., monomers not allowing increased crosslinking of polysaccharide and polymer chains to each other, i.e. monomers by any double bonds in one molecule / AND. A. Berlin, Chim. Wood. 2, 3 (1985)]. In this context, spits of their preparation are also being developed. Most notably, the method for obtaining such copolymers by radical scavenging polymerization of the cellulose and its derivatives (Z). A. Rogovin, Vysokomol. sosd, 13. ' 437, (1971)],

Polymers of this kind and generally are prepared by adding a defined amount of a radical polymerization initiator to the aqueous solution of polyaccharide with the respective double bond-containing monomers, which may be potassium permanganate, cerium salt, peroxides, or polymerization initiated ultraviolet or x-ray radiation. The initiators cause the polysaccharide to form "radicals" (E. N. Zilbererman, A.A. Starkov, E.V. Alexandrovska, High. Soled., 17, 744-746, (1975)) suitable for radical polymerization of the respective polysaccharide monomer.

Polysaccharide polymers formed with monomers containing double bonds are used as emulsifiers, oils and fibers (3P 558), coagulants for the treatment of fall waters (OP16635), flocculants / BG 15198 /, or as materials with increased sorption capacity / 5B 1503198, OE 252 6501].

Copolymers of oligo and / or polysaccharides with vinyl monomers according to the invention have now been prepared by copolymerization of 0.01 to 99.98% by weight. allyl oligo derivative and / or bopolysaccharide with a degree of substitution of 0.01 to 3.0, 0.01 to 99.98% by weight. % vinyl monomer and 0.01 to 90.0 wt. ethers of the formula I, IX or III R1 * * V - C - OOC - R2 - COO - C - R1 II II (I) ch2 ch2 R3 - OOC - R2 - COO - R3 (IX) R3 - C - CONH - R2-NHCO-C-R3

Wherein R is hydrogen, alkyl of 1 to 8 or halogen, R is alkylene of 1 to 8, phenyl or hydroxyalkylene of formula CnH2n-. the whole is 2 to 6 and the whole is 1 to n-2 and R 3 is vinyl or allyl, the presence of a polymerization initiator and optionally a suspension stabilizer at a temperature of at most 80 ° C. In the case of free-radical copolymerization of the components in the block, the corresponding components - allyl derivative of oligo and / or polysaccharide, vinyl monomer and crosslinker are dissolved in water, or if the allyl derivative of saccharide is not soluble in water both in the organic solvent and the solvent. homonomers such as methanol, ethyl alcohol, dioxane, and the like. The solution obtained is evacuated to a pressure of 150 to 10,000 Pa, a radical polymerization / anpr. N, N, N ', N ' -tetramethylethylenediamine) and the reaction mixture are incubated without stirring at a temperature of max. CS 272 491 B1 2

In radical copolymarization in suspanium, the individual components are first suspended in water and the thus prepared suspension is mixed with the aqueous phase, which is formed by an organic solvent such as n-heptane, octene, isooctane, gasoline, chloroform, toluene and / or dissolves. To the suspension! a suspension stabilizer is added to which the organic substance, preferably oxyethylated astaryl sorbitol (TWEEN), alkylaminooxides / ethoxones /, polyvinylpyrrolidone, polyvinyl alcohol, liquid at the polymerization temperature with the monomer and allyldarea saccharide, then the polymarization initiator, preferably ammonium bisulphate, and the suspension polymerized to mixing at a maximum temperature of 80 ° C. It is also possible that the components are dissolved in the water-insoluble material, which is an organic solvent, a liquid at temperature polymarization, having the properties of a good solvent for a copolymer, such as cyclohaxanol, n-octanol or cyclohaxylamine. The suspension stabilizer in this case is an organic compound, preferably octane, haptan, or hydrocarbons having 10 to 16 carbon atoms, which is a liquid at the temperature of the polymerization mixture with the allyldareate monomer and the initial component of the poor solvent for the copolymer. A polymarization initiator, preferably azobisbutyronitrile in an amount of 0.001 to 0.01% by weight, is added to the suspension and the suspension is blotted to a maximum of 80 ec.

The copolymers of the oligo and / or / and / or polysaccharide allyldarivatives 8 with vinyl monomers of the present invention can be used as sorbates with affinity for the oligo and polysaccharide hydrolase, lactins and antibodies in a variety of configurations (batch alignment, collagen chromatography, electrophoresis, etc.). In addition, they can be used as carriers of biologically active substances, anynes and other bialkines.

Polymers so far prepared are suitable as affinity sorbents for the praparation of bio-bialkine-type molecules with specificity to the respective polysaccharide, the incorporated dopolymer, mainly because they are mostly water soluble. In addition, there is a random nature of radical eye formation on the polysaccharide chain and it is difficult to define the degree of cross-linking of the polysaccharide with the monomers in the copolymer.

The amount of saccharide in the pradmatic copolymer can be controlled by the ratio of allylated saccharide to vinyl monomer. The degree of sizing from which the porosity of the formed polymer depends, the permeability and the degradability of the specific carbohydrate hydrolysates can in turn be controlled by the number of allylic compounds per molecule of saccharide and the addition of bifunctional crosslinkers.

By appropriately selecting the medium, the ratio of its components, the temperature and the amount of initiators, it is possible to prepare the preparation to produce the desired defined copolymers in the formomomogenic particles. Hydrophobicity, rasp. the hydrophilicity of their surface can be controlled by selecting the appropriate type of vinyl monomer. The particle size is determined by the mixing rate of 8 µl, the amount of stabilizers, initiator and temperature.

These examples illustrate, but illustrate, the invention. EXAMPLE 1 6 parts of acrylamide, 2 dials of methylene bieakrylamide and 0.05 parts of mannan isolated from Candida albicans, allylated to eubetitation 0.01 were dissolved in 46 parts by weight of water. The solution was evacuated for 5 minutes on a water pump and, after addition of 0.02 N, N, N, N 2 -tatra-tathatyl-nylindiamine and 0.08 parts of ammonium parsulfate, was incubated for 20 min on a water bath at 40 ° C. A block polymer was formed which was mixed with water on a mixer and washed several times with ethanol and dried. Casticaboli average size 0.05 to 0.15 mm. However, a solid block polymer may also be used (e.g., for bialkovine-binding carbohydrate electrophoresis). Example 2

In a vacuum vessel equipped with a cooler and a temperature control

Claims (5)

3 CS 272 491 B1 poured the organic phase in toluene 52 parts by weight, 20 parts chloroform, polyoxyethylated sorbitol aster / TWEEN 40/2 parts, with the addition of 9 parts acrylamide, 4 parts methylene bisacrylamide, 1 part by stirring. of methylene starch with a degree of substitution of 0.1 and 13 parts of 0.05 mol / l triethanolamine-hydrochloric acid, pH 7.0, the suspension was stirred in a nitrogen atmosphere for 15 min and added to a temperature of 40 ° C. 0.5 part Ν, Ν, Νί N * -tetramethyl-ethylenediamine and 0.01 part of ammonium persulfate. With vigorous stirring for 200 rpm, the mixture was incubated for 5 h. The polymer obtained was blended in a mixer and methanol, filtered, rinsed with methanol, water and dried, and the particles were in the range of 0.1 to 0.3 mm, good filterable, 90% conversion. EXAMPLE 3 75 parts of water (w / w) containing 14.3 parts of n-octanol, 4.8 parts of glycidyl methacrylate, 2.5 parts of ethylenedimethacrylate, 1 part were poured into a reaction vessel equipped with a stirrer, a low condenser and a thermometer. starch substitution of 3.0 and 0.1 parts of azidobisbutyronitrile was bubbled through, the mixture was purged with 15 mindusics and the reaction vessel was sealed with a low temperature condenser, stirred at 300 rpm, and stirred at 50 ° C for 12 hours. % and an average particle size of 200 to 500 µm. The resulting polymer was worked up by filtration and washing with methanol and water. Finally, it was dried. Example 4 The procedure was as in Example 3 except that cyclohexanol (13.3 parts) and lauryl alcohol stabilizer (1 part), initiator / water concentrations and reaction conditions as in Example 3 were used as inert phase. 93 particle size 0/1 'to 0.3 mm *, $ Example 5 A copolymer of allylmanane with acrylamide prepared as described in Example 1 was charged to a 1 cm diameter column. The column was washed with 0.05 mol / l phosphate buffered saline, pH 7.0, and 5 ml blood serum of rabbits immunized with Candida albicans wall was applied to the column. The antibody titer was determined by ELISA, using β-galactosidase, and the antibody yield was 92%. The purified antibodies exhibited 1 band in electro-foresis according to Laemli, U.K., London, 227,680 (1970) The invention finds application in biotechnology especially for the isolation and purification of protein binding binders of lectin, oligo and polysaccharide hydrolase types as well as saccharide antigens, as well as in medicine in the identification of these whites by gel electrophoresis or Affinity Chromatography CLAIMS 1. Copolymers of oligo and / or polysaccharide allyl derivatives and vinyl monomers obtainable by copolymerizing a solution of the mixture from 0.01 to 99.98% by weight. allyl oligo and / or polysaccharide derivatives having a degree of substitution of 0/01 to 3.0, 0/01 to 99.98% by weight; % vinyl monomer and 0/01 to 90 wt. of the formula (I) / (II) or (III) (R) C - OOC - R 2 - COO - C - R 1 II II ch 2 ch 2 (i) CS 272 491 B1 4 R 3 - OOC - R 2 - COO - R 3 (II) R 3 - C - CONH-R 2 -HNCO-C-R 2 JI II (III) ch 2 ch 2 where hydrogen, alkyl of 1 to 8 carbon atoms, or halo, R 2 is alkylone by carbon number 1 to 8, phenylene or hydroxyalkylene of the general formula the number 2 to 6 and jn is 1 to n-2 and R3 is a vinyl or alkyl group in the presence of a polymers initiator and optionally a suspension stabilizer at a temperature of not more than 80 ° C. 2. Preparation of copolymers according to claim 1, characterized in that ammonium persulphate with N, N, N / N, N-tetramethylenediamine is used as the radical copolymerization initiator, 3. A process for preparing copolymers according to claim 1, wherein an oxyethylated sorbitol ester is used as the suspension stabilizer. 4. A process for preparing copolymers according to claim 1, wherein a n-alkane having from 8 to 16 carbon atoms is used as the suspension stabilizer. 5. A process for preparing copolymers according to claim 1, wherein azobisbutyronitrile is used as the polymerization initiator.