CS263374B1 - 2-hydroxy-3-chloropropyldiate-crosslinked polyhydroxyethyl methacrylate - Google Patents

Abstract

2-hydroxy-3-chloropropyl derivative of fused poly-hydroxyethyl methacrylate (formula I), which is capable of binding ligands, especially of the type of carbohydrates and amines. It is prepared from fused poly-hydroxyethyl methacrylate by reaction with chloromethyloxirane in a dioxane environment and in the presence of perchloric acid at temperatures of 95 to 102 °C. Its application is in the field of biotechnology and medicine in the binding of biologically active substances, in affinity chromatography of enzymes and nucleic acids and in the immobilization of enzymes.

Description

263374 2

The present invention relates to a novel 2-hydroxy-3-chloropropyl-cross-linked polyhydroxy-ethylmethacrylate compound.

Polyhydroxyethyl methacrylates (SPHERON gels) are Czechoslovak sorbents made by radical suspension polymerization of a mixture of ethylene dimethacrylate, 2-hydroxyethyl methacrylate in cyclohexane or dodecanol (Lim.D., Coupek J., Krivankova M., Pokorny S., Csl. AO 150 819 (1973)]. By varying the composition of the dispersed organic phase, it is possible to vary the size and volume, the specific surface area, the content of reactive groups and other parameters (Svec F., Held J., Coupek J., Kala J., Angew. Makromol. Chem., 48, 135 (1975)]. Crosslinked polyhydroxyethyl methacrylates are used! as materials for gel chromatography / Vondruška M., Ludwig M., Mladek M., J. Chromatogr. 116, 457 (1976)]. The primary hydroxyl groups can be modified by any means to produce cross-linked polyhydroxyethyl methacrylate derivatives using ion exchange resins (Mikeš O., Strop P., Čoupek J., J. Chromatogr. 153, 23 (1978)], chelate ion exchangers and selectively sorbents / Slovák Z., Toman J., Z. Anal. Chem. 278, 115 (1976)], affinity chromatography carriers (Filka K., Coupek J., Kocourek J., Biochim. Biophys. Acta, 539, 518 (1978)] or the immobilization of enzymes and other biologically active agents (Coupek J., Turkova J., Valentova 0, Labsky J., Kalal J., Biochim. Biophys. Acta, 481, 289 (1977)]. Currently in n.p. LACHEMA Brno produces cross-linked polyhydroxyethyl methacrylates and their derivatives under the trade name SPHERON with different grain and different porosity. Three derivatives are known: diethylaminoethyl, carboxymethyl and its hydrazide, aroinoaryl, p-nitrophenoxycarbonyl-methyl, aminoaryl, oxine (8-hydroxyquinoline bound by azo), salicyl, thio. Of these, aminoaryl, hydrazide, p-nitrophenoxycarbonylmethyl and 1,2-epoxypropyl can be used as carriers of biologically active substances. Their disadvantage is that prior to binding of the biologically active substance requiring further explosive activation (aroinoaryl and hydrazido derivatives, they are converted into reactive, explosive azoderivatives after reaction with nitrous acid), they contain a hydrophobic chromophoric group which can be sorbed non-specifically to the support and distorted the binding results (p-nitrophenoxycarbonylmethyl derivatives), or contain a reactive group, which over time can be resolved by reaction with air oxygen or air humidity (1,2-epoxypropyl derivatives).

The present invention is solved by the present invention, which is based on the 2-hydroxy-3-chloropropyl-derivative of cross-linked polyhydroxyethyl methacrylate of the general formula (I).

CH, I 3C -I

CO i 0

I? 2 CH2 ?.

Whatever

I c - CH, CH. CH, I 3-Ο-Ι COi

O

I CH,

CH 2 OH, CH 2 OH, CH 2 OH - CH 2 OH - Cl CH, CH,

I 3-> C-CO 1

I CH, I 2CH,

2OH CH. CH3

- c -I what

I o

I CH, I 2CH,

Whatever

I -o-CH, (I)

Since it is a cross-linked product, the exact index value n cannot be determined.

In contrast to the previously synthesized derivatives, its use has the advantage that it does not require activation prior to ligand binding and does not release sorbable chromophore. The amount of ligand bound can be monitored by acid-base titration. The compound of the present invention is substantially more stable than the corresponding 1,2-epoxypropyl derivative of the compounded polyhydroxyethyl methacrylate, in which the content of reactive epoxide groups decreases over time. At the same time, the reactivity of the chlorine in the molecule of the present invention is sufficient to allow binding through primary, optionally secondary, hydroxyl and amine groups, optionally hydrazine groups. In addition, while the 1,2-epoxypropyl derivative of crosslinked polyhydroxyethyl methacrylate reacts due to the high reactivity of oxirane with polyfunctional protein-type reactants with multiple types of functional groups simultaneously, the compound of the invention reacts more selectively in a mildly alkaline environment.

The new chemical, 2-hydroxy-3-chloropropyl propylate polyhydroxyethyl methacrylate, is prepared from known entangled polyhydroxyethyl methacrylate by reaction with chloromethyl oxirane in a diosine medium in the presence of perchloric acid. Acid catalysis ensures high efficiency of the reaction with the suppression of unwanted by-products. EXAMPLE 1 2 g of ground poly-hydroxyethyl methacrylate (Spheron-300, 0.063-0.100 mm) were suspended in 20 ml of dioxane. Chloromethyloxirane (20 ml) and a 1 ml mixture of HCI (70%) and dioxane (5 ml) were added sequentially. Finally, 1 ml of water is added to the mixture and the mixture is heated to reflux with stirring at 95 to 97 ° C for 4 hours. Stopping the reaction and cooling to 20-25 ° C were added 20 mL of acetone to the mixture. After stirring and filtering off, the mixture is suspended in 20 ml of acetone, filtered and washed with 20 ml of acetone and dried in air for 24 hours. Product weight: 2.0 g, chlorine content 7.50% by weight. Example 2

The procedure is as in Example 1 except that 30 ml of chloromethyloxirane and 1.5 ml of water and 2.0 ml of HClO2 (70%) are used. The reaction time at 102 ° C was 8 hours. Yield: 1.8 g, chlorine content 8.7% by weight. EXAMPLE 3 Application 2 g of 2-hydroxy-3-chloropropyl-derivative of cross-linked polyhydroxyethyl methacrylate (Spheron-300, 0.020-0.040 mm) prepared according to Example 1 is suspended in 5 ml of 0.1 mol / l carbonate. sodium, pH 10.0 and allowed to stand at room temperature for 2 hours. Thereafter, 5 ml of a 10% mannan solution isolated from the Saccharomyces cerevisiae cell walls in tomist buffer is added and incubated on a shaker at 40 ° C for 6 hours. The support is then washed with 2 ml of 1N NaCl, water, ethanol, acetone and dried. The bound mannan content was determined by the phenolic method and was 13.5 µmol per gram dry carrier. The carrier thus prepared can serve for the isolation and purification of mannanases, mannosidases or lectins. Specific namanose from different biological sources. EXAMPLE 4 Application 2 g of 2-hydroxy-3-chloropropyl propylate poly-hydroxyethyl methacrylate prepared according to Example 1 was suspended in 5 ml of 0.1 mol / l sodium carbonate pH 9.0 and quenched at room temperature. h. 5 ml of bull pancreas trypsin solution of 20 mg / ml and enzyme activity of 670 nkat.mg (determined on N-benzoyl-arginine-ethyl ester as substrate at 25 ° C according to Kédy et al.) were then added. Biochemistry 4, 2,303, (1965) in the same buffer and the suspension was incubated at room temperature with stirring for 24 hours, then the carrier was washed with 2 mol / l sodium chloride, TRIS-HCl buffer, pH 8.2a the immobilized enzyme activity as well as the protein concentration in the supernatant after immobilization was determined From the protein loss in the supernatant after immobilization, it was concluded that 25 mg protein per g carrier (dry) and enzyme activity was 469 nkat.mg-1 immobilized protein on the carrier, i.e., the immobilization efficiency, based on the enzyme activity of the test, was used to isolate its inhibitor from seracids, or to specific protein cleavage, or to synthesize peptide linkage.

The new chemical 2-hydroxy-3-chloropropyl derivative of cross-linked polyhydroxyethyl methacrylate has utility in biotechnology amedicine for the incorporation of biologically active substances such as sucrides or amines in the affinity chromatography of enzymes, lectins and nucleic acids and the immobilization of enzymes.

Claims (2)

I 263374 4 PKBCMET OF THE INVENTION 2-Hydroxy-3-chloropropyl derivative of a stranded polyhydroxyethyl methacrylate containing repeating units of formula I ch 3 - C - co I o CH 2 CH 2 o I - I - CH, CH, CH, 3 C - CH , CO 10 I CH 1 t 2 CH 2 o I CH 1 I 2 CH - OHI CH2 - Cl CH, i - L, ch3 C - ch2 - C - what CO I 1 0 0 I CH, | 2 CH, 1 CH, | 2 CH 1 2 OH 0 i 1 1 η -Ο-3 ch 3 (I). Severografia, n. P., MOST Price 2,40 Kcs