CS217458B1 - Process for preparing mercaptohydroxypropylcellulose - Google Patents

Abstract

The essence of the invention consists in the fact that cellulose with 5 to 15% moisture is catalytically etherified in the first step with epichlorohydrin at a temperature of 80 to 95 °C for 2 to 4 hours at molar ratios of the andhydroglucose unit of cellulose: epichlorohydrin: carbonic acid or hydrochloric acid of 1:3 to 5: 0.03 to 0.04, after which in the second step, by reaction with sodium thiosulfate or by reaction with sodium hydroxide and with sodium thiosulfate, it is converted to thiosulfate hydroxypropylcellulose, which is reduced in the appropriate step by thiols in aqueous buffer solutions. The invention has applications in the isolation and purification of enzymes and proteins containing thiol groups, purification of peptides, synthesis of adsorbents for affinity chromatography, reduction of disulfide bonds in proteins, reversible immobilization of enzymes through their cysteine residues in industrial practice or in research.

Description

2

The present invention relates to a process for the preparation of mer-captohydroxypropylcellulose, a useful use in enzyme engineering. The preparation of the water-insoluble thiol group-containing carrier has been discussed in the preparation of mercaptone deoxycellulose. The best properties for their application to enzyme engineering may have polytols, in which the SH-sphcupine spheres bound by "bridge" (spacers), in the case of mercaptohydroxypropylcellulose, would be hydroxypiropyl moiety, such a derivative of agarose supplied by Pharmacia FineChemicals, Uppsala (Sweden) pdd labeled Thiopropyl-Sepiharose 6, B (at a price of 357 Swedish crowns for 10 g).

The process for preparing such a derivative, which results from the preparation of the oxira derivative and the use of all chemicals from the foreign exchange, is not suitable for cellulose (U.S. Pat. No. 201,363). Better possibilities are given by acid catalyzed etherification of cellulose with 1-chloro-2,3-epoxypropane (epichlorohydrin) using fluorofluoride catalysts. However, there are some variations from the hillfort of cellulose choice (P. Gemeiner, J. Zemek, Coileiot.Czech. Cheim. Commun., V 46 (1981)). This remains, while maintaining good conversions, eliminates the use of lanorganic acids (HCIO 4, HCl) as "acid" catalysts. The major benefits here are mainly the possibility of using mafcro-H + Z (CH 2 -CHCH 2 Cl - -> cd ^ OCH 2 GH '). (OH) CH 2 Cl (1)) of poirical pearl cellulose, which has undeniable advantages over traditional cellulose materials (MS AO 20 20 363; MS AO No. 206 346 and MS No. 206 378). In a further embodiment, the most preferred cetetation is thiosulfate derivatives, in this case the reaction of fluorhyldroxypropylcellulose with sodium thiosulfate (No. AO No. 203 818) to form thiosulfate hydroxypropylceululose. The unconventional conversion yields the preparation of the thiosulfate derivative through oxirane. However, the process is less energy intensive. The reduction of thiosulphate derivatives with thiols is thus used to prepare Thiopropyl-Sepharoise 6B as mercaptone deoxycellulose.

The above-mentioned disadvantages are eliminated by the present invention, wherein the first stage catalyzes the cellulose 5 to 15% moisture of epichloryl hydrinium at a temperature of 80 ° to 95 ° C for 2 to 4 hours of the anhydroglucose unit cellulose: epichlorohydrin: alkyl chlorinate or acid hydrochloric acid 1: 3 to 5: 0.03 to 0.04, then in the second stage by reaction with sodium thiosulphate or with sodium hydroxide and sodium thiosulphate to form thiosulphate hydroxypropyl cellulose, which in third stage is reduced by thiol in buffers.

The known acid catalyzed etherification method makes it possible to use both traditional forms of cellulose (powdered, fibrous or microcrystalline), their cross-linked modifications, or macroporous pearl cellulose. The most reactive form is epichlorohydrin cross-linked powder cellulose i (P. Gemeiner et al., Collect. Czech. Ghem. Commun. 45, 2847 (1980)). Here, at the lowest temperature (80 ° C), it reaches the highest (degree of sulbstation / dissolution (DSCi = 0.67). Perl cellulose requires removal of water before use, best equilibration in acetone and ether (J. Peška et al. Chem., Technol., 419 (1978)) The exchange of chlorine with thiosulfate groups requires a temperature of 95 ° to 105 ° C for 15 to 60 hours, depending on the chlorine content. In the first stage, 1 hour, in the second stage 6 to 16 hours, if the crosslinked cellulose is, for direct exchange, it accounts for 8.74% of sulfur, 8.15% sulfur over the oxirane process, the more preferred pearl i (2 * 9.3%) and cellulose (19.4%) cellulose are the more preferred pearl and (2 * 9.3%) matrices of the total conversion to mercapitohydroxypropylcellulose. the content of SH groups (0.43 relsp.33 mmoles SH / g), and the thiosulfate derivative derivatives of thiols in aqueous buffers are more effective more alkaline medium (pH 9), however, it is also necessary to use an effective thiol-protective reagent such as tri-n-ibutylphosphine. If high demands on the content of SH groups in the final product are not required, it is sufficient to work at pH 8 and use cheaper Chelaton 3 and thioglypholefin. The advantage of the proposed method for the preparation of mercapto-hydroxypropylcellulose is that it makes it possible to produce preparations of home-made inexpensive silicones by a simple technological process. EXAMPLE 1 1.6 ml of water and 17.6 ml of epilohorohydrin were added to 9 g of dried cellulose, stirred for 30 min at room temperature, and 0.2 ml of 60% brick chlorosulphate was added incrementally. intermittent mixing. The product (8.6 g) was washed with water and acetane; contained 4.6% chlorine. 5 g of this chlorohydroxy-propylcellulose was resuspended in 15 ml of 4.4 M sodium sulfate and maintained at 100 ° C for 15 hours with occasional mixing. Washing with water and acetone, the product (4.95 g) contained 4.05% sulfur. This was resuspended in 27 ml of 50 mM sodium tetraborate containing 1.15% v / v tributylphosphine, added with 3.7 ml of 2-mercaptoethanol and stirred for 30 minutes at room temperature and pH for 30 minutes after adjusting pH for 9 minutes. 9. The product (4.3 g) was washed rapidly with 1 mM Chelaton 3, water and acetone; it contained 2.3% sulfur and 0.33 mmol SH-groups / g. Example 2

Chlorhydroxypropylcellulose (11.9 g; 10.6% chlorine) was prepared by the procedure of Example

Claims (1)

3 du 1, with the result that the powdered cellulose cross-linking degree = 10 per volume of 3.6 ml water / g and a temperature of 80 ° C was used. 5 g of this intermediate was suspended in 15 ml of 20 M sodium thioisulfate and kept at 100 ° C for 40 hours with occasional mixing. After washing with water and acetone, the product (5.25 g) contained 8.74% sulfur. After resuspension in 51 ml of 50 mM tetralborate sodium containing 1.2 v / v tributylfoisinone, 9 ml of β-mercaptoethanol was added and after adjusting the pH to 9, the pulp suspension was stirred for 30 minutes at pH 9 and pH 9. Product (4.45 g) sapremyl rapidly with 1 mM Ghelaton, water and acetone: contained 5.8% sulfur and 0.19 mmole SH-iskupm / g. Example 3 Chlorohydroxypropyl-cross-linked cellulose (5g) prepared according to Example 2 was mixed with 5 ml of 0.485 M sodium hydroxide at room temperature for one hour, washed with water, 50 ml of 2.3 M sodium thiosulfate in 0.5 M phosphate buffer. 6.3 and aspirated. After resuspension in 2.3 M sodium thiosulfate in 0.5 M phosphate buffer pH 6.3 (25 mL), it was stirred overnight (16 h) at room temperature, washed with water, acetone and filtered off with suction. . 8.15% sulfur (5.7 g) was resuspended in 53 ml of 50 mM sodium tetralborate containing 1.2% trilobutylphosphine, 9.25 ml of 2-mercaptoethanol were added and the suspension was stirred after adjusting the pH to 9sa 30 min at room temperature and pH 9. The product (4.45 g) was washed rapidly with 1 mM Ghelaton 3, void and acetone: containing 5.07% sulfur and 0.16 mmol SH-is-pine / g. EXAMPLE 4 Chlorohydroxypropyl peroxy cellulose (32.6g, 6.0% chlorine) was prepared according to the procedure of Example 1, except that 9.55g of dry pearl cellulose, pre-dried with acetone and ether, was added. 1.05 ml of water and the temperature was 90 ° C. 14.5% of the intermediate was washed slowly SUBJECT Method of preparation of mercapto-hydroxypropyl cellulose, characterized in that 5 to 15% moisture of cellulose was catalytically etherified by epiohlorohydrin at 80 to 95 ° C for 2 to 4 hours with anhydreglucose ratios. cellulose units: epichlorohydrin: acid with 5.7 M sodium thiosulphate (250 mL), aspirated to 30 mL of this same thioisulfate solution. The suspension was maintained at 20 ° C with occasional mixing, the product (13.7 g) was washed with water and aspirated, containing 7.8% sulfur. After a rapid wash with 50 mM sodium tetraborate (200 mL), aspirate and resuspend in 50 mM tetraborate containing 1.2% v / v tributylphosphine (38 mL), 7.5 mL 2-mercaptoethanol was added and after adjusting the pH to 9, the suspension was stirred at room temperature and pH 9 for 30 minutes. The product (11 g) was washed with 1 mM Ghelaton 3 and 0.1 M acetic acid containing 1 mM Ghelaton 3 in which it was also stored at 4 ° C. The product contained 5.1% sulfur and 0.43 mmoleSH-1 / g of sudden weight. EXAMPLE 5 The procedure of Example 2 except that hydrochloric acid (0.4 ml) and etherification with epichlorohydrin were added as catalyst. the mercaptohydroxypropyl cross-linked cellulose contained 2.32% sulfur and 0.075 mmol SH-sfcu-pine / g. EXAMPLE 6 The procedure of Example 1, except that 4.2 ml of 80% thioglyphic acid was used to reduce thiosulfate hydroxypropyl powdered cellulose, 50 mM sodium tetraborate contained 1 mM Chelaton 3 and the pH was maintained at 8. The product contained 2.4% sulfur and 0.25 mmol SH-groups / g. The invention is applicable wherever it is necessary to isolate or purify enzymes and other thiol-containing proteins, isolate molecular thiol compounds, purify (synthetic) peptides, synthesize adsorbents for affinity chromatography, reduce disulfide bonds in the protein, reverse not immobilize enzymes via iclcysteine residues, either in industry or research. Crude or hydrochloric acid 1: 3 to 5: 0.3 to 0.04, then converted to thiosulphate hydroxypropylcellulose by reaction with sodium thiosulphate or with reactive sodium hydroxide and sodium thiosulphate, the third stage reduces the flame-retardant buffers.