CS203818B1 - Process for preparing mercaptodeoxycellulose - Google Patents

Description

The invention relates to a process for the preparation of mercaptodeoxycellulose from chlorodeoxycellulose.

Water-insoluble carriers containing thiol groups have a wide range of applications in various areas of enzymatic engineering. They are most commonly used in the Thiol Enzyme Affinity Chromatography (Methods in Enzymology 34, Affinity Techniques) 1974 and in the immobilization of "enzymes". R. Zaborsky, Immobilized Enzymes, CRC Cleveland 1974 /. Except for the polyacrylamide cross-linker (Enzacryl Polythiol, Koch-Light), polytioles exclusively based on polysaccharides are produced today, mainly agarose, e.g. , all Pharmacia Fine Chemicals, Uppsala.

The preparation of these thiol-containing polysaccharides is based on the activation of the polysaccharide by cyanogen bromide [Activated Thiol-Sepharose 4B, Upplands Grafiska AB (1974), respectively. from O-alkylation of agarose with bis-epoxides, -halohydrinini / R. Axen, H. Drevin, J. Carlsson, Acta Chem.

Scand. B 29/1975/471; H. Nishikawa, P. Bailon, J. Solid-Phase Biochem. 1 33 /. These methods use unusual polysaccharides (agarose, dextran) and foreign exchange chemicals (eg: cyanogen bromide, glutathione, 2,2'-dipyridyldisulfide, bisepoxides, dithiothreitol).

Moreover, in cellulose, these methods of preparation do not give satisfactory results [J, Zemek, L. Kuniak,

P. Gemeiner, PV-7358 (78). On the other hand, chlorodeoxycellulose (CD-cellulose), readily obtainable under mild reaction conditions with relatively high degrees of substitution (DS 0.3) (T), appears to be a suitable starting material for the preparation of thiol-polysaccharides. L. Vigo, C.W. Welch, Textile J. Res. 40 (1970) (109). The chlorine in this CD-cellulose can be replaced by conventional nucleophilic substituents, e.g. -amino, -mercapto, -hydroxy and others. Thus, in the case of chlorodeoxydextran, substitution with thiosulfate is described to form thiosulfate-deoxydextran (TSD-dextran) / A. F.

Kolova et al., Cell. Chem. Technol. 12 (1978). Such TSD-dextran is then converted to mercaptodeoxydextran (MD-dextran) by alkaline cleavage under argon.

Our research in this field has shown that CD-cellulose can also be converted to thiosulfate-deoxycellulose satisfactorily. Its reduction by thiols such as thioglycolic acid or 2-mercaptoethanol in weakly alkaline medium and in the presence of chelating agents leads to the formation of mercaptodeoxycellulose.

SUMMARY OF THE INVENTION In the first step, the CD-cellulose is converted in an aqueous sodium tipulphate solution at a temperature of 90 to 105 ° C for 35 to 45 hours at a molar ratio of CD of the polysaccharide: thiosulfate of 1:10 to 15 for hydromodule. up to 15 to thiosulphate-deoxycellulose, then in the second step after washing the product to a negative reaction to the thiosulphates and drying, an aqueous thioglycolic acid buffer or a solution of sodium thiosulphate are added. 2-mercaptoethanol at pH 7.7 to 7.9 containing 1 to 5 mM Chelaton 3 at a molar ratio of the thiosiran group of the polysaccharide: thiol of 1:15 to 20 and a hydromodule of 10 to 15 and allowed to act for 20 to 40 minutes at a temperature of 20 to 30 C.

CD-cellulose prepared according to I. L. Vigo, C.M. Welch, Textile Res, J, 40/1970/109 with a DS of 0.3 to 0.6 is sufficiently suitable for the preparation of MD-cellulose. Such CD-cellulose has a markedly increased heat resistance.

The introduction of the thiosulfate group can be carried out with sodium thiosulfate at a temperature of 90 to 105 ° C for 35 to 45 hours. The resulting product contains 0.4 to 0.6 mmol of thiosulfate groups per g dry formulation.

The reduction of the thiosulfate group to thiol is accomplished by treatment of thioglycolic acid or 2-mercaptoethanol in aqueous buffers of pH 7.7-7.9 containing Chelaton 3.

After reduction, the product is washed rapidly with a buffered aqueous solution containing Chelaton 3, distilled water containing Chelaton 3 and acetone. The resulting product contains 0.25 to 0.5 mmol of thiol groups per g dry formulation.

The advantage of the proposed process for the preparation of mercaptodeoxycellulose is that the preparations make it possible to produce from domestic cheap raw materials by a relatively simple technological process.

Examples of design

Example 1 g of chlorodeoxycellulose (CD-cellulose) containing 1.8 mmol of Cl / g was swollen in 100 ml of a 2.1 M aqueous sodium thiosulfate solution. The reaction mixture is then held at 100 ° C for 40 hours with occasional stirring. The product was washed with water to a negative reaction to the thiosulfate ions, acetone and allowed to dry at 40 ° C. The dry product is suspended in 80 ml of a 0.1 M aqueous sodium bicarbonate solution containing 5 mM Chelaton 3. While stirring, 10 ml of thioglycolic acid are added and the pH is adjusted to 7.8 with concentrated sodium hydroxide. The suspension is allowed to stir for 30 minutes at room temperature. The product was quickly washed on a glass filter with 0.1 M aqueous sodium bicarbonate solution containing 5 mM Chelaton 3, distilled water containing 1 mM Chelaton 3 and acetone, each time to a negative reaction to the thiols. The product is dried at 40 ° C.

The preparation contains 2.4 Z sulfur and 0.45 mmol / g titratable thiol groups.

E2SSÉS! S2 ^{: +} Chelaton 3 - sodium salt of ethylenediaminotetraacetic acid

Example 2

The procedure of Example 1, except that 10 ml of 2-mercaptoethanol is used in the reduction of CD-cellulose thiosiran. The dry product contains 2.85 Z sulfur and 0.33; mmol / g titratable thiol groups.

Example 3

The process according to Example 1 is the difference that the starting CD-cellulose is a preparation containing 3.2 Ram of Cl / g and in the first stage 3.85 M aqueous sodium thiosulfate solution. The dry product contains 2.5 Z sulfur and 0.25 mmol / g titratable groups.

The invention has application wherever it is necessary to isolate or purify thiol-group-containing enzymes and other proteins, and where it is preferable to immobilize the enzymes via their cysteine residues, whether in industrial practice or in research.

Claims (1)

Process for the preparation of mercaptodeoxycellulose characterized in that in the first stage chlorodeoxycellulose is reacted with an aqueous solution of sodium thiosulphate at a temperature of 90 to 105 ° C for 35 to 45 hours at a molar intake of the chlorodeoxy CD of the polysaccharide group: thiosulphate 1:10 to 15 at hydromodule 10 to 15, then in the second step, after washing the product to a negative reaction to the thiosulfates and drying, an aqueous buffer of thioglycolic acid or 2-mercaptoethanol of pH 7.7 to 7.9 containing 1 to 5 mM Chelaton 3 at a molar ratio is added thiosulfate groups of the polysaccharide: thiol 1:15 to 20 and hydromodule 8 to 12 and allowed to act for 20 to 40 minutes at 20 to 30 ° C and finally washed quickly on the filter with an aqueous buffer containing 1 to 5 mM Chelaton 3, distilled water containing 0.5 to 1 mM Chelaton 3 and. acetone, always in a negative reaction to the thiols.