CH653348A5 - SORBENTS FOR SACCHARIDES AND SACCHARID-CONTAINING POLYMERISATES AND METHOD FOR THE PRODUCTION THEREOF. - Google Patents

Description

The invention relates to sorbents for saccharides and polymers containing saccharides, e.g. Glycoproteins, as well as a process for the production of such substances.

Proteins that are able to selectively form complexes with sugars, polysaccharides and glycoproteins and that have the general name “lectins” have been isolated from a whole range of plant and animal sources. They are characterized not only by their origin, but above all by their specific affinity for the sugar substances and their derivatives.

By binding the lectins to a suitable carrier, specific adsorbents for saccharides are formed. The condition for such a binding reaction is to maintain the active sorption center in the lectin molecule, which must not be blocked during the reaction. The sorbents containing lectins are used in analytical and preparative affinity chromatography and in selective precipitation techniques used to isolate biologically active fractions.

Lectins were already on polysaccharides from dextran or. Agarose type bound using the cyanogen bromide method and for a whole range of isolation techniques in the separation of glycoproteins (K. Aspberg, J. Porath, Acta Chem. Scand, 24 1970, 1839), dextrans (KO Lloyd, Arch Biochem. Biophys. 137, 1970, 460)

or cells (G.H. Edelman, U. Rutishauser, C.F. Millete, Proc. Nat. Acad. Sei. USA, 68, 1971, 2153). An advantage of this process is its selectivity and speed. However, carriers of the dextran or polysaccharide type also have certain disadvantageous properties. These properties include, above all, large swelling differences in solutions with variable ionic strength and pH, low mechanical strength of particles and the resulting difficulties due to clogging of the columns to be filled with such materials at elevated pressure values and through reduced flow rates of the mobile phase. Last but not least, a relatively low resistance to hydrolysis and the action of microorganisms can stand in the way. Binding of the lectin molecules in the cyan bromide method requires using a highly toxic

Activation agents of relatively low effectiveness to work. The bond created with the cyanogen bromide method is not stable enough for certain purposes. Their successive hydrolytic cleavage with a simultaneous decrease in sorption capacity was determined.

The substances according to the invention eliminate the aforementioned disadvantages of the previously known sorption materials. The starting compounds are preferably processed according to the procedure according to Czechoslovak copyright certificate no. 206 823.

The present invention relates to selectively acting polymeric sorbents for saccharides and saccharide-containing polymers as defined in claim 1.

The method for producing these sorbents is defined in claim 2. Sodium borohydride is preferably used as the reducing agent.

The starting compounds of the process according to the invention are oxidized with periodate, as a result of which the C-C bond between two carbon atoms with vicinally bound hydroxyl groups is cleaved in molecules of the covalently bound saccharides with the formation of two aldehyde groups which are considerably reactive. The polymer activated by oxidation reacts with lectins to form a Schiff base. The speed of this reaction depends on the concentration of the components and on the temperature, and is strongly influenced by the pH of the reaction medium. The unreacted active aldehyde groups are reduced by the action of sodium borohydride. The resulting sorbents are characterized by high capacity.

The effects of the substances according to the invention are comparable with known and available saccharides bound with lectins by the cyan bromide method if the adsorbing capacity of the sorbent is selected for the comparison. The method according to the invention is essentially lighter from a technological point of view, does not require any special measures for working with highly toxic activating agents and enables simple control of the degree of conversion of binding reactions with high reproducibility. In addition, the non-oxidized saccharide molecules bound to the carrier give this carrier a hydrophilic character or increase its original hydrophilicity. In this way, the probability of non-specific interactions of the components of the systems to be inhibited is reduced when the sorbents are used in affinity chromatography.

The subject matter of the invention is to be explained in more detail by the following exemplary embodiments.

example 1

15 g of copolymer of 2-hydroxyethyl methacrylate and ethylene methacrylate with a molecular weight cut-off limit of 1,000,000, which also contained 16% covalently bound D-galactose (eg “Separon” HEMA-1000-Gal), was swollen in 150 ml of water for 20 hours . The liquid was then suctioned off and 346 ml of 0.1 M NaI04 were added. The oxidation was carried out at 25 ° C. for 100 minutes. The product was washed with water until the negative reaction to oxidizing agent (Mn **), equilibrated with a buffer in which the binding of the lectin was carried out (acetate buffer with different pH) and used in the subsequent binding reaction.

140 ml of acetate buffer (pH 7.8) was used to dissolve 2.4 g of lectin from Canavalia ensiformis DC (Concanavalin-A)

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and the equilibrated oxidized carrier from the previous step was added to this solution in the form of thick slurry together with 250 mg of D-glucose. The mixture was stirred in a refrigerator at 4 ° C for 24 hours. 25 mg of NaBH4 were then added, and after 20 minutes a further dose of 25 mg of NaBH4 was added. The mixture was left in contact with sodium borohydride for a total of 40 minutes. Afterwards, the sorbent was washed with acetate buffer (pH 7.3) and further washed three times with 200 ml of Tris-HCl buffer in 1 mol NaCl (pH 8) and with acetate buffer in 1 mol NaCl (pH 4). The sorbent treated in this way was transferred to a column and washed with a buffer in which a sorption effect against saccharide derivatives was demonstrated. The amount of concanavalin bound was 15 mg per gram of the dry vehicle.

The effect of the bound lectin was determined by sorption of a soluble polyacrylamide derivative with polymerized allyl-a-D-glucoside [V. Horejsi et al., Biochim. Biophys. Acta 538, 293 (1979)] proved by the following process:

0.3427 g of soluble polyacrylamide polymers with 9.4% by weight of glucose in the starting buffer were added to a column of 1 g of support with bound lecton after equilibration with acetate buffer (pH 7.3). When 102 ml of buffer was eluted, the sugar content was monitored to zero. A solution of 0.5 mol of methyl a-D-glucopyranoside (30 ml) was then brought to the column, whereupon the column was washed with the starting buffer (pH 7.3). The eluate was collected, dialyzed and lyophilized. 0.016 g of polymer was determined gravimetrically. Due to the material balance, a 3.1% loss of glycosylated polyacrylamide standard was found in the chromatography.

Example 2

The sorbent was prepared and treated analogously to Example 1. Its effect has been demonstrated by selective sorption of ovalbumin. Repeated use was also investigated. 1 g of sorbent, according to Example 1, with bound concavalin-A was equilibrated by acetate buffer (pH 7.3) and subsequently by Tris-HCl buffer, pH 7.8 and 0.15 M NaCl. 49 mg of ovalbumin in 5 ml of start-up buffer were added to the column and the system was washed with buffer until there was a negative reaction to proteins (UV absorption 280 nm) while simultaneously collecting the eluate. Then a 0.1 M solution of methyl-a-D-glucopyranoside (50 ml) was prepared. The eluate was collected, dialyzed and lyophilized. The yield in the eluate was 7.8 mg. The unbound protein content was determined to be 40 mg, loss 2.44%.

The column was then washed with 20 ml of Tris-HCl buffer, pH 7.8 and 1 M NaCl content and with 20 ml of acetate buffer, pH 4 and 1 M NaCl content, with Tris-HCl buffer, pH 7, 8 equilibrated with 0.15 M NaCl content and the entire process repeated.

In the second cycle, the protein content in the eluent was found to be 7.6 mg after washing with methyl-a-D-glu-copyranos'd, in the third cycle with 7.8 mg and in the fourth cycle with 7.6 mg.

Example 3

The sorbent was prepared analogously to Example 1, with the exception that instead of the copolymer with the molecular weight cut-off limit of 1,000,000 daltons, a copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate of 300,000 molecular weight was used as the carrier. When the effect was tested by glycosylated polyacrylamide, a 4.6% loss (0.3470 g batch, 0.0154 g in the eluent) was found. The starting sorbent contained 18% by weight of covalently bound glucose.

Example 4

The oxidized carrier described in Example 3 was used to bind the lectin from Ricinus communis. 1.9 g of lectin were dissolved in 70 ml of acetate buffer (pH 7.3) containing 220 mg of galactose and 10 g of the oxidized carrier were added. The elimination of the unreacted active aldehyde groups was carried out with 25 mg NaBH4. Response time: as indicated in Example 1.

The effect was tested by polyacrylamide with 9.4 percent by weight sugar. Batch amount: 0.3412 g; Yield in the eluent: 0.0154 g; Losses: 3.8%.

Example 5

Binding of the Ricinus communis lectin was carried out analogously to Example 4, with the exception that the oxidized carrier had a molecular weight cutoff of 300,000 daltons.

When testing the effect of polyacrylamide with bound D-glucose, 0.3400 g of polymer were made up per 1 g of the support column. Yield in the eluent: 0.0171 g; Veluste: 4.75%.

Example 6

4.2 g of spherical cellulose was allowed to swell to 50% by weight in 50 ml of dioxane saturated with hydrogen chloride gas within 20 hours. After this period, 2.0 g of D-galactose was added to the reaction mixture and it was then shaken at laboratory temperature for 24 hours. The contents of the vessel were then poured into 5 liters of water and the product was washed repeatedly with water to a neutral pH after suction through a filter to give a negative reaction to sugar.

4.0 g of spherical cellulose with bound D-galactose (11.7% by weight of bound D-galactose analytically determined) was left to swell in distilled water for 20 hours. The water was then filtered off and 100 ml of 0.1 M NaIOé was added to the wet gel. The oxidation was carried out for 1 hour at the laboratory temperature with stirring. The sodium periodate solution was then filtered off and the gel on the column was washed through with distilled water until the conductivity of the eluate corresponded to that of the distilled water used. Borate buffer of pH 8.5 and, after 1 hour, 500 mg of concanavalin-A (lectin from Canavalia ensiformis) were then added to the cellulose gel. The lectin was inhibited with methyl-a-glucopyranoside. After a residence time of 70 hours at 4 ° C., the unreacted active groups were reduced at 4 ° C. with 20 mg NaBH4 with stirring for 20 minutes. The gel was then washed alternately with acetate buffer pH 4.1 (1 M NaCl) and Tris-HCl buffer pH 9 (1 M NaCl). In the final phase before application, the gel was stored in acetate buffer pH 6 (1 M NaCl) containing 10-3 M MnCl and 10 -3 M CaCl2. The bound protein content was 9.0 mg per millimeter of the wet gel.

Example 7

The aminoglycosyl derivative of cellulose was prepared analogously to Example 6, with the exception that in the

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Reaction 0.8 g of N-acetyl-D-glucosamine used, and that the product was not oxidized. Only the amount of amino sugar bound to the cellulose support was determined.

Example 8

A similar experiment as in Example 7 was carried out with the carrier consisting of hydroxyethyl methacrylate copolymer with a molecular weight cut-off limit of 1,000,000 (see Example 1). The content of the bound amino sugar was 8.9 percent by weight, which was twice the amount compared to Example 7.

Example 9

4.0 g of soluble polyacrylamide gel with polymerized allyl-aD-glucoside (manufactured according to V. Horejsi, P. Smo-lek and J. Kocourek: Biochim. Biophys. Acta, 538 (1978), 293 to 298) oxidized in 100 ml 0.1 M NaI04 for 1 hour at laboratory temperature. At the end of this time, the reaction mixture was diluted to 250 ml and dialyzed against distilled water. After removal of the periodate from the reaction mixture by dialysis, the gel was lyophilized. The lyophilisate was dissolved in 20 ml of distilled water. The solution was 500 mg of lectin from Canavalia ensiformis, inhibited with methyl ^ a-D-

-glucopyranoside, added. The mixture reacted within 70 hours at 4 ° C, whereupon the remaining aldehyde groups were reduced by reaction with NaBH4, similar to that in Example 6. The resulting reaction mixture - after thickening by lyophilization - was separated by preparative gel chromatography on dextran gel into a lectin fraction and a fraction containing lectin bound to polyacrylamide with polymerized allyl-a-D-glucoside.

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Example 10

4.0 g of polyvinyl alcohol (125,000 molecular weight and 87 to 89% degree of hydrolysis), which by D-glucose according to the Czechoslovak copyright certificate No. 195 044 15 was substituted, was oxidized analogously to Example 9.

Example 11

The sorbent was prepared analogously to Example 1, with the exception that instead of the copolymer 20 of ethylene dimethacrylate and hydroxyethyl methacrylate, a copolymer of hydroxyethyl acrylate with ethylene diacrylate of 500,000 Dalton molecular weight cut-off was used. A 5.3% loss was found by testing the effect of polyacrylamide with copolymerized allyl-a-D-glu-25 coside.

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Claims (3)

653348 1. Sorbents for saccharides and saccharide-containing polymers, e.g. Glycoproteins, characterized in that lectins are covalently bound to carriers which are selected from oxidation products of polysaccharides, of glycosylated hydroxyalkylacrylate polymers, of glycosylated hydroxyalkyl methacrylate polymers, of polyacrylamide containing glycosylated monomer units or of glycosylated polyvinyl alcohol. 2. A process for the preparation of sorbents according to patent claim 1, characterized in that the polymers containing covalently bonded saccharides to be oxidized to the oxidation products mentioned are oxidized with sodium or potassium periodate, then the polymers thus activated with lectins at a temperature of 0 to 40 ° C can react in buffer solution from pH 7 to 12, and then the active aldehyde groups remaining after the reaction are eliminated from the polymer by the action of reducing agents. 2nd PATENT CLAIMS 3. The method according to claim 2, characterized in that sodium borohydride is used as the reducing agent.