CS206823B1 - Sorbents for saccharides,glycoproteins and polymers,containing saccharides and method of their manufacture - Google Patents

Abstract

Lectines are covalently bonded on vehicles selected from the group comprising oxidized polysaccharides, oxidized glycosilized hydroxyalkylacrylates or hydroxyalkylmethacrylates, oxidized polyacrylamide and polyvinylalcohol to form sorbents. The vehicles may be oxidized by the action of sodium or potassium periodate. Following the bonding of the lectines, remaining active aldehydic groups in the vehicle may be eliminated by the action of a reductant such as sodium borohydride.

Description

The invention relates to sorbents for carbohydrates, glycoproteins and polymers containing carbohydrates and processes; production.

Proteins that are capable of selectively forming complexes with sugars, polysaccharides and glycoproteins, and which bear the common name lectins, have been isolated from a variety of plant and animal sources. They are characterized not only by their origin, but also by their specific affinity for sugars and their derivatives.

By binding the lectins to a suitable carrier, specific adsorbents for carbohydrates are formed, the binding reaction being conditioned by the maintenance of active sorption. centers in the lectin molecule, which in the binding reaction; must not be blocked. Lectins containing sorbents also find use in analytical and preparative affinity ^! chromatography and selective precipitation techniques used in the isolation of biologically and ac; fractions. ;

Lectins were bound to dextrano-polysaccharides agarose type of cyanogen bromide. method and used for a variety of isolation and glycoprotein separation techniques (K. Aspberg = J. and J. Porath, Acta Chem. Scand. 24 (1970) 1839); dextrans (KO Lloyd, Arch. Biochem. Biophys. j L137 (1970) 460) or cells (GH Edelman, U. ii Rutishauser and CF Millete, Proc. Nat. Acad. Sci. J. USA, 68 (1971) 2153) . The advantage of the process is its high selectivity and speed. However, polysaccharide carriers of the dextran or polysaccharide type also have some disadvantageous properties, including, in particular, large differences in swelling in solutions of variable ionic strength and pH, low mechanical strength of the particles, and hence difficulties in clogging columns loaded with these materials at elevated pressures and flow rates. mobile phase speeds. Last but not least, the resistance to hydrolysis and the action of microorganisms may be an obstacle. Method for Binding Cyanogen Lectins The method represents the need to work with high ^! a toxic activating agent whose activity is relatively low. The bond formed in the cyanogen bromide process is not sufficiently stable for some purposes, its gradual hydrolytic cleavage was observed, accompanied by a decrease in sorbent bent capacity.

The compositions of the present invention overcome these drawbacks of the prior art sorption materials. The covalent bonding of the sugar molecules can preferably be carried out according to the procedure of MS. No. 195,044, utilizing the acid-catalyzed reaction of saccharides with hydroxyl-containing polymers in dioxane or tetrahydro-furan. In addition to hydrophilic and synthetic polymers (macroporous hydroxyalkylacrylates and hydroxyalkylmethacrylates), this reaction can be used in part!

hydrolysed cross-linked polyvinyl acetate and others) as well as a number of natural or semi-synthetic hydrophilic polymers (polydextrans, agarose polymers, cellulose, etc.) because the reaction itself has to be carried out in an anhydrous environment, which excludes possible hydrolysis of glycosidic or peptide bonds.

The resulting glycosyl derivatives of the polymers are subjected to periodate oxidation to degrade the C-C bond between the two carbon atoms with the vicinally bonded hydroxyls in the covalently bonded carbohydrate molecules to form two aldehyde groups, which are highly reactive. The oxidation-activated polymer reacts in the next step with lectins to form a Schiff base. The rate of this reaction depends on the concentration of the components and the temperature and is greatly influenced by the pH of the reaction medium. Unreacted aldehyde functional groups are reduced by the action of sodium borohydride. The resulting sorption or precipitation materials are characterized by high capacity.

The present invention provides selectively acting polymeric sorbents for carbohydrates, glycoproteins, and carbohydrate-containing polymers.

They are characterized in that lectins are covalently bound on carriers selected from the group of oxidized polysaccharides, oxidized glycosylated hydroxyalkyl acrylates or hydroxyalkyl methacrylates, oxidized polyacrylamide and polyvinyl alcohol.

The process for producing these sorbents is characterized in that the polymers containing cobalent-bound carbohydrates are oxidized with sodium or potassium periodate, followed by reacting the activated polymers with lectins at 40 ° C in buffer solutions of pH 7 to 12, leaving the remaining aldehyde after the reaction. functional groups are eliminated by the action of reducing agents, preferably sodium borohydride.

In their action, the compositions of the invention are comparable to known and available lectin-based polysaccharide materials by the cyanogen bromide process, the adsorption capacity of the sorbents being considered as an efficiency criterion. The process according to the invention is considerably easier from a technological point of view, does not require special measures for working with highly toxic activating agents and allows simple regulation of the degree of conversion of the coupling reaction at high reproducibility. In addition, the non-oxidized carrier-bound carbohydrate molecules lend it hydrophilic character or enhance its original hydrophilicity. This reduces the likelihood of non-specific interactions of the components of the partitioned systems using sorbents in affinity chromatography.

The subject matter of the invention is illustrated in the following examples without being limited thereto.

Example 1 g of 2-hydroxyethyl methacrylate-ethylene methacrylate copolymers with a molecular weight cut-off of 1,000,000 containing 16% covalently bound galactose (Separon HEMA1000-Gal) were swelled in water (150 ml) for 20 hours. Subsequently, the liquid was aspirated by addition of 346 ml of 0.1 M NaJO ₄ . The oxidation was carried out for 100 minutes at 25 ° C. The product was washed with water to a negative reaction to oxidizing substances (Mn ⁺⁺ ), equilibrated with lectin-binding buffer (acetate buffers of different pH) and used in the following coupling reaction.

140 ml of acetate buffer pH 7.8 was used: to dissolve 2.4 g of lectin from Canavalia ensiformis DC (concanavalin) and to the solution was added an equilibrated oxidized carrier from the previous step as a thick slurry together with 250 mg D-glucose. The mixture was stirred at 4 ° C for 24 hours. Then 25 mg NaBH ₄ was added and after 20 minutes another dose of 25 mg NaBH ₄ was added. The mixture was left with sodium borohydride for a total of 40 min. The sorbent was then washed with acetate buffer pH 7.3 and then 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 so treated was transferred to the column and washed with buffer in which a sorption effect against carbohydrate derivatives has been demonstrated. Amount per bound concanavalin 15 mg / g dry carrier.

The effect of bound lectin was verified by sorption of a synthetic polymer (acrylamide) containing covalently bound carbohydrates (glucose, galactose) as follows:

A column of 1 g of lectin-bound carrier after equilibration with an acetate buffer pH 7.3 was loaded with 0.3427 g of a soluble polyacrylamide polymer containing 9.4 wt% glucose in the starting buffer. The elution with 102 ml of buffer monitored the sugar content up to zero. The column was then loaded with a solution of 0.5 mol methyl-alpha-D-glucopyranoside (30 mL) and then the column was washed with a pH 7.3 start buffer. The eluate was collected, dialyzed and lyophilized. Gravimetric determination: 0.016 g of polymer was determined. On the basis of the material balance,

3.1% loss of glycosylated polyacrylamide standard.

Example 2

The sorbent was prepared and processed analogously to Example 1. Its effect was verified by the selective sorption of ovalbumin, and the reuse was evaluated. 1 g of the sorbents of Example 1 bound with concanavalin was equilibrated in acetate buffer pH 7.3 and then equilibrated in Tris HCl buffer pH 7.8 + 0.15 M NaCl. The column was loaded with 49 mg of ovalbumin in 5 ml of starting buffer and the system was washed with buffer up to; to a negative protein reaction (UV absorbance 280 nm) while collecting the eluate. Then, a 0.1 M solution of Me-alpha-D-glucopyranoside (50 mL) was seeded. 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. losses represent 2.44%.

The column was then washed with 20 ml of Tris HCl buffer pH 7.8 + 1 MNaCla20 in acetate buffer 4-1 M NaCl pH 4, equilibrated with Tris HCl buffer + 0.15 M NaCl pH 7.8 and the procedure repeated.

In the second cycle, the protein content of the eluent was determined after washing with Me-alpha-D-glucopyranoside 7.6 mg for the third 7.8 mg and for the fourth 7.6 mg.

Example 3

The sorbent was prepared in a manner analogous to Example 1 except that copolymers of 2-hydroxyethyl methacrylate with ethylenedimethacrylate with a molecular weight exclusion limit of 300,000 were used as the carrier instead of a copolymer with an exclusion limit of 1 million daltons. 6% loss (0.3470 g applied, 0.0154 g in eluent). The starting sorbent contained covalently bound glucose at 18% by weight.

Example 4: The oxidized carrier of Example 3 was used to bind lectin from Ricinus communis. 1.9 g of lectin were dissolved in 70 ml of acetate buffer pH 7.3 containing 220 mg galactose and 10 g of the oxidized carrier were added. Elimination of unreacted aldehyde functional groups was performed with 25 mg NaBH ₄ , the reaction time was the same as in Example 1.

The effect was tested with polyacrylamide having a sugar content of 9.4% by weight. Loaded 0.3412 g, eluent yield 0.0154 g, loss 3.8%.

Example 5

The lectin binding from Ricinus communis was performed analogously to Example 4 except that the oxidized carrier had a molecular weight exclusion limit of 300,000 daltons.

To verify the effect of the glucose-bound polycrylamide, 0.3400 g of polymer was applied to 1 g of column, yield in eluent 0.0171, loss 4.75%.

Example 6

4.2 g of spherical cellulose were allowed to swell for 20 hours in 50 ml of dioxane saturated with HCl gas to a content of 16% by weight. After this time, 2.0 g of D-galactose was added to the reaction mixture, and the mixture was shaken at room temperature for 24 hours. After this time, the contents of the vessel were poured into 5 liters of water and the product was sucked on the filter repeatedly with water to neutral pH and negative reaction to sugar.

4.0 g of spherical cellulose with bound D-galactose (content of bound D-galactose was determined by analysis)

11.7% by weight) was swelled for 20 hours in distilled water. The water was filtered and 100 ml 0.1 was added to the wet gel

M NaJO ₄ . Oxidation at room temperature is 206823 l for 1 hour with stirring. The NaJO ₄ solution was then filtered off and the gel was washed on the column with distilled water until the conductivity of the eluate was equal to that of the distilled water used. Borate buffer pH 8.5 was added to the cellulose gel and after 1 hour 500 mg concanavaline A (lectin from Canavalia ensiformis). The lectin was inhibited by methyl α-D-glucopyranoside. After standing at 4 ° C for 70 hours, unreacted active functional groups were reduced at 4 ° C to 20 mg NaBH ₄ with stirring for 20 minutes. The gel was then washed alternately with pH 4.1 acetate buffer (1 M NaCl) and TRIS-HCl buffer 9 (1 m NaCl). In the final phase prior to use, the gel was stored in a pH 6 acetate buffer (1 M NaCl) containing 10 ^-3 M MnCl ² and 1 S ³ M CaCl 2. The bound protein content was determined to be 9.0 mg / ml wet gel.

Example 7

The aminoglycosyl cellulose derivative was prepared in a manner analogous to Example 6, except that 0.8 g of N-acetyl-D-glucosamine was taken into the reaction and the product was not oxidized, but only the amount of bound amino sugar on the cellulose carrier was determined.

Example 8

An analogous experiment to Example 7 was carried out with a carrier consisting of a hydroxyethyl methacrylate copolymer with a molecular weight cut-off of 1,000,000, see Example 1. The bound amino sugar content was 8.9% by weight. is twice that of Example 7.

Example 9

4.0 g of soluble polyacrylamide gel with polymerized allyl glucoside (prepared according to V. Horejsi, P. Smolek and J. Kocourek, Biochim. Biophys, Acta, 538 (1978) 293-298) was oxidized in 100 ml of 0.1 M NaJO ₄ for 1 hour at room temperature. After this time, the reaction mixture was diluted to 250 mL and allowed to dialyzed against distilled water. After the periodate was removed from the reaction mixture by dialysis, the gel was lyophilized. The lyophilisate was dissolved in 20 ml of distilled water. 500 mg of methyl-α-D-glucopyranoside-inhibited lectin from Canavalia ensiformis was added to the solution. The mixture was reacted for 70 hours at 4 ° C, then the remaining aldehyde groups were reduced by treatment with NaBH ₄ as in Example 6. After concentration by lyophilization, the final reaction mixture was separated by preparative dextran gel chromatography on the lectin fraction and the lectin-containing fraction bound to the lectin. glycosylated polymer acrylamide.

Example 10

4.0 g of polyvinyl alcohol of molecular weight

125,000 and a degree of hydrolysis of 87-89%, which was substituted with D-glucose according to U.S. Pat. No. 195044 was oxidized analogously to Example 9.

They did

The sorbent was prepared by a procedure analogous to Example 1 with the difference that instead of ethylene glycol dimethacrylate copolymer having hydroxyethylmet ------ _{(hylakryíátu} ethylendiakrylátem with the exclusion <limit of molecular weight 500,000 daltons. When verification glycosylated polyacrylamide effect: the sorption was found Loss 5.3%.

hydroxyacrylate copolymers were used

Claims (2)

SUBJECT OF THE INVENTION Sorbents for saccharides, glycoproteins and carbohydrate-containing polymers, characterized in that lectins are covalently bonded to carriers selected from the group of oxidized polysaccharides, oxidized glycosylated hydroxyalkyl acrylates or hydroxyalkyl methacrylate, oxidized polyacrylamide and polyvinyl alcohol. 2. A process for preparing sorbents according to claim 1, characterized in that the polymers containing covalently bound carbohydrates are oxidized with sodium or potassium periodate, followed by reacting the activated polymers with lectins at a temperature of 0 to 40 [deg.] C. in buffer solutions of pH 7. 12 to 12, wherein after the reaction the remaining aldehyde functionalities from the polymer are eliminated by treatment with reducing agents, preferably sodium borohydride. Printed by Moravian Printing Works, provoit 12, Leninova 21, Olomouc