RU2203730C1 - Sorbent and method of preparation thereof - Google Patents

Abstract

FIELD: sorbents. SUBSTANCE: sorbent suitable for use in chromatographic separation of racemates is based on cyclodextrine derivatives covalently immobilized on substrates, their chiral selectors being per-6-amino derivatives of alpha-, beta-, and gammacyclodextrine or their acetylated analogues. Invention also provides sorbent preparation method. EFFECT: extended range of substances to be separated. 6 cl, 1 tbl, 6 ex

Description

 The invention relates to sorbents for the separation of racemates of optically active compounds and can be used to isolate individual isomers of various derivatives of amino acids and coumarin and to control the enantiomeric purity of amino acids, pesticides, biologically active compounds.

 Chromatographic sorbents for the separation of racemic mixtures are known which are obtained by immobilization by adsorption on a mineral support of hydrophobic derivatives of optically active amino acids (US 4851382, B 01 J 20/22, 1989).

 A disadvantage of the known sorbents is the lack of a covalent bond between the carrier used and the chiral selectors. The latter are easily washed out of the chromatographic column with organic solvents and therefore the column can only be used in aqueous media and for the separation of only water-soluble racemates.

 For chromatographic separation of the optical isomers of pharmaceuticals, a sorbent is known containing a carrier with covalently linked chitosan derivatives as chiral selectors (A. Senso at al., J. Chromatogr., V. 839, 1999, p. 15-21, "Chitosan derivatives as chiral selectors bonded on allyl silica gel). The method for producing the above sorbents involves many steps, including the preparation of silica gel with grafted allyl functional groups, the preparation of chitosan with 10-undecenoyl and benzoyl or phenylaminocarbonyl groups, and finally copolymerization the double bond of the carrier and the chiral selector. These sorbents provides separation of the optical isomers due to Van der Waals and dipole interactions.

 The disadvantage of the above sorbents is their relatively low selectivity.

 The closest in technical essence and the achieved result is a sorbent containing a carrier and a chiral selector based on derivatives of β-cyclodextrin (β-CD), namely β-CD crosslinked by epichlorohydrin and modified with 2,3-epoxypropyltrimethylammonium chloride. A method of producing a known sorbent consists in adsorption immobilization of an epichlorohydrin-β-CD copolymer on silica due to ionic interactions, but without the formation of a covalent bond with the carrier.

 The optical isomers of warfarin and a number of dansyl derivatives of amino acids are separated on this sorbent due to the formation of inclusion complexes of enantiomers with cyclodextrin (N. Thuaud et al., J. Chromatogr., V. 555, 1991, p. 53-64, "Retention behavior and chiral recognition of β-cyclodextrin-derivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids ").

 The disadvantage of the sorbent is again the gradual loss by the carrier of a chiral selector.

 The present invention is the development of a stable sorbent suitable for the separation of a wide range of substances.

 The problem is solved by the described sorbent for the separation of racemates of optically active compounds containing a carrier and a chiral selector selected from the group of per-6-amino derivatives of α-, β- or γ-cyclodextrin or their acetylated analogues.

 The problem is also solved by the described method of producing a sorbent for the separation of racemates of optically active compounds, including covalent immobilization of a cyclodextrin derivative on a carrier, which is carried out by sequential interaction of the aminated carrier first with a condensing agent, then with a reagent from the group: per-6-amino-α-cyclodextrin , per-6-amino-β-cyclodextrin, per-6-amino-γ-cyclodextrin, and then with metal borohydride. Additionally, the resulting sorbent is subjected to acetylation. Preferably, silica is used as a carrier and glutaraldehyde is used as a condensing agent. Preferably, the interactions are carried out in sodium phosphate buffered saline.

 Below are examples of the method for producing the sorbent and examples for its use.

Example 1. To 5 g of Lichrosorb Si-100 (Merk) suspended in 25 ml of 0.1 M sodium phosphate buffer with a pH of 6.5 add 0.25 ml of 3-aminopropyl-triethoxysilane. The reaction mixture is heated to 95 ^o C and incubated for 2 hours with stirring at the same temperature. Then, the aminated Lichrosorb Si-100 is washed on a glass porous filter with water. The obtained aminated Lichrosorb Si-100 is suspended in 20 ml of 0.01 M sodium phosphate buffer solution with a pH of 7, 5 ml of 25% glutaraldehyde are added and stirred for 1 hour at 20 ^° C, after which the obtained product 1 is washed with water on a porous filter. 0.2 g of amino-β-cyclodextrin (amino-β-CD) is dissolved in 50 ml of 0.1 M sodium phosphate buffer with a pH of 6.0 (controlling the pH value, which should be in the range of 6.5-7.0 ) 5 g of product 1 are suspended in the resulting solution, the reaction is carried out for 16 hours with stirring at 20 ^{° C.,} and the resulting sorbent 2 is washed with water. The concentration of amino groups in product 2 is 150 μM per 1 g, which corresponds to 25 μM amino-β-cyclodextrin per 1 g.

5 g of product 2 are suspended in 50 ml of 0.3 M sodium phosphate buffer pH 6.0 and 90 mg of NaBH ₄ are added. The reaction is carried out for 6 hours with stirring at 20 ^o C. The resulting sorbent with peramino-β-cyclodextrin (sorbent A) is separated and washed on a porous filter successively with water, ethanol and acetone and dried.

Example 2. Analogously to example 1, sorbent A is obtained. After treatment with NaBH _4, 5 g of sorbent A are suspended in a mixture of 20 ml of water, 4 ml of methanol and 8 ml of acetic anhydride. The reaction mixture was stirred at 20 ^{° C. for} 8 hours. The resulting sorbent B with peracetylamino-β-cyclodextrin is separated and washed with water on a porous filter. No free amino groups were found in sorbent B.

Example 3. Analogously to example 1, product 1 is obtained. 0.2 g of peramino-α-CD is dissolved in 50 ml of 0.1 M sodium phosphate buffer with a pH of 6.0 (controlling the pH, which should be in the range of 6.5- 7.0). 5 g of product 1 are suspended in the resulting solution and the reaction is carried out for 16 hours with stirring at 20 ^° C. The resulting product 3 is filtered with a glass porous filter and washed with water.

5 g of product 3 are suspended in 50 ml of 0.3 M sodium phosphate buffer pH 6.0 and 90 mg of NaBH ₄ are added. The reaction is carried out for 6 hours with stirring at 20 ^o C. The resulting sorbent with peramino-α-cyclodextrin (sorbent C) is separated and washed on a porous filter successively with water, ethanol and acetone and dried.

Example 4. Obtain sorbent C analogously to example 3. After treatment with NaBH _4, 5 g of sorbent C are suspended in a mixture of 20 ml of water, 4 ml of methanol and 8 ml of acetic anhydride. The reaction mixture was stirred at 20 ^{° C. for} 8 hours. The resulting sorbent D with peracetylamino-α-cyclodextrin is separated and washed with water on a porous filter.

Example 5. Analogously to example 1, product 1 is obtained. 0.2 g of peramino-γ-CD is dissolved in 50 ml of 0.1 M sodium phosphate buffer with a pH of 6.0 (controlling the pH, which should be in the range of 6.5- 7.0). 5 g of product 1 are suspended in the resulting solution and the reaction is carried out for 16 hours with stirring at 20 ^° C. The resulting product 4 is filtered using a porous filter and washed with water.

5 g of product 4 are suspended in 50 ml of 0.3 M sodium phosphate buffer pH 6.0 and 90 mg of NaBH ₄ are added. The reaction is carried out for 6 hours with stirring at 20 ^o C. The resulting sorbent with peramino-γ-cyclodextrin (sorbent E) is separated and washed on a porous filter successively with water, ethanol and acetone and dried.

Example 6. Analogously to example 5, sorbent E is obtained. After treatment with NaBH _4, sorbent E is suspended in a mixture of 20 ml of water, 4 ml of methanol and 8 ml of acetic anhydride. The reaction mixture was stirred at 20 ^{° C. for} 8 hours. The obtained sorbent F with peracetylamino-γ-cyclodextrin is separated and washed with water on a porous filter.

 Thus, sorbents with per-6-aminocyclodextrins covalently immobilized on a support (A, C, E), which are cyclic analogues of chitosan, and with co-covalently immobilized per-6-acetylaminocyclodextrins (B, D, F), which are cyclic analogues of chitin, were obtained.

 The proposed sorbents allow the separation of the isomers of optically active derivatives of amino acids with different structure modifiers of the amino group - 5-dimethylaminonaphthalen-1-sulfonyl (Danil), N-9-fluorenylmethoxycarbonyl (FMOK), 2,4-dinitrophenyl (DNF), 3,5- dinitropyridyl (DNP), phthalyl (Phthalyl), carboxybenzoyl (CBZ), o-phthalaldehyde (OFA), benzoyl (Benzoyl), butoxycarbonyl (BOK) and some coumarin derivatives: warfarin and kumochlor (see table. In addition, derivatives of amino acids with butoxycarbonyl are separated on a new sorbent, which cannot be separated either on the prototype sorbent based on cyclodextrin or on an analog sorbent based on chitosan derivatives. Chromatographic separation of racemates of various optically active compounds was carried out on the obtained sorbents. Some results of the separation on the obtained sorbents (sorbent A) are presented in the table.

 Thus, the problem of expanding the circle of separable compounds was solved by using new chiral selectors combining the separation mechanisms of optical isomers characteristic of linear and cyclic oligosaccharides, i.e. due to the combination of various mechanisms of separation of optical isomers, namely, due to the formation of inclusion complexes and due to the van der Waals and dipole interactions. Also, the stability problem of the obtained sorbents was solved due to the use of silica as a carrier and covalent immobilization of chiral selectors on it.

Claims (6)

 1. Sorbent for the separation of racemates of optically active compounds, containing a carrier and a chiral selector based on derivatives of cyclodextrins, characterized in that as chiral selectors it contains per-6-amino derivatives of α-, β-, or γ-cyclodextrin or their acetylated analogues.  2. A method of producing a sorbent for the separation of racemates of optically active compounds, including covalent immobilization of derivatives of cyclodextrins on a carrier, characterized in that the immobilization is carried out by sequential interaction of the aminated carrier with a condensing agent, then with a reagent selected from the group: per-6-amino-α β-cyclodextrin, per-6-amino-β-cyclodextrin, per-6-amino-γ-cyclodextrin, and then with metal borohydride.  3. The method according to p. 2, characterized in that the obtained sorbent is additionally subjected to acetylation.  4. The method according to any one of the preceding paragraphs, characterized in that silica is used as a carrier.  5. The method according to any one of the preceding paragraphs, characterized in that glutaraldehyde is used as the condensing agent.  6. The method according to any one of the preceding paragraphs, characterized in that the interaction is carried out in sodium phosphate buffer solutions.

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