RU2541524C2 - Method of obtaining thin-layer chiral plates for planar chromatography of stereoisomers and their racemic mixtures - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to method of obtaining thin-layer chiral plates for planar chromatography of stereoisomers and their racemic mixtures, which includes non-covalent binding of glycopeptides antibiotic eremomycin with silica adsorbent with silicagel binding by method of impregnation in alkaline water solution with pH 8.0÷10.0 at room temperature in one stage.

EFFECT: increase of method efficiency.

2 cl, 3 tbl, 5 ex

Description

The proposal relates to the field of analytical chemistry, and in particular to a method for producing thin-layer plates for planar chromatography (TLC, PTX), and can be used for chiral recognition of stereoisomers and racemic mixtures of natural and synthetic organic compounds in analytical chemistry, pharmaceuticals, medicine, ecology, biochemistry and other areas.

The most important characteristic of chiral sorbents is a chiral selector fixed on the surface of the sorbent. Known methods of grafting selector by immobilization methods (chemical - covalent binding) [J. Chromatogr. A. 2002. V. 897. P. 113-129] or impregnation (physical - non-covalent binding) [J. Planar Chromatogr. 2004. V. 17. P. 173-176]. The enantioselectivity of the chiral selector lies in its ability to form complexes with a racemate of two diastereomeric pairs that differ from each other in thermodynamic stability [J. Chromatogr., A., 2001, V. 906. pp. 3-33].

It is known that there are no universal chiral phases for the analysis and separation of the entire variety of optical monomers [J. Org. Chem., 1981, V. 46, No. 16, pp. 125-134].

Currently known methods for producing chromatographic chiral phases (sorbents) for high performance liquid chromatography (HPLC) [SU 1429016, 1998; US 4,919,803, 1990; US 5439979, 1995; US 62656115, 2001], capillary electrophoresis [RU 2348455, 2006], thin layer chromatography [US 54399792, 1995].

Known methods for impregnation of planar layers using (+) - tartaric and (+) - ascorbic acids as chiral selectors [J. Pharm. Biomed. Anal., 2000. V. 21, P. 1143-1147]; L-lysine, L-arginine, erythromycin [Biomed. Chromatogr. 2001.15, P. 155-165].

Machery-Nagel (Durön, Germany) chiral thin-layer plates are known for production, consisting of hydrophobic silica gel (RP-18), with polyvinyl alcohol as a binder, impregnated (2S, 4R, 2'K.8) -N- (2-hydroxy- dodecyl) -4-hydroxyproline and divalent copper ions - Chiraplate plates [J. Chrom., 1988, V.448, pp. 11-30].

Known methods for producing chiral TLC plates (Merck, Germany) impregnated with L-serine [J. Pharm. Biomed. Anal. 2000. V. 21, P. 1143-1147]; L-arginine (Sigma, USA) [J. Chromatorg. A. 1996. V. 721. P. 369-372].

At present, a new class of enantioselective selectors is known - macrocyclic glycopeptide antibiotics (thiostrepton, rifamycin B, vancomycin) [Anal.Chem, 1994, V.66, pp.1473-1484].

Known methods for producing chromatographic phases with chiral agents of a new type with an impregnated macrocyclic antibiotic erythromycin for the separation of the enantiomers of amino acids, vancomycin and verapamil [J. Chromatography A. 1996. V. 736. R. 235-238]. A method for producing a sorbent for the separation of optical isomers by HPLC with an immobilized glycopeptide antibiotic eremomycin is described [RU 2255802, 2005].

The disadvantage of these analogues is the fact that the known chromatographic phases for column HPLC, capillary electrophoresis and planar chromatography obtained by different methods of chemical (immobilization) and physical (impregnation) surface modification of silica matrices with known chiral agents have selective enantioselectivity, in addition, their process getting multi-stage and complex. A significant drawback of these analogues is also manifested when trying to use them as adsorbents for TLC plates in planar chromatography and is due to the fact that the effect of a polymer or inorganic binder in a silica matrix on the quality of chiral TLC plates is not taken into account.

The closest in technical essence and the achieved result is a method of obtaining [RU 2012143015, 2012] chiral planar plates for thin layer chromatography of optical isomers with chemically grafted (immobilized) glycopeptide antibiotic eremomycin produced according to the known protocol [RU 2255802, 2005]. A method for producing the known silica gel chiral phase for TLC consists in covalently bonding (immobilizing) eremomycin on the surface of a silica matrix with a silica sol binder. The method of immobilization consists in the fact that first silica with an inorganic binder in an aqueous buffer solution is treated with γ-glycidoxypropyltrimethoxysilane, and then glycopeptide antibiotic is inoculated with silica and inorganic binder (silica sol), modified epoxy groups at the first stage of the process.

A significant disadvantage of the known prototype method is the complex multi-stage process of chemical (covalent) binding of the chiral selector by surface assembly, which takes a considerable (up to 14 days) time to obtain the final product and consists in processing the silica matrix with acid to hydroxylate its surface, followed by washing it sequentially with water and alcohol treatment with organosilanes (γ-glycidoxypropyltrialkoxysilane) for 7 days to form a spacer containing a glycidoxypropyl group on the surface of silica gel; covalent (by immobilization method) inoculation of the glycopeptide antibiotic eremomycin in an alkaline aqueous solution (pH 8-11) for 7 days.

The technical task of the proposed solution is to develop a low-temperature fixation of the chiral agent - the glycopeptide antibiotic eremomycin by non-covalent binding of molecules to the surface of a silica sorbent by impregnation to obtain thin-layer plates with silica sol binder for planar chromatography of stereoisomers and their racemates.

A positive result is the simple technology of non-covalent (physical) binding of the chiral selector of eremomycin due to π-π interactions and hydrogen bonds with the surface of the silica matrix and the silica binder planar layer of TLC plates to obtain a universal chiral layer for the separation of stereoisomers of different classes of organic compounds and their racemic mixtures by high performance planar chromatography.

The technological result is achieved in the developed method due to the fact that such a phase is characterized by stability in aqueous-organic solutions and, due to the presence of a large number of asymmetric centers and functional groups, demonstrates high enantioselectivity with respect to various classes of chiral compounds.

This task and technical result are achieved due to the fact that the method of producing thin-layer chiral plates for planar chromatography of stereoisomers and their racemic mixtures by impregnation is characterized by the non-covalent binding of the glycopeptide antibiotic eremomycin with the following structural the formula

,

,

in this case, silica gel with silica sol activated with 0.2 M H ₂ SO ₄ at a temperature of 25 ° C for 20-60 min and washed with demineralized water to pH 5-7 is used as an adsorbent, and the non-covalent binding of the glycopeptide antibiotic eremomycin to the adsorbent surface is carried out by the method impregnation in an alkaline aqueous solution at pH 8.0-12.0.

The method is also characterized by the fact that the process is carried out at a temperature not exceeding 25 ° C in one stage for 10 minutes - 1 day.

The essence of this method of obtaining chiral planar plates is that the chiral agent of the glycopeptide antibiotic eremomycin is fixed on the surface of silica by impregnation (physical fixing) in aqueous alkaline solutions (with pH 8-10) at room temperature (20-25 ° C) s stirring (60-120 rpm) of the solution for 1-24 hours.

The essence of this method is described below by examples of its experimental implementation.

Example 1. Silica gel planar plates with a silica sol binder were activated with 0.2 M sulfuric acid at 25 ° C for 40 min to achieve the maximum degree of surface hydroxylation (α _{he =} 4.6 ÷ 4.9 OH groups / nm ² ) and washed with demineralized water to pH = 5 ÷ 7.

Eremomycin with a concentration of 1 mg / ml was dissolved in demineralized water at a temperature of 25 ° C, adjusted to pH 8.56 with a 1M sodium hydroxide solution. TLC plates were immersed in the resulting solution with stirring (60 rpm) for 10 min at a temperature of 25 ° C. Then TLC plates were dried in air at a temperature of 20-25 ° C for 24 hours.

The data on the elemental analysis of the silica layer with a chiral selector are given in table 1. The chromatographic separation data of racemic mixtures of standard L- and D-amino acids are given in Table 2.

Example 2. The method according to example 1, the impregnation time of 30 minutes The data on the elemental analysis of the silica layer of TLC plates are given in table 1. The results of the separation of the racemic mixture of amino acids are shown in table.2.

Example 3. The method according to example 1, the impregnation time of 60 minutes The data on the elemental analysis of the silica layer of TLC plates are given in table 1. The results of the separation of the racemic mixture of amino acids are shown in table.2.

Example 4. The method according to example 1, the impregnation time is 1 day. The data on the elemental analysis of the silica layer of TLC plates are given in table 1. The results of the separation of the racemic mixture of amino acids are shown in table.2.

Example 5. The method according to example 1, the impregnation time of 2 days. The data on the elemental analysis of the silica layer of TLC plates are given in table 1. The results of the separation of the racemic mixture of amino acids are shown in table.2.

The obtained data for plates impregnated during the day (Example 4) practically do not differ from the data on grafting the selector for 2 days (Example 5) and are comparable with the results obtained by immobilizing the plates for 14 days, which is confirmed by the high enantioselectivity of eremomycin by in relation to the reference stereoisomers of amino acids in both cases. Comparative characteristics of the separation of the enantiomers of amino acids on immobilized and impregnated eremomycin plates are given in Table 3.

The best results for the separation of racemic mixtures of amino acids were achieved for plates with an impregnation time of 1 day (Rs 5.48 for phenylalanine and 6.03 for tryptophan); The enantiomers of amino acids on the plates impregnated with eremomycin for 10 and 30 minutes are practically not separated (Table 2).

Table 1. Summary of elemental analysis of plates impregnated with eremomycin (n = 3) Impregnation time %, FROM %, N %, N E (μmol / g) 10 min 0.80 ± 0.03 0.79 ± 0.02 0.03 ± 0.01 9.1 30 minutes 1.35 ± 0.03 0.84 ± 0.03 0.07 ± 0.01 15.4 60 min 2.55 ± 0.05 0.90 ± 0.02 0.17 ± 0.02 29.1 1 day 6.78 ± 0.06 1.52 ± 0.06 0.86 ± 0.03 77.4 2 days 6.78 ± 0.06 1.52 ± 0.06 0.86 ± 0.03 77.4

Table 2. The results of the separation of the racemic mixture of amino acids on plates impregnated with eremomycin Amino acids Time (min) Rf (L) N (L) Rf (d) N (D) α Rs Phenylalanine 10 0.9 1568 0.84 1366 1.07 ± 0.03 0.66 ± 0.03 thirty 0.84 1366 0.77 1653 1.09 ± 0.04 0.84 ± 0.04 60 0.8 1784 0.65 1840 1.23 ± 0.05 2.20 ± 0.11 day 0.74 2385 0.47 2463 1.57 ± 0.08 5.48 ± 0.27 2 days 0.74 2385 0.47 2463 1.57 ± 0.08 5.48 ± 0.27 Tryptophan 10 0.85 5595 0.81 5081 1.05 ± 0.04 0.88 ± 0.04 thirty 0.77 4591 0.72 4014 1.07 ± 0.04 1.10 ± 0.06 60 0.75 6273 0.68 5156 1.10 ± 0.04 1.85 ± 0.09 day 0.5 4356 0.34 3581 1.47 ± 0.10 6.03 ± 0.30 2 days 0.5 4366 0.34 3583 1.49 ± 0.10 6.05 ± 0.30 α is the coefficient. selectivity, Rs - resolution factor

Table 3. Comparative characteristics of the separation of enantiomers of amino acids on immobilized and impregnated eremomycin plates Amino acids Eremomycin Immobilized Plates (6 days) Plates impregnated with eremomycin (1 day) α Rs α Rs phenylalanine 1.59 ± 0.05 5.92 ± 0.30 1.37 ± 0.0 4.56 ± 0.23 tryptophan 1.36 ± 0.04 4.44 ± 0.22 1.33 ± 0.09 4.68 ± 0.23 leucine 1.21 ± 0.05 3.05 ± 0.15 1.23 ± 0.06 3.40 ± 0.17 methionine 1.16 ± 0.05 2.47 ± 0.12 1.20 ± 0.06 3.96 ± 0.20 alanine 1.15 ± 0.04 1.91 ± 0.10 1.28 ± 0.06 3.09 ± 0.15 valine 1.26 ± 0.05 2.96 ± 0.15 1.42 ± 0.07 5.04 ± 0.25 aspartic 1.24 ± 0.04 1.50 ± 0.08 1.00 0.00 acid threonine 1.00 0.00 1.00 0.00 lysine 1.00 0.00 1.00 0.00 serine 1.00 0.00 1.08 ± 0.04 0.80

The data presented indicate that the proposed technical solution meets all the required criteria protected by a patent.

Thus, the registered method can significantly simplify the procedure for introducing the glycopeptide antibiotic eremomycin as a chiral agent into the silica layer of thin-layer wafers by using the low-temperature impregnation method - physical, non-covalent binding of the chiral selector to the sorbent.

The set of essential features of this method shows the originality, novelty, applicability of thin-layer chiral plates for planar chromatography of stereoisomers and their racemic mixtures.

Claims (2)

1. A method of producing thin-layer chiral plates for planar chromatography of stereoisomers and their racemic mixtures (impregnation method), characterized in that they produce non-covalent binding of the glycopeptide antibiotic eremomycin with the following structural formula to the surface of the adsorbent fixed on a TLC plate.

,
in this case, silica gel with silica sol activated with 0.2 M H ₂ SO ₄ at 25 ° C for 20-60 min and washed with demineralized water to a pH of 5–7 is used as adsorbent, and the non-covalent binding of the glycopeptide antibiotic eremomycin to the adsorbent surface is carried out by the method impregnation in an alkaline aqueous solution at pH 8.0 ÷ 10.0. 2. The method according to claim 1, characterized in that the process is carried out at a temperature not exceeding 25 ° C in one stage for 10 min ÷ 1 day.