RU2255802C1 - Sorbent for resolution of optical isomers and a method for preparation thereof - Google Patents

Abstract

FIELD: chromatographic sorbents.

SUBSTANCE: invention relates to chromatographic sorbents, which can be used for analysis and preparative purification of optically active compounds. A novel sorbent for resolution of isomers of optically active compounds is developed containing, as chiral selector, macrocyclic glycopeptide antibiotic eremomycin, vancomycin, ristomycin A, teicoplanine, or their aglycons. Method of immobilization of macrocyclic glycopeptide antibiotics is also developed, which resides in that silica gel in aqueous buffer solution is first treated with 3-glycidoxypropyl(trialkoxy)silane and then, in alkaline aqueous or water-organic solution, above-indicated macrocyclic glycopeptide antibiotic is grafted to epoxy group-modified silica gel.

EFFECT: increased selectivity in enantiomer resolution and simplified preparation procedure.

9 cl, 12 dwg, 5 tbl, 9 ex

Description

The invention relates to sorbents for chromatography and can be used for analysis and preparative purification of optically active compounds.

Sorbents for the separation of optically active compounds based on silica gel with grafted optically active compounds for the separation of enantiomers are known. Known, for example, a sorbent containing quinine derivatives on the surface of silica gel (SU 1429016, 1988):

Known sorbents containing grafted to silica groups:

or

(SU 1132965, 1132966, 1985).

Known chiral optically active sorbent containing an optically active polymer covalently bonded to a solid carrier, the polymer is a network polymer containing optically active derivatives of dicarboxylic acids, diamines, diols or hydroxyacids, preferably derivatives of tartaric acid, and the carrier is an organic or inorganic material. A method of obtaining this sorbent involves fixing derivatives of tartaric acid on the surface of the carrier, which is produced by network polymerization through hydroxylation in the presence of hydrosilane and hydrosiloxane and is fixed on the carrier in the presence of a catalyst (RU 2121395, 1998).

A known sorbent for the separation of racemates of optically active compounds, containing a carrier and a chiral selector - per-6-amino derivatives of α-, β-, or γ-cyclodextrin or their acetylated analogues. The method for producing this sorbent includes covalent immobilization of chiral selectors on a carrier, which 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-cyclodextrin, per-6-amino-β-cyclodextrin , per-6-amino-γ-cyclodextrin, and then with metal borohydride (RU 2203730, 2003).

However, known sorbents possess enantioselectivity only to certain classes of substances or have insufficient selectivity for complete separation of enantiomers.

The closest in technical essence and the achieved result is a sorbent based on silica gel with chemically grafted glycopeptide antibiotics such as vancomycin, teicoplanin, teicoplanin aglycone, ristomycin, the structural formulas of which are shown in Figs. 1-4, as well as the method for its preparation described in ( US 6669842, 2003).

Figure 1 illustrates the structural formula of vancomycin.

Figure 2 illustrates the structural formula of teicoplanin.

Figure 3 illustrates the structural formula of teicoplanin aglycon.

Figure 4 illustrates the structural formula of ristomycin A.

Sorbents show selectivity in the separation of a wide range of enantiomers in both aqueous-organic and non-aqueous eluents. However, enantioselectivity is not always high enough.

A method of producing known sorbents consists in the interaction of an antibiotic with an organosilicon modifier, followed by chemical bonding of the resulting compound to the surface of silica gel.

A disadvantage of the known method for producing sorbents with grafted glycopeptides is that the modification of the antibiotic and the grafting of its derivative to silica gel is carried out at a temperature of 90-105 ° C, which can lead to the destruction of the antibiotic molecule. An uncontrolled change in the structure of the chiral selector molecule during grafting can lead to an uncontrolled change in the selectivity of the sorbent. In addition, the disadvantage of this method is the use of absolute organic solvents.

The objective of the present invention is to increase the selectivity of the sorbent in the separation of optical isomers and the development of a method that allows for the reproducible production of selective sorbent and simplification of the technology.

The problem is solved by the described sorbent for chromatography of optical isomers, which contains silica gel grafted through a spacer with a chiral selector - a glycopeptide antibiotic, in which the spacer is represented by a group of the formula:

Preferably, it contains eremomycin or its aglycon as a glycopeptide antibiotic.

The sorbent may also contain antibiotics selected from the group: vancomycin, ristomycin A, teicoplanin or their aglycones.

The problem is also solved by the described method of producing a sorbent for chromatography of optical isomers, which includes the chemical immobilization of a glycopeptide antibiotic using an organosilicon modifier on the surface of a silica gel, which is carried out by first treating the silica gel with 3-glycidoxypropyltrialkoxysilane and then with an alkaline aqueous-organic antibiotic solution at appropriate temperature with an antibiotic-organic buffer solution above 40 ° C.

Preferably, eremomycin or its aglycon is used as an antibiotic.

It is also possible to use antibiotics selected from the range: vancomycin, ristomycin A, teicoplanin or their aglycones.

The following are specific examples of the method.

Example 1

An example describes a technique for immobilizing eremomycin, which was obtained using the producer strain Amicolatopsis orientalis subsp (RU 2110578, 1998). Its structural formula is shown in figure 5.

101.1 g of Kromasil KR100-5-SIL silica gel was suspended in 500 ml of a 0.1 M sodium acetate solution adjusted to pH 5.5 with glacial acetic acid. To the resulting suspension was added 78 ml of 3-glycidoxypropyltriethoxysilane. The reaction mixture was intensively stirred for 2 hours, and then left without heating and stirring for 4 days. At the end of the reaction, the modified silica gel was washed with water, ethanol, acetone and filtered. Dried at a temperature of 105 ° C. According to elemental analysis, the carbon content is 5.2%.

Received silica gel modified with epoxy groups. Then 1 g of the macrocyclic antibiotic - eremomycin - was dissolved in 15 ml of distilled water. The pH was adjusted to 8.75 by dropwise adding a 1 M aqueous KOH solution. The resulting solution was mixed with 3 g of epoxy grafted silica gel. The resulting reaction mixture was heated at 40 ° C. with stirring for 14 hours. After completion of the reaction, the sorbent was filtered and washed successively with water, acetonitrile, methanol and diethyl ether. Dried in an oven at 50 ° C for 4 hours. According to elemental analysis, the carbon content is 9.5%.

Thus, silica gel with a grafted chiral selector was obtained:

Silica gel - Si- (CH ₂ ) ₃ O-CH ₂ -CH (OH) -CH ₂ - Eremomycin

Example 2

Epoxy-modified silica gel was prepared as in Example 1.

3 g of eremomycin was dissolved in 25 ml of distilled water. The pH was adjusted to 8.52 by dropwise adding a 1 M aqueous KOH solution. The resulting solution was mixed with 9.2 g of epoxy grafted silica gel. The resulting reaction mixture was heated at 40 ° C. with stirring for 14 hours. After the reaction, the sorbent was filtered and washed successively with water, methanol and acetone. Dried in an oven at 50 ° C for 4 hours. According to elemental analysis, the carbon content is 9.5%.

Example 3

Epoxy-modified silica gel was prepared as in Example 1.

1.2 g of ristomycin A was dissolved in a mixture of 15 ml of distilled water and 4 ml of ethanol. The pH was adjusted to 8.59, dropwise adding a 1 M aqueous KOH solution. The resulting solution was poured onto 5 g of epoxy grafted silica gel. The resulting reaction mixture was heated at 40 ° C. with stirring for 14 hours. After the reaction, the suspension was filtered and washed with water, methanol, acetone. Dried in an oven at 50 ° C for 4 hours. According to elemental analysis, the carbon content is 13.1%.

Example 4

30.1 g of KSK-G silica gel was suspended in 150 ml of a 0.1 M sodium acetate solution, adjusted with glacial acetic acid to pH 5.5. To the resulting suspension was added 24 ml of 3-glycidoxypropyltriethoxysilane. The reaction mixture was intensively stirred for 2 hours, and then left without heating and stirring for 4 days. At the end of the reaction, the solid phase was washed with water, ethanol, acetone and filtered. Dried at a temperature of 105 ° C. According to elemental analysis, the carbon content is 4.8%.

1 g of vancomycin was dissolved in a mixture of 15 ml of distilled water and 4 ml of ethanol. The pH was adjusted to 8.50, dropwise adding a 1 M aqueous KOH solution. The resulting solution was poured onto 5 g of epoxy-modified KSK-G. The resulting reaction mixture was heated at 40 ° C. with stirring for 15 hours. After the reaction, the suspension was filtered and washed with water, methanol, acetone. Dried in an oven at 50 ° C for 4 hours. According to elemental analysis, the carbon content is 8.3%.

The sorbents obtained in accordance with the present invention were tested in the separation of optical isomers under the following conditions.

The sorbents obtained in Examples 1–4 were packaged by the suspension method into 4.0 × 250 mm stainless steel columns and optical isomers were separated using high performance liquid chromatography.

Chromatographic analysis was carried out on an KNAUER HPLC chromatograph (Germany) comprising: a K-1001 pump, a K-2501 spectrophotometric detector, a Jetstream column thermostat, with the ability to control temperature in the range of 5-85 ° C with an accuracy of 0.1 ° C, manual valve -doser with a loop of 20 μl. The sample volume is 5-20 μl. Chromatographic peaks were detected in the range 210-280 nm in accordance with the absorption maxima of the separated compounds.

Chromatograms were recorded and the retention factors of the separated components k, α selectivity and R _S resolution were calculated using the Multichrom software and hardware complex (Ampersend, Russia).

As the mobile phase, water-acetonitrile and water-methanol buffer solutions, methanol with the addition of triethylamine and glacial acetic acid were used. The yield order of the L- and D-isomers was determined using standard samples of optically pure compounds.

Example 5

Table 1 presents the composition of the mobile phases, and in table 2 the results of chromatographic separation of amino acids and some of their derivatives on a column with the sorbent obtained in example 1, inoculated with eremomycin.

Table 1 Eluent code The composition of the eluent 40 40% CH ₃ OH - 60% H ₂ O 60-3.8 60% CH ₃ OH - 40% aq. buffer (CH ₃ COOH pH 3.8) 60-4.1 60% CH ₃ OH - 40% aq. buffer (1% TEAA pH 4.1) 85 85% CH ₃ OH - 15% aq. buffer (CH ₃ COOH ₄ , 0.025M) 20-4.1 20% CH ₃ OH - 80% aq. buffer (0.2% TEAA pH 4.1) 40-4.1 40% CH ₃ OH - 60% aq. buffer (0.2% TEAA pH 4.1) twenty 20% CH ₃ OH - 80% aq. buffer (NaH ₂ PO ₄ , 0.1 M) 42-58 42% CH ₃ CN - 58% H ₂ O

Example 6

Table 3 presents the results of the chromatographic separation of amino acids and some of their derivatives on a column with a sorbent obtained in Example 3 with grafted ristomycin A with mobile phases selected from those shown in Table 1.

Table 3 Compound Mobile phase k ' _L k ' _D α R _s DL-Trp 20-4.1
40-4.1
twenty 1.81
1.23
2,58 2.85
2.05
3.64 1,57
1.66
1.41 3.78
3.67
2.99 DL-Phe 20-4.1
40-4.1
twenty 0.67
0.57
0.84 1.34
1.20
1,69 2.00
2.10
2.01 3.69
3.30
2.80 DL- (2-thienyl) -Ala 20-4.1
40-4.1
twenty 0.78
0.73
1.15 1.06
1,07
1.46 1.36
1.46
1.27 2.19
2.70
1.94 DL-Cit 20-4.1
twenty 0.11
0.20 0.18
0.34 1,63
1.70 0.88
1.78 DL-Met 20-4.1
40-4.1
twenty 0.24
0.29
0.32 0.43
0.54
0.60 1.79
1.82
1.87 2,30
2.95
3.31 DL-Ala twenty 0.09 0.33 3.66 3.16 DL-Val twenty 0.16 0.58 3.62 3.40

Example 7

The example presents the results of the separation of atenolol and phenoprofen on a column with the sorbent obtained in example 4 with immobilized vancomycin.

The results for the separation of atenolol enantiomers were obtained in the system of 100% CH ₃ OH - 0.01% CH ₃ COOH - 0.01% (C ₂ H ₅ ) ₃ N and phenoprofen in the system 10% THF - 90% 0.1 M NaH ₂ PO ₄ pH 6.5.

The separation results are presented in table 4.

Table 4 Compound k ' _L k ' _D α R _s atenolol 1.34 1.48 1.10 0.95 phenoprofen 1.22 1.32 1,08 0.91

Example 8

Table 5 presents the comparative characteristics of the sorbents obtained according to example 1 and example 3, and the sorbent obtained in a known manner with grafted ristomycin A (from Ekborg-Ott KH, Liu Y., Armstrong DW // Chirality, 1998, v.10, p.434.). Separation was carried out in an eluent of the same composition: 50% CH ₃ OH - 50% H ₂ O.

Table 5 Compound Column with a sorbent according to example 1 Column with a sorbent according to example 3 Column with a sorbent obtained in a known manner α R _s α R _s α R _s DL-DOPA 4.28 10.03 2,50 5.11 1.33 1.4 DL-Tyr 5.05 9.91 2.66 4,50 1.30 1,52 DL-Trp 1.81 4.30 2.03 4.65 1.23 1.55 DL-PhGly 3.09 6.42 3.78 8.25 1.23 1,52 DL-Asn 1 0 1,53 2,35 1.17 1,56 DL-Met 2.23 4.19 2.13 4.22 1.15 1,52 DL-α-amino-n-butyric acid 3.39 4.80 1.30 1.42 1.26 1,56 DL-Cys 2.02 3.45 1.34 1,54 1.18 1.4 DL-Leu 2.54 3.64 5.88 8.30 1.15 1.45 DL-Val 3.35 4.09 3.48 5.58 1.23 1.55 DL-norVal 2.65 3.76 3.08 4.80 1.25 1,58 DL-Ala 2,37 2.94 2,56 3.15 1.14 1.45

From the obtained results it is seen that the claimed sorbent and the claimed method of immobilization of macrocyclic glycopeptide antibiotics on silica gel show higher enantioselectivity results than the known one.

Example 9

The antibiotic teicoplanin and aglycons of eremomycin, ristomycin A, vancomycin and teicoplanin were immobilized by the method described in example 3. The obtained sorbents showed the ability to separate the optical isomers of various compounds.

Chromatograms of the separation of some compounds are presented in Fig.6-12.

6. Chromatogram of separation of DL-DOPA on a column with immobilized eremomycin. Eluent: 20% CH ₃ OH - 80% 0.1 M NaH ₂ PO ₄ (pH 5.5); 0.7 ml / min. Detection: UV 280 nm. Temperature: 22 ° C.

7. Chromatogram of DL-Met separation on an immobilized eremomycin column. Eluent: 20% CH ₃ OH - 80% 0.1 M NaH ₂ PO ₄ ; 0.7 ml / min. Detection: UV 210 nm. Temperature: 5 ° C.

Fig. 8. Chromatogram of DL-His separation on an immobilized eremomycin column. Eluent: 20% CH ₃ OH - 80% 0.1 M NaH ₂ PO ₄ ; 0.7 ml / min. Detection: UV 210 nm. Temperature: 22 ° C.

Fig.9. Chromatogram of DL-Asp separation on an immobilized eremomycin column. Eluent: 20% CH ₃ OH - 80% 0, l M NaH ₂ PO ₄ ; 0.7 ml / min. Detection: UV 210 nm. Temperature: 22 ° C.

Figure 10. Chromatogram of DL-PhGly separation on a column with immobilized ristomycin A. Eluent: 50% CH ₃ OH - 50% H ₂ O; 0.7 ml / min. Detection: UV 220 nm. Temperature: 22 ° C.

11. Chromatogram of DL-Asn separation on a column with immobilized ristomycin A. Eluent: 20% CH ₃ OH - 80% 0.1 M NaH ₂ PO ₄ ; 0.7 ml / min. Detection: UV 210 nm. Temperature: 22 ° C.

Fig. 12. Chromatogram separation DL-Atenolol on a column with immobilized vancomycin. Eluent: 100% CH ₃ OH - 0.01% TEA - 0.01% HOAc; 0.7 ml / min. Detection: UV 254 nm. Temperature: 22 ° C.

Claims (10)

1. Sorbent for chromatography of optical isomers containing silica gel with a chiral selector glycopeptide antibiotic grafted through a spacer, characterized in that the spacer is represented by a group

2. The sorbent according to claim 1, characterized in that as an antibiotic it contains eremomycin or its aglycon. 3. The sorbent according to claim 1, characterized in that it contains ristomycin A or its aglycon as an antibiotic. 4. The sorbent according to claim 1, characterized in that as an antibiotic it contains vancomycin or its aglycon. 5. The sorbent according to claim 1, characterized in that it contains teicoplanin or its aglycon as an antibiotic. 6. A method of producing a sorbent for chromatography of optical isomers, including chemical immobilization of a glycopeptide antibiotic using an organosilicon modifier on the surface of silica gel, characterized in that the silica gel is first treated with 3-glycidoxypropyltrialkoxysilane and then with an aqueous or aqueous-organic alkaline buffer solution at a temperature not higher than the corresponding antibiotic 40 ° C. 7. The method according to claim 6, characterized in that eremomycin or its aglycon is used as an antibiotic. 8. The method according to claim 6, characterized in that ristomycin A or its aglycone is used as an antibiotic. 9. The method according to claim 6, characterized in that vancomycin or its aglycon is used as an antibiotic. 10. The method according to claim 6, characterized in that teicoplanin or its aglycone is used as an antibiotic.

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