SU1705303A1 - Method of glycopeptides synthesis - Google Patents

Abstract

The invention relates to glycopeptides. in particular, the preparation of compounds of common phla: CH, OH «v. BUT NHAc UNHAc CHjCH-SUSH The invention relates to the synthesis of glycopeptides that can be used in medicine, immunology and veterinary medicine as immunomodulators. The purpose of the invention is to increase the yield and simplify the process. The drawing shows a diagram explaining the method. Example 1. Preparation of M-acetyl-glucosaminyl-1-4) -m-acetylmuramyl - (/ 9 1-4) -M-acetylglucosaminyl- (ft where l is 1 or 2; NHR is the residue of an amino acid or peptide of f-ly L-Ala-Dei-Glu; L-Ala-Del-Gln or L-Ala, used in immunology and veterinary medicine as immunomodulators. The goal is to increase the yield of the target product and simplify the process. It is carried out by N-acetyl sugar (or its salts with an organic base) with a protected amino acid or peptide in the presence of a condensing agent - acpentafluorophenyl carbonate or di (4-chloro-2,3.5,6-tetrachlorophenyl) carbonate with and molar ratio (M, 3) :( 1-1.2): 1 at 0-20 ° C, followed by cleavage of the protective groups and chromatographic purification of the target product. Condensation is carried out in the presence of a base in a solvent (cimethylformamide). These conditions allow They use more accessible substances, provide a better ecology of the process, increase the yield of the target product by 10–25%, reduce the duration of the process from 28 to 5–10 hours, and reduce the number of chromatographic purifications. 1 tab., 1 Il. 1-4) -m-acetylmuramyl-1-alanyl-0-iso-glutamic acid (IV). 9.74 g (10 mM) of tetrasaccharide II. () was dissolved in 25.0 ml of distilled dimethylformamide, 4 ml of M-methylmorpholine was added, stirred for 10-15 minutes, and the solution was poured into 200 ml of ethyl acetate. The precipitate formed is filtered off, washed with 4x50 ml of ethyl acetate, 2x50 ml of hexane, dried and yielded 11.65 g (99%) of M-methyl morpholine salt of tetrasaccharide (V). h | About SP W about SA

Description

11.65 g (10 mM) of compound V is dissolved in 100 ml of dimethylformamide, 4.0 g (10 mm) of diphentafluorophenyl carbonate (Ilia) are added and stirred at 18-20 ° C for 1 h. Then the solution is poured to the reaction mass. 5.2 g (10 mM) of L-alanyl-0-isoglutamic acid dibenzyl ester (VI) trifluoroacetate and 2 g (20 mM) of triethylamine in 15 ml of dimethylformamide. The mixture is stirred for 5 hours, the solvent is evaporated, 200 ml of demineralized water are added to the remaining oil, the solution is extracted with 2x50 ml of hexane, 5 g of palladium black and 4 ml of formic acid are added to the aqueous layer and a stream of hydrogen gas is passed through for 1.5 hours. After separation of the catalyst, the filtrate is evaporated to half the volume and applied to a 1 liter column containing seondex A-25 anion exchange resin in acetate form. The column is washed with 2.5 L of demineralized water, then the glycopeptide is eluted with a gradient of water — 0.15 M acetic acid. After evaporation of the fractions containing the glycopeptide IV and freeze-drying the residue, 7.5 g IV (61.3%, calculated as tetrasaccharide II (p-2)) are obtained.

Example 2. Preparation of N-acetylgluco-zaminyl-m-acetylmuramyl-halyl-O-isoglutamine: (GMDP) (VII).

5 g (10.0 mM) of disaccharide I (p-1) is dissolved in 100 ml of distilled dimethylformamide; 4.4 g (11.0 ΩM) of compound (Pai 1.2 ml (11, ΩM) M-methyl ml-morpholine Stir at 18–20 ° 30 min. And a solution of 4.6 g (11.0 mmol) of L-alanyl-O-iso-utamine y-benzyl ether (VIII) trifluoroacetate and 2.4 ml of N-methylmorpholine is poured. 20 ml i of dimethylformamide. After 4.5 hours, the solvent is evaporated. The treatment is then carried out as described in Example 1. After evaporation of the fractions containing GMDP and freeze-drying the residue, 4.27 g (57.0%) of GMDP VH containing the main substances 99.5% (filed by EC X).

Example 3. Preparation of compound VII.

1.5 g p. O mm) of compound I (p-1) is dissolved in 30 ml of distilled dimethyl formamide; 1.3 g (3.0 mmol) di- (4-chloro-2.3.5, 6-tetrafluorine n and l) -carbonate (1Mb) and 0.45 ml (3.0 mM) of triethylamine. After 2 hours, a solution of 1.3 g (3 mM) of compound VIII and 0.9 ml (6 mM) of triethylamine and 5 ml of dimethylformamide is added. The mixture is stirred for 8 hours. The solvent is evaporated and further processing is carried out as described in Example 1. 1.4 g (62.2%) of GMDP are obtained, with a basic substance content of 99.1% (by HPLC).

Example 4 Preparation of Compound VII 1.2 g (20 mM) of N-methylmorpholinium salt of disaccharide II () is dissolved in 250 ml of dimethylformamide, 8.0 g (20 mmol) is added

Ilia compounds and stirred for 30 minutes. A solution of 8.5 g (20 mM) of compound VIII and 4 ml (40 mM) of N-methylmorpholine in 20 ml of dimethylformamide are added to the reaction mass. After 5 h, the solvent is evaporated. Further processing is carried out as described in Example 1. 9.7 g (64.7%) of GMDP are obtained, with a basic substance content of 98.9% (by HPLC).

Example 5 Preparation of Compound VII

60 g (0.1M) of N-methylmorpholine salt II (p-1) are loaded into a 15-liter reactor with a jacket and a mechanical stirrer and dissolved with stirring in 10 l of a mixture of dimethylformamide-acetonitrile 1: 4. The reaction mass is cooled to 5-10 ° C and a solution of 48 g (0.12 M) of Ilia compound in 0.2 L of dimethylformamide is added over 30 minutes with stirring from a measuring device,

The cooling is removed, stirring is continued for 1 hour, then a solution of 51 g (0.12 M) of trifluoroacetate VIII in 0.2 l of acetonitrile containing 26 ml is slowly poured into the reaction mixture from a mernik for 30 minutes

(0.24 M) N-methylmorpholine. Stirring is continued for 5 hours, the solvent is evaporated, the gummy residue is dissolved in 1 l of dimeralized water and extracted with ethyl acetate 2x400 ml. Further processing

carried out as described in example 1, taking into account the scaling of the synthesis. 48.7 g (62%) of glycopeptide VII are obtained.

Example 6. Preparation of N-acetylglucosaminyl-N-acetylmuramyl-b-alanine

(Ix).

Similar to the experiment described in Example 3, from 0.5 g (1 mM) of disaccharide II (p-1), 0.43 g (1 mm) of compound II16, 0.45 ml (3 mmM) of triethylamine and 0.35 g (1 mM) L-alanine benzyl ester p-toluenesulfonate (X) gives 0.31 g (55%) of N-acetyl-glucosaminyl-N-acetylmuramyl-1-alanine (IX).

PRI me R. 7. Preparation of Compound VII. Similar to the experiment described in Example 2, from 5 g (10 mM) of compound II (p-1), 5.2 g (1.3 mM) of compound Ilia, 1.4 ml (13 mM) of N-methylmorpholine and 4.2 g (10 mM) of compound VIII with 2.2 ml (20 mM) of N-methylmormoline get 3.8 g (50.6%) of GMDP.

Example Preparation of Compound VII, Analogously to the experiment described in Example 2, from 5 g (10 mM) of compound II (p-1), 5.2 g (13 mM) of Ilia compound, 1.4 ml (13 mM) of N-methylmorpholine, 5.5 g (13 mM) of compound VIII

with 2.8 ml (26 mM) of N-methylmorpholine, 3.4 g (45.3%) of GMDP is obtained.

Example 9. Preparation of compound VII.

Similar to the experiment described in Example 2, from 1.0 g (2 mM) of compound II (p-1), 0.72 g (1.8 mM) of Ilia compound. 0.2 ml (1.8 mM) of N-methylmorpholine and 0.76 g (1.8 mm) of compound VIII with 0.4 ml (3.6 mmM) of N-methyl morpholine give 0.7 g (46.6%, calculated as II; 52 %, counting on UE) GMDP.

The ratios of the reagents and the yield of the target products in examples 1-9 are shown in the table.

All samples of glycopeptides were analyzed by HPLC (basic substance content 99%, see drawing). Melting points, specific rotation, and chromatographic mobility in a thin layer correspond to constants for substances obtained by the prototype method.

Thus, a new method for the synthesis of glycopeptides of formula I was developed, which differs from the prototype method by using more available symmetric carbonates of pentahalophenols of Formula II as a condensing agent, ensuring environmental protection by simply separating the condensing reagent from the reaction product by extraction from an organic solution solvent.

The implementation of the proposed method allows to increase the yield of glycopeptides by 10-25% and simplify the process of their production by reducing its duration from 28 hours by the prototype method to 5-10 hours and reducing the number of full-length chromatographic ones.

Claims (1)

Invention Formula The method of obtaining glycopeptides of General formula I - СЦЦОН- ОЦОН BUT INAS OSHE SNЈN-SH-SH where n-1; NHR is the residue of an amino acid or peptide of formula LAIa-Dlglu, LAIa-Dlgln or LAIa. the interaction of N-acetylamino sugar General formula II H CH, HE SNLN 1 O- frvtf NHAc 0 NHAe. . CH, UNCT where n-1 or 2, with a protected amino acid or peptide in an organic solvent in the presence of a condensing agent and an organic base at 0–20 ° C with cleavage of the protective groups and chromatographic purification of the target product, characterized in that, in order to increase the yield and simplify the process, condensing agent use symmetric carbonates of pentahalophenols of formula III thirty 35 where a) x - F; b) x-C1, and as N-acetylaminosaccharum, directly N-acetylaminosaccharide or its salt with an organic base and at a molar ratio of N-acetylaminosaccharide or its salt: symmetric pentahalophenol carbonate: a protected amino acid or peptide equal, 2) 1 -1.2), with the removal of the protective groups immediately after the reaction.  Compound 11 was previously converted to salt with N-methylmorpholine. V5 THE JUICE 80 90 100tto ep ppcxtfw