RU2210596C2 - Method of synthesis of cefazolin - Google Patents

Abstract

FIELD: antibiotics, microbiology. SUBSTANCE: invention relates to production of cefazolin relating to semisynthetic antibiotic of cephalosporin order. Method involves acylation of 3-mehylmercapto-(5-methyl-1,3,4-thiadiazolyl-2-yl)-7- aminocephalosporanic acid with tetrazole-1-acetic acid esters in an aqueous medium in the presence of immobilized enzyme from E. coli followed by fractional precipitation of 3-methylmercapto-(5-methyl-1,3,4-thiadiazolyl-2-yl)-7-aminocephalosporanic acid and cefazolin and by recycling of 3-methylmarcapto-(5-methyl-1,3,4-thiadiazolyl-2-yl)-7-aminocephalosporanic acid. Acylation is carried out at temperature 10-30 C and pH gradient 7.8-6.2 using 3-methylmercapto-(5-methyl-1,3,4-thiadiazolyl-2-yl)-7- aminocephalosporanic acid in the initial concentration 20-40 g/l and the excess of tetrazole-1-acetic acid = (1.5-4) : 1. Polyenzyme complex of peptide hydrolases from the E. coli strain FU-99-S with predominant content of cefazolin synthetase with activity ≥ 300 mcmole/g x min by cefazolin synthesis and ratio above 8:1 by synthesis and hydrolysis of cefazolin is used as a biocatalyst immobilized in polyacrylamide gel as biomass cells of producer of cefazolin synthetase or enzyme isolated from biomass cells. Method provides the enhancement of output, effectiveness and quality of the end product. EFFECT: improved method of synthesis. 5 cl, 3 tbl, 7 ex

Description

 The invention relates to the field of production of drugs, namely the production of cefazolin, a representative of the group of effective semi-synthetic cephalosporin antibiotics.

 Known methods for producing cefazolin by anylation of 3-methylmercapto- (5-methyl-1,3,4-thiadiazolyl-2-yl) -7-aminocephalosporanic acid (MMTD-7-ACA) with tetrazole-1-acetic acid esters (ETZUK) using as biocatalysts (BC) (enzymes from E. coli (see USSR author's certificate 1835827, 1990 [1] and USSR author's certificate 1817473, 1990 [2]) and fungus culture Arthrobacter viscosous sp. nov. (see Japan patent 54 -59397, 1979) [3].

 The analysis of the known method [3] indicates that the enzyme used in this method is not covalently bound to the carrier, which leads to leaching of the enzyme into the reaction mass, and, consequently, to loss of BK activity and contamination of the final product with protein impurities. Indeed, the method [3] provides a rather complicated scheme for the purification of cefazolin: after separation of the BA, the filtrate is passed through a column with a nonionic sorbent, then the eluate is subjected to film chromatography on silica gel, treated with activated carbon, and the solution thus purified is evaporated to precipitate cefazolin. The disadvantage of this method is the process at non-optimal for the synthesis of cefazolin pH values of 7.3-7.8.

 According to the method [2], a nonionic sorbent in combination with anion exchange resin is also used to extract cefazolin from the reaction mass. The synthesis of cefazolin is carried out in a continuous mode with the withdrawal of the solution from the bioreactor and its recycling through a system of series-connected columns filled with sorbents saturated with the original key amino acid (CA). The disadvantage of this method, in addition to the complex scheme of the process, is the low productivity of the process, due to the irrational choice of the initial concentrations of the reagents.

 As a prototype of the selected method [1], as the most technologically advanced and superior in efficiency to other known methods. It was also taken into account the fact that it uses an enzyme from the same family of microorganisms as in the claimed invention. According to the prototype method, immobilized penicillin amidase from E. coli (K.F.3.5.1.11) with a ratio of the activity of the synthesis and hydrolysis of cefazolin of 8: 1 is used for acylation. The degree of biotransformation of MMTD-7-ACC to cefazolin is 80-82%. After synthesis, the components of the reaction mixture are separated by fractional precipitation, and MMTD-7-ACC is used for repeated acylation. The degree of spacecraft utilization is 88%, which corresponds to a consumption coefficient of 0.86 kg / kg cefazolin. The content of the main substance in the product is not less than 95%.

This method has the following disadvantages:
- Low productivity of the process due to the low initial concentration of MMTD-7-ACC (6 g / l according to the examples). The synthesis begins with incomplete dissolution of the CA at a pH of 6.2.

- The use of BC with activity on the synthesis of cefazolin 200 ± 50 μmol / g • min leads to the need for a reaction at a temperature of 40 ^o C, which leads to a deterioration in the quality of the product due to the hydrolysis of MMTD-7-ACC and cefazolin C3 connection.

 The technical problem that the claimed invention solves is to increase the productivity and overall efficiency of the cefazolin production process, as well as to improve the quality of the final product.

где К₂ константа ионизации аминогруппы ММТД-7-АЦК; s^± - растворимость ее цвиттерионной формы.This problem is solved as follows. MMTD-7-ACC is acylated by ETZUK in an aqueous medium in the presence of CD based on either E. coli cells immobilized in polyacrylamide gel (PAAG), strain FU-99-S, or a peptide-hydrolase polyenzyme complex with a predominant content of cefazolin synthetase isolated from these cells modified with a bifunctional reagent - glutaraldehyde, and immobilized in SDS page. A biocatalyst is obtained with cefazolin synthesis activity of 360 ± 60 μmol / g • min (determination conditions: 30 ^° C, pH 7.5, [MMTD-7-ACC] = 0.075 M, [METZUK] = 0.145 M). The density of the suspension of BC in the reactor varies from 1 to 3.5% depending on the activity of the BC and the temperature of the reaction mass during synthesis, selected in the range from 10 to 30 ^o C. The initial concentration of MMTD-7-ACC 20-40 g / l, the molar ratio of ETZUK and MMTD-7-ATsK (X) - (1,5 ÷ 4): 1. The synthesis of cefazolin is carried out in a pH gradient of values, starting with pH, at which the dissolution of MMTD-7-ACA is ensured (7.3-7.8 depending on the initial concentration of spacecraft and the process temperature), and ending at pH 6.0-6, 3 (optimal for the synthesis of cefazolin). The decrease in pH during synthesis occurs due to the release of free acid - TZUK formed during the hydrolysis of ETZUK. However, with an excessive decrease in the pH of the reaction mixture, precipitation of MMTD-7-ACA may occur during the synthesis, which is undesirable. Therefore, the pH of the reaction mass must be maintained at such a level that the current concentration of MMTD-7-ACA (N _τ ) does not exceed its solubility (s), that is, that the ratio N _τ / s≤1 is observed:

where K _{2 is} the ionization constant of the amino group MMTD-7-ACC; s ^± is the solubility of its zwitterionic form.

 The calculation of the current concentration of MMTD-7-ACC at each time point (τ) is carried out using equations (2-7) of a mathematical model developed on the basis of the kinetic scheme for the synthesis of cefazolin product (P), according to which ETZUK is a substrate (S) acylating enzyme and MMTD-7-ACC by a nucleophile (N) competing with water at the stage of deacylation of ancyl enzyme.

где V₂ и V₃ скорость ферментативного гидролиза и ферментативного синтеза цефазолина, соответственно.

where V ₂ and V _{3 are the} rates of enzymatic hydrolysis and enzymatic synthesis of cefazolin, respectively.

a₁=1+N/k₁₂+P₂/k₁₁ (5)
a₂=1+N/k₁₂ (6)
a₃=N/K_N (7)
Здесь P₂ - побочный продукт процесса, ТЗУК.

a ₁ = 1 + N / k ₁₂ + P ₂ / k ₁₁ (5)
a ₂ = 1 + N / k ₁₂ (6)
a ₃ = N / K _N (7)
Here, P ₂ is a by-product of the process, PLC.

The values of the constants included in equations (2-7) and used to calculate the pH time dependence are shown in Table 1. These values were determined by the iterative method when comparing the experimental curves of cefazolin synthesis with the calculated curves obtained using equations of the mathematical model and kinetic and equilibrium constants found in model experiments. Figures 1-3 show, for example, the results of calculating the pH gradient, the degree of conversion of MMTD-7-ACC, and the parameter N _τ / s for several variants of the initial concentrations of reagents in the synthesis of cefazolin. As can be seen from Figures 2 and 3, when using a high excess of acylating agent, the degree of conversion of MMTD-7-ACC reaches 95-97%. The expenditure ratios of raw materials in kg per kg of cefazolin in this case are 0.90-0.95 for MMTD-7-ATsK and 1.3-1.5 for ETZUK (based on TZUK). The regeneration of TZUK, consisting in the conversion of the TZUK salt to an organic solvent - butanol - by azeotropic distillation of water after removal of inorganic salts and partial evaporation of the aqueous solution, allows to reduce the ETZUK expenditure coefficient to 0.50-0.55 kg / kg, however, the regeneration process is sufficient laborious. Meanwhile, when using a approximately 2-fold excess of the acylating agent, regeneration of TZUK is not required: the expenditure coefficient of ETZUK is about 0.9-1.0 kg TZUK per kg of cefazolin. As for MMTD-7-ACC, since its conversion to cefazolin with a 2-fold excess of the acylating agent is only 70-75%, to create an effective technology, the unreacted MMTD-7-ACC needs to be regenerated, which is carried out by simple deposition of it in isoelectric point. The expenditure coefficient of MMTD-7-ACC is about 0.8. The calculations show that a process variant with a 2-fold excess of the acylating agent is most preferred.

 In addition, for the selected ETZUK excess, there is also the preferred concentration range of MMTD-7-ACC (25-30 g / l), at which the calculated pH gradient from 7.3 to 6.2 is reached spontaneously. In other cases, the pH of the reaction mass is maintained by adding an ammonia solution in an automatic mode using computer control. The controlling parameter in this case is the pH of the reaction mixture, the calculation of which is carried out according to equation 1. At the end of the biotransformation, the BA is separated by filtration and used repeatedly and in the next cycle of cefazolin synthesis. The reaction mixture was acidified to pH 4.2-4.3, MMTD-7-ACA was separated by filtration, the paste was washed and used for repeated acylation.

 The filtrate after separation of MMTD-7-ACC or immediately after separation of BC (with a technology variant with a large excess of ETZUK without SC regeneration) is treated with aluminum oxide to remove colored impurities and cefazolin is precipitated by acidification of the solution to a pH of 1.4-1.5. If necessary, TZUKs are regenerated from the mother liquor after separation of cefazolin, as described below.

The total degree of utilization of MMTD-7-ACC is 90-95%, which corresponds to a consumption coefficient of 0.85-0.80 kg / kg of cefazolin. The expense coefficient ETZUK varies from 0.5 to 1.4. The content of the main substance in the target product is more than 97%. The proposed method is as follows. Prepare a solution of MMTD-7-ACC with a concentration of 20-40 g / l in 0.3 M phosphate-ammonia buffer (FAB), pH 7.5-8.0, at a temperature of 10-30 ^o C. After that, the solution MMTD -7-ATsK add a sample of ETZUK from the calculation of the molar ratio of substrate and spacecraft (1.5 ÷ 4): 1. The volume of the solution is adjusted to the calculated value by adding 0.3 M phosphate-ammonia buffer, pH 7.5-8.0, and the initial pH of the solution is set in the range 7.3-7.8.

The synthesis of cefazolin is carried out in a thermostatic reactor with a filter bottom equipped with a paddle mixer with a variable speed and a system for automatically maintaining the pH gradient according to a given program. A weighed sample of wet BC with cefazolin synthesis activity ≥ 300 μmol / g • min is loaded into the reactor from 10 to 35 g / l (depending on the selected process temperature) and a solution of the starting reagents. The reaction mass is incubated with stirring and automatically maintaining the pH gradient in the range (7.3-7.8) - (6.2-6.4) with a 2M ammonia solution for 45-60 minutes. The reaction mass is separated from the BC by filtration, cooled to 5 ^{° C.} , acidified with sulfuric acid to pH 4.2, and unreacted MMTD-7-ACA is precipitated for one hour. The precipitate was separated by filtration, washed with a small volume of water, squeezed thoroughly and used in the next cycle of cefazolin synthesis. In the case of regeneration of TZUK, the spacecraft regeneration stage is omitted. After separation of MMTD-7-ACA (or immediately after separation of BC), 1-2% w / v alumina is added to the mother liquor, the suspension is stirred for 30-40 minutes, and the spent sorbent is separated by filtration. The filtrate is acidified to pH 1.4-1.5 and cefazolin precipitated. After holding the suspension for an hour with stirring and cooling, the crystalline precipitate of cefazolin was separated by filtration, washed with cold water and dried in vacuum at 30 ^° C for 12 hours. The content of the main substance in the target product is more than 97%. To regenerate TZUK, the mother liquor after separation of cefazolin is neutralized by adding a powder of CaO or Ca (OH) ₂ and the precipitate of calcium sulfates formed is removed by filtration. Next, the solution was concentrated 5-7 times by evaporation in vacuo, the concentrate was treated with activated carbon (2% w / v) to clarify, butanol was added to it at a rate of 4 volumes per 1 volume of aqueous concentrate, and the azeotropic mixture was distilled off on a rotary evaporator at a residual pressure not less than 10 mmHg and a bath temperature of 40-45 ^o C. The evaporation is carried out until the water is completely distilled off: for this it is necessary to drive off at least 4/5 of the volume of the water-butanol mixture. At the end of the distillation, a suspension of the salts of TZUK in butanol is cooled to 5 ^{° C.} and filtered under vacuum. The paste is washed, dried and ETZUK obtained after esterification is used again for the synthesis of cefazolin. The expenditure coefficient TZUK taking into account the regeneration is 0.5-0.55 kg / kg cefazolin.

The advantages (distinguishing features) of the proposed method:
1. Significantly increased in comparison with the known methods [1], [2], [3] productivity and overall efficiency of the cefazolin production process, achieved due to the high initial concentration of MMTD-7-ACC, which became possible due to the organization of the synthesis process with a pH gradient values.

2. The ability to create highly effective CDs not only on the basis of an enzyme, but also on the basis of cells through the use of a highly active strain producing a specific enzyme. In turn, this allows to reduce the process temperature up to 10 ^o C, without increasing the reaction time and the density of the suspension of BK. Carrying out the process under milder conditions improves the quality of the product.

 3. The ability to remove colored impurities due to the introduction of an additional stage of purification of the cefazolin solution with aluminum oxide leads to an improvement in the quality of the product.

 The method is illustrated by the following practical examples.

Example 1. (25 g / l KA, X = 2)
Prepare a solution of substrates. 10.29 g (29.90 mmol) of MMTD-7-ACC is dissolved in 350 ml of 0.3 M FAB, pH 7.5 at a temperature of 30 ^o C, maintaining the pH of the solution at least 7.3 by adding 2 M ammonia solution . After that, 8.70 g (61.26 mmol) of methyl ETZUK (METZUK) was added to the MMTD-7-ACA solution and the volume of the solution was adjusted with 0.3 M FAB, pH 7.5 to 400 ml.

4.0 g of wet BK with a cefazolin synthesis activity of 360 μmol / g • min and a solution of the starting reagents are loaded into the reactor. The reaction mass is incubated at a temperature of 30 ^o With stirring for 50 minutes The pH of the reaction mass spontaneously decreases and reaches a value of 6.3 at the end of the process. The reaction mass is separated from the BC by filtration, cooled to 5 ^{° C.} , acidified with hydrochloric acid to pH 4.2, and unreacted MMTD-7-ACA is precipitated for one hour. The precipitate was separated by filtration, washed with 20 ml of water, squeezed thoroughly and used in the next cycle of cefazolin synthesis. 13.50 g of wet paste are obtained with a content of MMTD-7-ACC 21.3% and cefazolin 1.2%. After separation of MMTD-7-ACA, 8 g (2% w / v) of aluminum oxide are added to the mother liquor, the suspension is stirred for 30-40 minutes, and the spent sorbent is separated by filtration. The filtrate is acidified to pH 1.4-1.5 with a solution of sulfuric acid (1: 1) and cefazolin is precipitated. After holding the suspension for one hour under stirring and cooling, the cefazolin crystalline precipitate was separated by filtration, washed with 30 ml of cold water and dried in vacuum at 30 ^° C for 12 hours. 8.98 g (19.78 mmol) of cefazolin are obtained. The content of the main substance in the target product is 98.1%. The expenditure ratios of MMTD-7-ACC and TZUK are 0.828 and 0.874 kg / kg cefazolin, respectively.

 Examples 2 and 3.

The preparation of cefazolin is carried out analogously to example 1, except that ethyl and propyl esters of TZUK (EETZUK and PETZUK, respectively) are used. The temperature is 30 ^o C, the weight of the BC is 4.0 g. The results are presented in table 2.

 Examples 4-7.

 Solutions of substrates with a volume of 400 ml are prepared as described in Example 1, except that 0.3 M FAB is used, the pH is 7.5-8.0, and the pH of the stock solution is set in the range of 7.3 to 7.8.

The corresponding sample of wet BC and a solution of the starting reagents are loaded into the reactor. The reaction mass is incubated at a given temperature and stirring for 50-60 minutes, maintaining the pH of the reaction mass in accordance with the calculated gradient by adding an ammonia solution in automatic mode. At the end of the synthesis, fractional precipitation of the components is carried out as described in example 1. In example 6, the deposition step of MMTD-7-ACC is omitted. In examples 5-7, TZUK is regenerated from the mother liquor after separation of the cefazolin crystalline precipitate. To the stock solution of 420 ml, 13.9 g of CaO (94% basic substance content) are added with stirring until the pH of the suspension is about 7.5. The resulting precipitate of CaSO _{4 was} separated by filtration in vacuo and washed thoroughly, suspending it in 50 ml of water. Flushing attaches to the filtrate. The combined filtrate with a volume of 430 ml was evaporated on a rotary evaporator at a residual pressure of 10 mm Hg. and a bath temperature of 45 ^o C to 70 ml. The aqueous suspension of inorganic salts is filtered off, washed with water, 3 times 5 ml each, washing is added to the concentrate of TZUK. 1.6 g of activated carbon was added to a combined 80 ml concentrate, and the suspension was stirred for 20 minutes. The coal is filtered off, washed with 10 ml of water, squeezed thoroughly. 340 ml of butanol are added to a clarified aqueous concentrate of TZUK with a volume of 85 ml and the azeotropic mixture is distilled off on a rotary evaporator at a residual pressure of 10 mm Hg. and a bath temperature of 45 ^° C. The evaporation is carried out until the water is completely distilled off. The distillation of a 350 ml water-butanol mixture is divided in a separatory funnel: water is discarded, butanol-distillation is reused. VAT residue butanol suspension of salts TRZU with a volume of 70 ml is cooled to 5 ^o C and filtered under vacuum. The salt precipitate is washed with fresh butanol, 2 times 5 ml each, squeezed out and dried in a vacuum oven. A powder is obtained containing about 60% TZUK and 40% inorganic salts. The yield of TZUK during regeneration is about 95% of its content in the mother liquor after separation of cefazolin. The product is used for the synthesis of METZUK with a yield of 88-90%. The experimental results are presented in table 3.

Claims (5)

1. The method of producing cefazolin by acylation of 3-methylmercapto- (5-methyl-1,3,4-thiadiazolyl-2-yl) -7-aminocephalosporanic acid (MMTD-7-ACA) with tetrazole-1-acetic acid esters (ETZUK) in aqueous medium in the presence of an immobilized enzyme from E. coli, followed by fractional precipitation of MMTD-7-ACC and cefazolin and recycling of MMTD-7-ACC, characterized in that the acylation is carried out at a temperature of 10-30 ^o C in a pH gradient of 7.8-6 , 2, using an initial concentration of MMTD-7-ACC of 20-40 g / l, a molar excess of ETZUK (1.5-4): 1 and immobilized into polyacrylamide spruce (PAGE) multienzyme complex peptidogidrolaz E. coli strain FU-99-S with a predominant content tsefazolinsintetazy with activity on the synthesis of cefazolin ≥ 300 .mu.mol / g • min and the ratio of activity on the synthesis and hydrolysis of cefazolin than 8: 1.  2. The method according to p. 1, characterized in that the acylation is carried out preferably in a pH gradient of 7.3-6.2, established spontaneously, using the initial concentration of MMTD-7-ACC 25-30 g / l and an excess of ETZUK (1, 9-2.4): 1.  3. The method according to p. 1 or 2, characterized in that as the acylating agent is preferably used methyl ETZUK.  4. The method according to any one of paragraphs. 1-3, characterized in that as the biocatalyst using biomass immobilized in the SDS page biomass of cells of the producer of cefazolin synthetase.  5. The method according to any one of paragraphs. 1-3, characterized in that as the biocatalyst use immobilized in the SDS page cefazolinsyntas, isolated from the biomass of cells.

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