Enzymatic synthesis of amoxicilloic acids

Abstract

(αR,2R,4S)-2-[2(R)-Amino-2-(4-hydroxyphenyl)acetylamino]-4-carboxy-5,5-dimethyl-2-thiazolidinylacetic acid (amoxicilloic acid) (2) is the first product of the hydrolytic clevage of the semisynthetic penicillin amoxicillin 1, used for the treatment of bacterial infections. This explains the large number of analytical investigations on the identification of these compounds in biological solutions and industrial samples. Analysis of literature data showed that samples of amoxicilloic acid (αR,2R,4S)-2 and (αR,4S)-2 for analytical and chemical investigations were obtained by alkaline hydrolysis of amoxicillin 1, acidification or neutralization of the reaction mixture with subsequent dehidration [1, 2]. Preparative hydrolytic clevage of the β-lactam ring in natural and semisynthetic penicillins, carried out to obtain the valuable penicilloic acid 2, is a routine problem, which is complicated in the case of amoxicillin: a) the amphoteric properties amoxicilloic acids, caused by the presence of amino and carboxylic groups, excludes the extraction of the initial or final reaction products from aqueous solutions by organic solvents; b) the configurational instability in acid and basic media of the 2C chiral center in (αR,2R,4S)-2 which leads to the formation of the corresponding racemate (αR,4S)-2 [3, 4]; c) the formation in quantity of the similar substance 2',5'-dioxopiperazine isomer of amoxicillin 3 and amoxilloic acid 4 and also other products, makes it difficult to isolate and purify the valuable product (αR,2R,4S)-2. In this connection we have developed an effective method for obtaining analytically pure amoxicilloic acid (αR,2R,4S)-2 by hydrolytic clevage of the β-lactam ring of amoxicillin 1 in the presence of the immobilized penicillinase of Bacillus cereus at pH 8. The constant value of the pH of the solution, decreased as the result of formation of additional carboxyl groups, is maintained by the addition of 10-15% ammonia solution. Chromatographic monitoring of the end of the reaction is indicated by the decrease in the pH of the medium and the disappearance of the amoxicillin spot. After removal of the immobilized penicillinase by filtration, the filtrate was acidified to pH 5.5 with dilute hydrochloric acid, partially evaporated and maintained at 5°C for several hours. The finely crystalline precipitate was filtered and dried in the air. According to