Alternative Cefazolin Synthesis with a Cephalosporin-Acid Synthetase
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rnative Cefazolin Synthesis with a Cephalosporin-Acid Synthetase A. V. Sklyarenkoa, *, I. A. Groshkovaa, A. I. Sidorenkoa, and S. V. Yarotskya aNational
Research Center “Kurchatov Institute”, Moscow, 123182 Russia *e-mail: [email protected]
Received March 9, 2020; revised April 1, 2020; accepted April 22, 2020
Abstract—Two pathways of combined chemical and biocatalytic synthesis of the antibiotic cefazolin (CEZ) from 7-amino-cephalosporanic acid (7-ACA) with the immobilized recombinant cephalosporin-acid synthetase as the biocatalyst are compared. The first pathway involved chemical substitution with 2-mercapto5-methylthiadiazole to modify the 3-acetoxy group in 7-ACA with subsequent biocatalytic acylation of the amino group of the product, 7-amino-3-[2-methyl-1,3,4-thiadiazol-5-yl)-thiomethyl]-3-cephem-4-carboxylic acid (TDA), with the methyl ester of 1(Н)-tetrazolylacetic acid. An alternative pathway involved biocatalytic acylation of the 7-ACA amino group to form an intermediate (S-p CEZ) that was chemically transformed into CEZ at the next step without isolation from the reaction mix. Analysis and optimization of each of the biocatalytic processes showed that 7-ACA acylation had a number of important advantages over TDA acylation with respect to the process yield, final concentration of the product in the reaction mix, and the tolerance of the process conditions with respect to enzyme activity and stability. Given the obvious environmental advantages of the process of chemical S-p CEZ transformation into CEZ over the process of TDA production from 7-ACA, we conclude that the second pathway of combined chemical and biocatalytic CEZ synthesis is preferable. Keywords: cefazolin, biocatalytic synthesis, biocatalysis, cephalosporin-acid synthetase, solubility, 7-aminocephalosporanic acid DOI: 10.1134/S0003683820050130
INTRODUCTION Cefazolin ((6R,7R)-3-[(5-methyl-1,3,4-thiadiazol2-yl)thiomethyl]-8-oxo-7-[(1H-tetrazol-1-yl)acetylamino]-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid, CEZ) is among the most important representatives of the class of cephalosporin-acids, which includes more than fifteen semisynthetic parenteral β-lactam antibiotics [1, 2]. This antibiotic is included in the list of basic pharmaceuticals recommended by the World Health Organization. The current volume of CEZ production worldwide is approximately 1000 metric tons per year (calculated for free acid) [3]; its production bases on chemical synthesis. The precursor 3-(acetyloxomethyl)-7-amino-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid (7-amino-cephalosporanic acid, 7-ACA) is transformed into 7-amino-3-[2-methyl-(1,3,4-thiadiazol5-yl)-thiomethyl]-3-cephem-4-carboxylic acid (TDA) via substitution of the 3-acetoxy group mediated by 2mercapto-5-methylthiadiazole (MMTD) in the presence of strong acids in a nonaqueous medium [4, 5]. The TDA amino group is subsequently acylated by activated derivatives of 1(Н)-tetrazolylacetic acid (TzAA) in a nonaqueous or a water-organic solvent medium at
a low temperature (–40°С) via the
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