Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst.

A method for the synthesis of ß-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of 7-amino-3-(5-methyl-l,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (TDA) using immobilized cephalosporin-acid synthetase (IECASA) from recombinant E. coli strain VKPM B-12316 has been developed. A stepwise pH gradient designed on the basis of investigations on the solubility of components was applied for synthesis. This helped in avoiding the precipitation of TDA in the reaction when its initial concentration was high (150-200 mM). Thus, under optimal conditions a high yield of CEZ (relative to TDA) of 92-95% was obtained. Where the final reaction mixture contained 65-85 mg/mL of CEZ, 4-5 mg/mL of unreacted TDA, and 40-60 mg/mL of the by-product, 1(H)-tetrazolylacetic acid (TzAA). Testing of optimized CEZ synthesis using IECASA in a batch reactor has proved sufficiently high operational stability of the biocatalyst, with its residual activity after the 25th cycle accounting for about 83 ± 2% of its starting value. The half-inactivation period of IECASA was estimated as 85 cycles of CEZ synthesis.

Mild reaction conditions for the terminal deuteration of alkynes.

Routinely employed syntheses of terminally deuterated alkynes often utilize strong bases (i.e., LDA, n-BuLi, or Grignard reagents) or low (i.e., -78 °C) or elevated (i.e., 56 °C) reaction temperatures; furthermore many of these procedures afford average yields and in some cases less than optimum deuterium incorporation. Herein we report the application of alternative extremely mild reaction conditions that readily afford quantitative yields of terminally deuterated alkynes in a matter of minutes with exceptional isotope incorporation at ambient temperature.

Metalloantibiotics: synthesis and antibacterial activity of cobalt(II), copper(II), nickel(II) and zinc(II) complexes of kefzol.

Kefzol (kzl), a beta-lactam antibiotic, possesses various donor sites for interaction with transition metal(II) ions [Co(II), Cu(II), Ni(II) and Zn(II)] to form complexes of the type [M(kzl)2]Cl2 and [M(kzl)Cl], with molar ratio of metal: ligand (M:L) of 1:2 and 1:1 respectively. These complexes were prepared and characterized by physicochemical and spectroscopic methods. Their IR and NMR spectra suggest that kefzol potentially acts as a bidentate, tridentate as well as monoanionic tetradentate ligand. The complexes have been screened for antibacterial activity and results were compared with the activity of the uncomplexed antibiotic against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Proteus mirabilis. The metal complexes were found to be more potent against one or more bacterial species than the uncomplexed kefzol.

[Enzymatic synthesis of beta-lactam antibiotics. I. Cefazolin]. / Fermentativnyi sintez betalaktamnykh antibiotikov. I. Tsefazolin.

Production of cefazolin by acyl transfer enzymatic synthesis with immobilised cefazolin synthetase from Escherichia coli as a biocatalyst acting in accordance with the mechanism including formation of the acyl-enzyme complex was shown possible. The process kinetic parameters and the ratio of the maximum conversion of the key amino acid and the initial concentrations of the substrate and nucleophile were determined. Correlation of the calculated and experimental data on the cefazolin yield in the enzymatic synthesis was good. The main physico-chemical properties of the substrates and the reaction products i.e. dissociation constants and solubility were investigated. The complex of the physico-chemical studies makes it possible to design a highly efficient technological process for production of cefazolin including not only the stage of the enzymatic synthesis but also the stage of separation of the reaction mixture components.

The effect of 2-mercapto-5-methyl-1,3,4-thiadiazole on enzymatic synthesis of cefazolin.

The effect of unreacted residual 2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD), the reagent for 3-[5-methyl-1,3,4-thiadiazole-2-yl]-7-aminocephalosporanic acid (M-7-ACA) synthesis, on the enzymatic acylation of M-7-ACA by the methyl ester of 1,2,3,4-tetrazol-1-acetic acid (MeTzAA) to produce cefazolin (CEZ) was studied. In the two-step process of synthesizing CEZ from 7-aminocephalosporanic acid (7-ACA), one of the key parameters controlling the overall CEZ yield was the ratio of MMTD to 7-ACA in M-7-ACA synthesis. The increase of the ratio showed opposing effects by increasing the M-7-ACA yield in the first step, while decreasing CEZ yield in the subsequent enzymatic reaction by the inhibitory effect of the increased content of MMTD as an impurity in the M-7-ACA preparation. It was revealed that the decrease of CEZ yield in the enzymatic reaction was caused by the selective retardation of the rate of CEZ synthesis reaction by a typical competitive inhibition, while not affecting the rate of MeTzAA hydrolysis reaction. The optimum MMTD-to-7-ACA ratio rendering the highest overall CEZ yield over 7-ACA was 1.2:1.

Synthesis and mechanisms of decomposition of some cephalosporin prodrugs.

The delta-3 and delta-2 methyl esters of cefazolin were synthesized. The kinetics and mechanisms of degradation of the methyl esters and the delta-3 and delta-2 isomers of pivaloyloxymethyl prodrug esters of the new cephalosporin ceftetrame (Ro 19-5247) were investigated in buffer systems and in human plasma in vitro. The major hydrolytic products of all the delta-3 and delta-2 esters were the inactive delta-2 cephalosporin free acids. The following reaction scheme describes the in vitro hydrolysis of these compounds: [formula: see text]. In addition, there was evidence of opening of the beta-lactam ring to form cephalosporoic acid when the methyl ester of cefazolin was studied in human plasma and in the presence of penicillinase. For the methyl esters, the processes represented by k12, k21, and k20 were operative in buffers; in human plasma, the processes represented by k12, k21, and k20 were operative in addition to cephalosporoic acid formation. For the isomers of the cephalosporin prodrug ester Ro 19-5248 only k12 and k20 were operative in buffers; in human plasma all pathways were operative and there was no evidence of cephalosporoic acid formation. In all cases, the processes represented by k12, k21, and k20 were subject to general and/or specific base catalysis.