Influence of Substrate Structure on PGA‐Catalyzed Acylations. Evaluation of Different Approaches for the Enzymatic Synthesis of Cefonicid

The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different β‐lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (v_s) and the rate of hydrolysis of the acylating ester (v_h1). 7‐Aminocephalosporanic acid (7‐ACA) and 7‐amino‐3‐(1‐sulfomethyl‐1,2,3,4‐tetrazol‐5‐yl)thiomethyl‐3‐cephem‐4‐carboxylic acid (7‐SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R‐methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7‐SACA. Cefonicid ( 8 ) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7‐ACA, a “one‐pot” chemo‐enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D ‐amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes.