Postsynthetic Structure Modification

Most antibiotics in therapeutic use are synthesized or modified exclusively by the means of chemistry and are derived from the established compound classes, which have been known for decades. Chemical derivatization thus has also been applied to substances that are of microbial origin and that therefore are termed semi-synthetic.

Among the most recent semi-synthetic antimicrobials are Aventis' streptogramin derivative Synercid and the erythromycin derivatives clarithromycin (Klacid) from Abbott (Abbott Park, Illinois) and roxithromycin (Rulid) and the brand new ketolide telithromycin (Ketek), both from Aventis (Paris, France) [86].

Despite these recent successes and increasing efforts, the yields of therapeuti-cally useful entities emerging from such chemical derivation programs are continuously decreasing.

For this reason, large-scale derivation methods (combinatorial chemistry) are integrated in the search for innovative antibiotics as are biotechnological approaches for structure modification. Biocatalytic procedures, which have been shown to be useful for generating novel antibiotic structures like Loracarbef, a novel p-lactam compound from Eli Lilly (Indianapolis, Indiana) highly active against various p-lactamase producing species [258, 259], are also about to replace continuously chemical production processes. At several universities and corporations, screening studies have been initiated to find appropriate enzymes for compound conversions, as it has been successfully established in production processes for penicillin and cephalosporin. (A review discussing criteria like resource and energy consumption, emissions, health risk potential, area use, and environmental effects for the evaluation of the eco-efficiency of biotechnological processes is given by Saling [260]).

1.1.7.1 b-Lactam Side-Chain Cleavage

6-APA and 7-ACA as the key intermediates for the production of semisynthetic penicillins and cephalosporins (see Section 1.1.3.2) are obtained by removal of the acyl side chains.

There is a large body of patents existing for chemical and enzymatic splitting procedures, but enzymatic processes have been more successful for economical as well as for ecological reasons.

Enzymatic 7-ACA splitting procedures [for general review, see 261] have been developed and commercialized by companies like Asahi Chemical, Hoechst, and Novartis. The replacement of the hitherto employed chemical deacylation processes like the imino ether (Figure 1.1-3) or the nitrosyl chloride method [262] resulted in a cost reduction of 80% and a decrease of the waste volume by a factor 100 from 31 t to 0.3 tons per 1-ton 7-ACA. Chlorinated hydrocarbons like dimethyl aniline and methylene cloride as well as heavy metal ions can be completely avoided. Instead of zinc salt formation, multiple silylation, formation of the imino chloride, imino ether, and finally an imino ether hydrolysis, the side chain is removed in two enzymatic steps (Figure 1.1-3).

Cephalosporin C is first oxidized and deaminated by a D-amino acid oxidase (DAO), which can be obtained from various fungal species, like the yeasts Trigo-nopsis variabilis and Rhodotorula gracilis or the ascomycete Fusarium solani. The resulting a-keto-adipyl-7-ACA, upon decarboxylation, is converted into glutaryl-7-ACA (G-7-ACA). DAO is a flavoenzyme containing flavin adenine dinucleotide as the prosthetic group and catalyzes oxidation of D-amino acids to their corresponding keto acids. In a second step, the glutaryl side chain of G-7-ACA is deacyl-ated by a glutarylamidase from Pseudomonas diminuta [263]. The molecular data of other potentially suitable enzymes and genes from various sources are given by Isogai [264]. It is noteworthy that the enzymatic splitting process could have only been rendered economical and therefore commercially employable through a significant increase of glutarylamidase yield on the fermentation level by using a gene-recombinant E. coli-strain.

A recombinant amidase from E. coli is also the most commonly employed enzyme for 6-APA production by deacylation of penicillin G [265], a process that has been established now for decades [266]. With an annual turnover of 30 tons, the E. coli penicillin amidase is one of the most widely used biocatalysts, despite h2n

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