Cephalosporins, biosynthesis synthesis

G Banko, S Wolfe, AL Demain. Cell-free synthesis of delta-(L-alpha-aminoadipyl)-L-cysteine, the first intermediate of penicillin and cephalosporin biosynthesis. Biochem Biophys Res Commun 137 528-535, 1986. 

Cephalosporin C Synthesis in Acremonium Chrysogenum. Biosynthesis (Figure... 

The biosynthesis of the p-lactam antibiotic penicillin (Fig. 65), and also of cephalosporin, involves incorporation of L-valine and the question arises as to which of the two diastereotopic terminal methyl groups of the valine occupies which position in the penicillin. (In the case of cephalosporin, the question is as to which methyl group is incorporated into the six-membered ring and which becomes the methylene group of the carbinyl acetate.) The problem has been solved by two groups 65d,141) by synthesis of specifically 13C methyl labeled valine (cf. Fig. 42, and p. 35) which was then biosynthetically incorporated in the antibiotics. The position of the 13C in the resulting antibiotic molecules was determined by 13C NMR spectroscopy. 

The main aspect for monitoring intracellular components was to find the bottlenecks in cephalosporin synthesis. For this it was important to measure the activities of the relevant enzymes in the biosynthesis. After tests in the past [49, 51] and new investigations [22] it was known that the use of protease inhibitors in the disintegration buffer was necessary for routine analysis of activities in crude material. The expense for such measurements was very high and so the main point was the detection of ACVS- and IPNS-activity. But all other important activities were monitored as well. 

The synthesis of the carbapenam-3-carboxylic acid 36 <03JA15746> as well as a study on carbapenem biosynthesis have been documented <03JA8486>. The cephalosporin derivative 37 has been prepared and its use as a novel fluorogenic substrate for imaging P-lactamase gene expression demonstrated <03JA11146>. The nucleus of the carbacephem antibiotic loracarbef has been synthesized in a highly efficient and enantioselective fashion from 25,3iS-2-amino-3-hydroxy-6-heptenoic acid, which was derived from enzyme-catalyzed... 

Although cephalosporin C is divisable into a-aminoadipic acid, cysteine, and valine, the actual mechanism whereby Cephalosporium sp. incorporates the three amino acids into cephalosporin C has not been established, Arnstein and Morris isolated 8 (a-aminoadipyl) cysteinyl valine from mycelia of Penicillium chrysogenum and suggested that the tripeptide is a precursor in all penicillin biosynthesis.. This same tripeptide also appears to be found in the intracellular pool of Cephalosporium sp.- The final postulated step in the biosynthesis of penicillin is an acyl transfer reaction, or the production of 6-aminopeni-cillanic acid if precursor is not added. Cephalosporium sp. apparently do not produce sidechain amidases or acyl transferases, and no 7-ACA has been reported found in the fermentation. Thus, to obtain clinically useful antibiotics, chemical manipulation of cephalosporin C is necessary. Synthesis of many 7-acyl derivatives was possible once a practical cleavage reaction made available large amounts of 7-ACA from cephalosporin C. of these derivatives, sodium cephalothin was the first... 

The elucidation of the mechanism of biosynthesis of penicillin stemmed from the discovery that isotopically labelled cysteine and valine were used in the assembly of penicillin by Penicillium chrysogenum (Amstein and Grant, 1954 Amstein and Clubb, 1957). Cysteine and valine together with a-aminoadipic acid are used by Cephalosporium acremonium to synthesise penicillin N (8.27) and cephalosporin C (8.28). Evidence was accumulated that a tripeptide, h-(f.-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV) was formed as an intermediate. Since this tripeptide is not transported into mycelial cells, it must be synthesised intracellularly and synthesis of penicillin from the isotopically labelled tripeptide was demonstrated using a cell-free system. Clearly, ACV is not produced by a ribosomal synthesis of a protein followed by proteolytic processing. The enzyme involved, ACV synthetase, not only forms the two peptide bonds but also epimerises the valine residue. Thus, incubation of [2-2H]-valine with purified ACV synthetase completely removed deuterium... 

There are also a large number of antibiotics structurally unrelated to penicillins and cephalosporins. These compounds exert their antimicrobial activity by inhibiting protein biosynthesis. In 1947 chloramphenicol (see Figure 3) was isolated from cultures of Streptomyces venezuelae. It is a broad-spectrum bacteriostatic agent that interferes with protein synthesis by binding to bacterial ribosomes. The use of chloramphenicol in humans is hmited because of the drug s toxicity. It inhibits liver enzymes and suppresses red blood cell formation. 

Synthesis of samples of L-cysteine 134 stereospecifically labeled with tritium was first achieved by ourselves in connection with studies on the biosynthesis of the -lactam antibiotics penicillin and cephalosporin (133, 134). The key steps in this synthesis were conversion of the imines 130 and 130, = H, to... 

Kim MG, Lee SB (1996) Penicillin acylase-catalyzed synthesis of P-lactam antibiotics in water-methanol mixtures effect of cosolvent content and chemical nature of substrate on reaction rates and yields. J Mol Catal B Enzym 1 201-211 Kohsaka M, Domain AL (1976) Conversion of penicDlin N to cephalosporin(s) by ceU-free extracts of Cephalosporium acremonium. Biochem Biophys Res Commun 70(2) 465-473 Koreishi M, Tani K, Ise Y et al. (2007) Enzymatic synthesis of P-lactam antibiotics and /V-fatty-acylated amino compounds by the acyl transfer reaction catalyzed by peniciUin V acylase from Streptomyces mobaraensis. Biosci Biotechnol Biochem 71(6) 1582-1586 Kupka JY, Shen, YQ, Wolfe S et al. (1983) Partial purification and properties of the alpha-ketoglutarate-linked ring expansion enzyme of beta-lactam biosynthesis of Cephalosporium acremonium. FEMS Microbiol Lett 16 1-6... 

Biosynthesis Tlie biosynthesis proceeds by a non-ri-bosomal process via a dipeptide of D-a-aminoadipic acid (D-a-A AA) and Lside chain which is exchanged in the further course of the synthesis of P., in contrast to that of the cephalosporins. The numerous natural and synthetic P. differ in the substituents on the side chain and the carboxy group for nomenclature, see the table. 

LACTAM ANTIBIOTICS - deals with the chemical and biochemical aspects of the -lactam antibiotics. Topics selected include the synthesis, biosynthesis, chemical reactivity, anti-microbial activity, pharmacodynamics, metabolism, toxicology, detection and analysis, and pharmaceutical applications of the penicillins, cephalosporins, and related compounds. 

This chapter summarizes the recent developments in the field of the synthesis of nuclear analogues of penicillins, cephalosporins, monocyclic -lactams and other penicillin and cephalosporin analogues. An account is given of the available data on other -lactams of biological interect and certain attempts to synthesize some of these molecules with the help of our recent knowledge on biosynthesis. 

The multistep chemical conversion of penicillin G to 7-ADCA (Fig. 16) has recently been replaced by a 2-step biosynthesis (Fig. 18). This is a major step forward to shorten the industrial synthesis of cephalosporins and this has already been implemented. The 7-N-adipoyl-ADCA is obtained directly through fermentation with a modified Penicilium chrysogenum followed by a simple enzymatic removal of the amino substituent. Again, various reagents such as silylat-ing agents, phosphor halides, pyridine and DMF, and some halogenated solvents have been replaced by biocatalysts in aqueous medium. 

All the reactions required for the synthesis of the tripeptide lld-ACV, which, as already mentioned, are common to the biosynthesis of penicillins and cephalosporins. 

A. are chemically heterogeneous Important components include amino acids, often nonproteogenic D-amino acids (e.g. in gramicidin), acetate/malonate units (griseofulvin, tetracycline), sugars and sugar derivatives (streptomycin), tetracyclic triterpenes (fusi-dinic acid, helvolinic acid, cephalosporin P,). There are also many Nucleoside A. (see). A. act by various mechanisms Many interfere with protein biosynthesis, but others, e.g. penicillin, inhibit cell wall synthesis in bacteria. 

A recent variation on the synthesis of cephalosporin by thiyl radical cycliza-tion was reported by Cabri and co-workers [36]. T ing advantage of the mechanistic studies carried out by Balwin s group on the enzymatic systems involved in the biosynthesis of penicillins and cephalosporins [37], the intramolecular thiyl addition promoted by Fe(III) and Mn(III) has been studied in detail. Some examples are summarized in equation (8). 

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