The biosynthesis of penicillins and cephalosporins

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 

IPNS contains Fe2+ (A/r 32000) and causes the loss of four hydrogen atoms 

Before it had been discovered that many penicillins could be made from appropi-ate tripeptides using IPNS, a semi-synthetic method was used to convert penicillin G (8.29) into 6-aminopenicillanic acid using a bacterial acylase followed by acylation of the free amino group. Examples of pharmaceutically important penicillins produced by this route include methicillin (8.41), ampicillin (8.42) and amoxycillin (8.43). There is a more important method of enzymically degrading penicillins than 

Strictly speaking, once IPNS has operated on a tripeptide substrate, any further changes are outside the domain of peptide chemistry and biochemistry and hence of this book. Nevertheless, for the sake of completeness the conversion of penicillins into cephalosporins is briefly mentioned. It has been mentioned above that the production of a cephalosporin may accompany the formation of a penicillin. This can occur, for example, if an extract of Cephalosporium acremonium is used as a source of IPNS. It has been shown that the formation of a cephalosporin results from a ring expansion of a penicillin. Thus penicillin N can be converted into the cephalosporin (8.44), which is also an antibiotic. 

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J Zhang, AL Demain. Purification from Cephalosporium acremonium of the initial enzyme unique to the biosynthesis of penicillins and cephalosporins. Biochem Biophys Res Commun 169 1145-1152, 1990. 

The relationship between the biosynthesis of the penicillin and cephalosporin nuclei [127] is shown in Fig. 8.25. The common intermediate in the biosynthesis of penicillins and cephalosporins is isopenicillin N (IPN), which in Penicil-lium is converted into penicillin G by replacement of the L-2-aminoadipyl side-chain with externally supplied phenylacetic acid, mediated by IPN acyl transferase (IPN AcT). In the cephalosporin-producing Ammonium chrysogenum, IPN is subjected to an enzymatic ring expansion. 

In 1998, Serianni and co-workers88 again resorted to 3 mm micro-NMR probe technology in a report dealing with the detection and quantitation of the cyclic and acyclic forms of 13C-labeled aldopentoses. In a very interesting biosynthetic application, Rowe and co-workers89 investigated the conversion of <5-(L-amino-adipoyl)-L-cysteinyl-D-valine (30) to isopenicillin-N (31), an important step in the biosynthesis of penicillins and cephalosporins. The authors utilized 3 mm micro-NMR probe capabilities in their work in the analysis of the biosynthetic products of mutants of the enzyme isopenicillin-/V synthase, the enzyme normally responsible for the conversion of 30 to 31. 

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. 

The technique of carbon-13 labelling in the study of antibiotic biosynthesis utilising NMR detection methods has been reviewed with specific reference to the -lactam antibiotics [204). The biosynthesis of penicillins and cephalosporins has recently been reviewed and stable isotopes have played a significant role in these studies [208]. 

Martin JF. Gutierrez S, Fernandez F], Belasco J, Fierro F, Marcos AT, Kosalkova K. Expression of genes and procession of enzymes for the biosynthesis of penicillins and cephalosporins. Antonie van Leeuwenhoek 1994 65 227-243. 

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). 

Due to the importance of penicillins and cephalosporins, which certainly saved the lives of hundreds of millions of people, especially the mechanism of the penicillin biosynthesis was intensively investigated (24). Although total syntheses of penicillins are known now for more than 50 years, this reaction in which both rings of the penicillin structure are formed in one step is still impossible to reproduce for... 

The biosynthesis of IPN (Schemes 48 and 49) is common to the production of penicillins and cephalosporins. In some organisms, exchange of the L-a-aminoadipoyl side chain of IPN results in the formation of the hydrophobic penicillins (mainly G and V) this step is catalyzed by a penicillin acyltransferase. In others, epimerization of the IPN L-configured side chain to the D-configured side chain of penicillin N <2000T7601>, catalyzed by IPN epimerase, followed by oxidative ring expansion leads to the cephalosporin family . 

Finally, there are a mixed bag of oxidases, catalysing ethylene formation in plants and many other diverse reactions, illustrated in Figure 13.20, by isopenicillin N-synthase, IPNS, which catalyses the cyclisation of the heterocyclic P-lactam ring. The importance of penicillin- and cephalosporin-related antibiotics in clinical medicine cannot be underestimated and has stimulated the study of their biosynthetic pathways. A key step in the biosynthesis of these antibiotics involves oxidative ring closure reactions of S-(L-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N, the precursor of penicillins and cephalosporins, catalysed by IPNS (Figure 13.20). The overall reaction utilizes the full oxidative potential of O2, reducing it to two molecules of H2O. As discussed earlier, these enzymes are technically oxidases and the four electrons required for dioxygen reduction come from the substrate. 

D. J. Hook, R.P. Elander, R.B. Morin, Recent developments with cell free extracts on the enzymic biosynthesis of penicillins and cephalosporins in Peptide-antibiotics . Biosynthesis and Function, H. Kleinkauf, H. von Dohren eds. de Gruyter, Berlin 1982, pp 84-100... 

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. 

The Rules Governing Medicinal Products in the European Community, Vol. IV, Guide to Good Manufacturing Practice for the Manufacture of Medicinal Products. Queener, S. W. (1990) Molecular biology of penicillin and cephalosporin biosynthesis. Antimicrobial Agents and Chemotherapy, 34,943—948. 

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... 

Warren SC, Newton GG, Abraham EP. Use of alpha-aminoadipic acid for the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp. Biochem J 103(3) 891-901, 1967a. 

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... 

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