Aminoadipyl side-chain

In organisms which produce cephalosporin and cephamycins, the configuration of the O -aminoadipyl side chain of (30) is D, while penicillin producers yield the l isomer. The exact point at which the configuration is inverted is unknown. Subsequent steps in cephalosporin biosynthesis are believed to involve ring expansion to deacetoxycephalosporin C (31), which may proceed by a mechanism analogous to the chemical pathway (see Section 5.10.4.2), followed by hydroxylation and acetylation at C-3 to produce cephalosporin C (32). 

After a strain improvement and development programme similar to, but more complicated than that of penicillin, the D-a-aminoadipyl side chain containing cephalosporin C was obtained by large scale fermentation. However, cephalosporin C could not be isolated as easily as penicillin G or V. Due to its amphoteric nature it is soluble at any pH in the fermentation broth. Several costly isolation procedures involving ion-exchange chromatography have been developed, as a result of which cephalosporin C is much more expensive than penicillin G. 

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

The building blocks for the biosynthesis of benzylpenicillin are three amino acids, a-aminoadipic acid, cysteine and valine, and PAA. The amino acids condense to a tripeptide, ring closure of which gives the penicillin ring structure with an cu-aminoadipyl side-chain, isopenicillin N. The side-chain is then displayed by a phenylacetyl group from PAA to give benzylpenicillin. 

By the addition of different acyl donors to the medium, different penieillins can be biologically synthesized. For example, penicillin V is made by a similar process to benzylpenieillin, but with phenoxyacetic add as the precursor instead of PAA. In the biosynthetic pathway, the a-aminoadipyl side-chain of isopeniciUin N is replaced by a phenoxyacetyl group. 

The generation of cephalosporin from penicillin is dependent on the epimeriza-tion of the L-a-aminoadipyl side chain of isopenicillin N to the D-a-aminoadipyl side chain of penicillin N, since penicillin N but not isopenicillin N is the substrate for ring expansion enzymes [31], This reaction is catalyzed by IPNE ([32] Table 1) and is coded for by the cefD gene (Fig. 1 [5]). While the activity of the IPNE from C. acremonium has been studied in cell-free extracts, it has not been purified to date [2], Jensen et al. [33] described a partial purification and charac-... 

A second purification strategy involves the substitution of the amine moiety on the a-aminoadipyl side chain at C-7. Two substituted derivatives, iV-2,4-dichlorobenzoyl CPC and tetrabromocarboxybenzoyl CPC, can be crystallized from the acidic aqueous solution. Alternatively, salts can be formed between the, /V-substituted derivatives and an organic base, such as dicyclohexylamine or dimethylbenzylamine, resulting in cephalosporin salts that are solvent extractable. Bristol-Myers Squibb uses a solvent-extractable process resulting in the isochlorobutylformate (ICBF) ester of CPC, termed cephalosporin D. Several extraction steps are usually necessary to achieve the desired final purity. iV-Substituted CPC salts containing small amounts of contaminants can be effectively converted to 7-ACA. 

The addition of suitable precursors (e.g., phenylacetic acid, phenoxyacetic acid) in the fermentation medium of P. chrysogenum allows the formation of specific penicillins (G, V, F, K, X) with nonpolar side chains < 1995JAN1195, 1998MI2001, 1999MI173>. Penicillins are the only /3-lactam products formed, while fermentation of Cephalosporium acremonium produces penicillin N (D-a-aminoadipyl side chain) together with cephalosporin derivatives (CHEC-11(1996), section 1.20.6.1) <1996CHEC-II(1B)623>. 

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. 

Various methods are known to produce 7-ACA from cephalosporin C (Ceph C) by removing the a-aminoadipyl side-chain. They can be classified into three types, the chemical process, a two-step enzymatic process and an enzymatic process in which the side-chain is directly removed from Ceph C. Today two processes are running commercially on an industrial scale, the classical chemical process and the modern two-step biocatalytic process (Fig. 2). Until now the favorable direct process is less effective, because of low conversion. 

In a first step the chiral center at the a-aminoadipyl side-chain has to be eliminated. Besides commercially available D-amino acid oxidase (DAO) from pig kidney for laboratory use, the enzyme is also synthesized by bacteria, yeasts and fungi. This enzyme can be used for the resolution of D,L-amino acids in various solutions, but of particular interest is its capacity for oxidative deamination of Ceph C. The first research was carried out by Glaxo Laboratories Ltd. [11] where DAO isolated from different fungal cells was used. Under the reaction conditions used, in the presence of oxygen as a co-substrate, the enzyme deaminates cephalosporin C to give a-ketoadipyl-7-ACA, ammonia and hydrogen peroxide (Fig. 5). 

Substitution of the amine on the a-aminoadipyl side chain of cephalosporin C (Figure 7), using many of the derivatization methods borrowed from classical peptide and amino acid chemistry, sufficiently alters the properties of cephalosporin C so as to, depending on the derivative, make it solvent extractable or insoluble in aqueous solutions. Figure 8 shows examples of many such derivatives which have been prepared and which have been reported to aid in the isolation of cephalosporin C (16-40). The utility of this approach lies in the fact that each of these cephalosporin C derivatives are able to be cleaved to 7ACA in yields equivalent to (and sometimes better than) yields achieved with cephalosporin C itself. 

It has been observed critically that the natural products usually exhibit a relatively lower level of antibacterial activity. Therefore, the articulate and judicial cleavage of the amide bond of the aminoadipyl side-chain present in cephalosporin C provides 7-amino-cephalosporanic acid (7-ACA), which is most ideally suitable for the synthesis of a wide range of semisynthetic cephalosporins via acylation of the C(7)-amino fimctional moiety as depicted under ... 

Cephalosporin C (33) is another important natural j -lactam antibiotic, isolated from fermentations involving Acremonium chrysogenum. It is related to penicillin G, and its synthesis is derived from the same metabolic intermediate, isopenicillin N (36) (Scheme 6.13). Unlike the penicillins, there is no therapeutic use for the natural product itself, and the manufacture of all useful cephalosporins requires the removal of the 7-aminoadipyl side chain to provide the 7-ACA (7-aminocephalosporanic acid) (37). This remains a chemical process in which NOCl or PClj is used to break the cxo-cyclic amide bond. 

The further potential of the technology is seen in a recent manipulation of the synthesis of cephalosporin C (35). If the two enzymes (Section 6.5) which are needed to remove the 7-aminoadipyl side chain are both transferred to A. chrysogenum, then the engineered strain will produce 7-AC A (37) directly. 

Guy Newton, who had come from Cambridge to Oxford as a doctoral student after a distinguished war record, then joined in a study of cephalosporin N. Our interest in this substance derived at first from the finding that some of its properties were those of a labile peptide, for we were already working on the thiazoline-containing peptide, bacitracin. By the end of 1953, cephalosporin N had been shown to be a new penicillin with a 8-(D-a-aminoadipyl) side chain, and it was later renamed penicillin N. It then seemed to us that an uncharacterized antibiotic named synnematin, which had been obtained in a crude form at the Michigan Department of Health from culture filtrates of Cephalosporium salmosynnematum, might be identical to penicillin N, and a direct com-... 

To explore the potentialities of semisynthetic cephalosporins it became imperative to find a method for the production of 7-ACA from cephalosporin C in a yield much higher than that obtained by acid hydrolysis. We confidently expected that an enzyme would be found that would catalyze the removal of the 8-(D-a-aminoadipyl) side chain the same view was expressed by Dr. Karl Folkers and Dr. Denkewalter who visited us in Oxford in 1959. In any event, widespread searches for such... 

Cleavage of the amide bond of the aminoadipyl side chain of cephalosporin C affords the 7-aminocephalosporanic acid (7-ACA) nucleus (180). This was first achieved in small yield by direct acid hydrolysis (151). The inability to cleave the side chain enzymatically as in the penicillins (6) resulted in much effort to find an efficient chemical method to provide (180), since this is the source of many clinically important semi-synthetic cephalosporins. Many of these compounds have advantages over the penicillins in terms of acid stability and resistance to P-lactamases. 

L-valine to form 5-(L-a-aminoadipyl)-L-cysteinyl-D-valine. This is then cyclised to isopenicillin N. In some microorganisms, such as Cephalo-sporium sp. and Streptomyces clavuligerus, the 8-(L-a-aminoadipyl)-side-chain is epimerised to the D-configuration and the resulting penicillin N is excreted. In Penicillium chrysogenum, and other fungi that produce hydrophobic penicillins, isopenicillin N is converted to one or more penicillins with a monosubstituted acetyl side chain derived from the appropriate monosubstituted acetyl-CoA precursor. Whether this happens by transacylation of isopenicillin N, or deacylation to 6-APA and subsequent reacylation, or by both processes, is not clear. Penicillin biosynthesis is summarised schematically in Fig. 4. 

In the course of studies on the Brotzu strain of Cephalosporium, Abraham and Newton detected small quantities of a second antibiotic, cephalosporin C. Painstaking work proved it to be chemically similar to penicillin N, but not a penicillin. It had pronounced gramnegative activity, was more stable to acid, and was not destroyed by penicillinase. It possessed the same a-aminoadipyl side chain as a new penicillin, but the nucleus was 7-aminocephalo-sporanic acid (7-AC A). 7-AC A contains a six-membered 1,3-dihydrothiazine ring instead of the five-membered thiazole ring in 6-APA. The structures of 6-APA and 7-ACA are shown in Fig. 24.31. 

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