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Now use Figure 6.10 to follow the rest of the discussion.
Figure 6.10 Biosynthetic pathways from isopenicillin N to penicillin G and cephalosporin C. Some strains have the ability to convert deacetylcephalosporin Ñ into cephamydn C.
ep im erase expan dase hydroxylase acyltransferase
Different strains of micro-organisms are responsible for the production of either penicillins or cephalosporins. In penicillin-producing strains, an acyltransferase enzyme system is present which can remove the side chain from isopenidllin N to give
6-aminopenicillanic acid (6-APA), and which can subsequently acylate 6-APA to generate various penidllins, the most important ones bring penidllin G and V(see section 6.3, Table 6.2).
Cephalosporin-producing strains are characterised by the presence of the enzyme epimerase, responsible for the conversion of the L-a-aminoadipyl side chain in isopenidllin N, into the D-a-aminoadipyl side chain in penidllin N. The next step is an oxidative ring expansion and involves the loss of two protons, it is catalysed by the enzyme expandase. Unaware of this phenomenon, chemists carried out the ring enlargement of the penicillin skeleton by non-enzymatic means, finding only much later that nature had been doing the same for a long time. Deacetoxycephalosporin Ñ thus obtained is hydroxylated to give deacetylcephalosporin C, using the enzyme hydroxylase (mono-oxygenase). Finally, deacetylcephalosporin Ñ is converted into cephalosporin Ñ with the aid of an acyltransferase.
From what you read in Section 6.3, does the enzyme IPN-acyltransferase exhibit a high degree of spedfidty? Given reasons for your answer.
6.6 Semi-synthetic penicillins
penidllin G In spite of its remarkable therapeutic usefulness and low toxidty, penicillin G appeared
resistance have had its limitations when resistant strains of bacteria emerged. Thus it became of
interest to consider produdng variants of this molecule with different activities.
Although a range of penidllins could be produced by directed biosynthesis using precursor feeding, this approach is limited by the toxidty of the precursors, the ability of the penidllin producing cells to take up the precursor and by the capability of the acyltransferase to transfer the acyl groups onto the 6-aminopenicillanic add moiety.
penidllin It was noted that many penicillin-resistant organisms produce enzymes that catalyse
acyiases the hydrolyses of the amide links in penicillin. These enzymes are penidllin acylases
P-lactamases [Hactamases depending upon the amide links they hydrolyse.
Production and diversification of antibiotics
Obviously, the production of penicillins that were not sensitive to this hydrolysis would be advantageous.
Ï Examine Figure 6.11 and see if you can suggest a strategy that might be adopted to produce modified penicillin.
The strategy we hope you identified is to first produce 6-aminopenidllanic add, then attempt to add different moieties to the 6-amino group. This can be achieved either chemically or enzymatically. In the following section we will consider the conversion of penicillin G into 6-aminopenicillanic acid and follow this by examining how
6-aminopenidllanic acid may be converted into ampidllin and amoxicillin.
6.6.1 Conversion of penicillin G into e-aminopenicillanic acid
penidlin In Figure 6.11 we indicated that penidllin acylases selectively hydrolysed the secondary
acyiases amide link, releasing 6-aminopenidllanic add (6-APA). Although these enzymes could be used to produce 6-APA from penidllin G, initially, the vulnerability and high costs of enzymatic deacylation were important reasons to search for alternative, chemical processes.
As can be seen in Figure 6.12, penicillin G contains two amide functionalities, of which the p-lactam linkage is extremely susceptible to basic and nudeophilic attack. Therefore, deavage of the phenylacetyl side chain could not be performed using classical base hydrolysis. The problem of selectivity was resolved by taking advantage of the fact that the amide bond to be hydrolysed is secondary rather than tertiary.
Figure 6.12 Penicillin G contains two amide linkages (circled in a). The amide linkage to the side chain is secondary and exists in two forms (shown in b).
Key factor in addressing this problem was the application of silyl chemistry in order to protect the penicillin C-3 carboxyl in situ, giving high yields at low cost. The process is illustrated in Figure 6.13. Examine this figure carefully so that you remember, at least in outline, how the chemical conversion takes place. Furthermore, the occurrence of various undesirable side-reactions, so easily met within penicillin chemistry, was successfully avoided by performing reactions with phosphorus pentachloride and alcohol at low temperatures. Thus an inexpensive one-stage process to 6-ÀÐÀ, now one of the world's largest selling P-lactam intermediates, was developed.