Penicillins ring expansion

Most of the new commercial antibiotics have resulted from semisynthetic studies. New cephalosporkis, a number of which are synthesized by acylation of fermentation-derived 7-amkiocephalosporanic acid, are an example. Two orally active cephalosporkis called cefroxadine and cephalexin are produced by a synthetic ring-expansion of penicillin V. 

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). 

It is possible to convert penicillin V or benzylpenieillin to a cephalosporin by chemical ring expansion. The first-generation cephalosporin cephalexin, for example, can be made in this way. Most cephalosporins used in clinical practice, however, are semisynthetics produced from the fermentation product cephalosporin C. 

Rasor and Tischer (1998) have brought out the advantages of enzyme immobilization. Examples of penicillin-G to 6-APA, hydrolysis of cephalospwrin C into 7-ACA, hydrolysis of isosorbide diacetate and hydrolysis of 5-(4-hydroxy phenyl) hydantom are cited. De Vroom (1998) has reported covalent attachment of penicillin acylase (EC 3.51.11) from E.Coli in a gelatine-based carrier to give a water insoluble catalyst assemblase which can be recycled many times, and is suitable for the production of semi-synthetic antibiotics in an aqueous environment. The enzyme can be applied both in a hydrolytic fashion and a synthetic fashion. 6-APA was produced from penicillin-G similarly, 7-ADCA was produced from desa acetoxycephalosporin G, a ring expansion product of penicillin G. 

There has also been extensive activity towards the replacement of the entire chemical route to 7-ADCA (Scheme 1.14) with a biocatalytic one. This is somewhat more complex than the above example, as the penicillin fermentation product requires ring expansion as well as side-chain hydrolysis in order to arrive at the desired nucleus. The penicillin nucleus can be converted to the cephalosporin nucleus using expandase enzymes, a process that occurs naturally during the biosynthesis of cephalosporin C by Acremonium chryso-genum and cephamycin C by Streptomyces clavuligems from isopenicUhn N (6-APA containing a 6-L-a-aminoadipoyl side chain). ... 

The common motif shared by non-heme iron oxygenases contains an active site, where two histidines and one carboxylate occupy one face of the Fe(ll) coordination sphere. These enzymes catalyze a variety of oxidative modification of natural products. For example, in the biosynthesis of clavulanic acid, clavaminic acid synthase demonstrates remarkable versatility by catalyzing hydroxylation, oxidative ring formation and desaturation in the presence of a-ketoglutarate (eq. 1 in Scheme 7.22) [80]. The same theme was seen in the biosynthesis of isopenicillin, the key precursor to penicillin G and cephalosporin, from a linear tripeptide proceeded from a NRPS, where non-heme iron oxygenases catalyze radical cyclization and ring expansion (eq. 2 in Scheme 7.22) [81, 82]. 

An interesting reaction related to the ring expansions of penicillin S-oxides (208) was observed in the thermal rearrangement of 2,2,4A-tetramethyl-thiethan-3-one 1-oxide to the five-membered thiolane ring 209. Oxidation attempts of these thermally unstable thietanone oxides led via ring opening to the more stable heterocycle. The reaction mechanism (Scheme 11) was... 

Cephalexin 7- (D-a-Aminophenylacetamido) cephalosporanic acid Aromatic alkylation, amination, imine formation, amidation (sidechain), fermentation, deamidation (penicillin nucleus), acid-catalyzed ring expansion... 

Deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), the enzyme isolated from Cephalosporium acremonium catalysing587 the ring expansion of penicillin N, 473, to deacetoxycephalosporin (DAOC), 474, and the hydroxylation of 474 to deacetylcephalosporin C(DAC), 475, which in vivo is acetylated by a different enzyme to give cephalosporin C, 476 (equation 284), converts also the unnatural substrate exomethylene cephalosporin C, 477a, directly to DAC, 475 (equation 285). 

Ring Expansion of Both Rings of Penicillin Sulfoxides... 

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-... 

The manufacture of several semisynthetic oral cephalosporin antibiotics involves the chemical ring expansion of penicillin V to 7-aminodeacetoxycephalosporanic acid (7-ADCA Fig. 2 [68]). This is a costly and potentially environmentally damaging process [69], Cloning of the C. acremonium cefEF gene (DAOCS/ DACS [47]) and the S. clavuligerus cefD (IPNE) and cefE gene (DAOCS [43]) opened the possibility for biosynthetic/enzymatic processes for production of 7-ADCA. 

Early in vitro data indicated that ring expansion of adipyl-6-APA or penicillin G was either nonexistent or barely detectable [45,70], Therefore, an alternative route to 7-ADCA through the enzymatic deacylation of DAOC (Fig. 3) requiring the fermentative production of DAOC at economically feasible levels was pursued. Elimination of DACS activity would allow C. acremonium to produce DAOC as its end product (Fig. 1). Before it was known that DAOCS and DACS activities in C. acremonium were catalyzed by a single bifunctional enzyme,... 

Figure 4 Metabolic engineering for ring expansion of penicillin G in P. chrysogenum expressing a modified S. clavuligerus DAOCS.

WK Yeh, SK Ghag, SW Queener. Enzymes for epimerization of isopenicillin N, ring expansion of penicillin N, and 3 -hydroxylation of deacetoxycephalosporin C function, evolution, refolding, and enzyme engineering. Ann NY Acad Sci 672 396-408, 1992. 

When isopenicillin N, penicillin G, penicillin V, ampicillin, carboxy-w-bu-tyl penicillin, and 6-amino-penicillanic acid (6APA) were tested as substrate analogs [3,5,12], no ring expansion was observed. 

Table 1 Effect of Cofactors on the Ring Expansion of Penicillin G Using Resting Cells...

Figure 2 Effect of a-ketoglutarate concentration on ring expansion of penicillin G by resting cells of Streptomyces clavuligerus. The concentration of a-ketoglutarate previously used for unsuccessful cell-free ring expansion of penicillin G was 0.64 mM [9], The reaction mixture contained 50 mM Tris-HCl pH 7.4 buffer, 1.8 mM FeS04, 4 mM ascorbic acid, and 2 mg/ml penicillin G. Dry cell weight was 12 mg/ml. Samples were taken at 2 hr and centrifuged (14,000 X g, 5 min) 200 pi were used in the bioassay. (From Ref. 14.)...

The above results [63] demonstrated the capacity of resting cells entrapped in polyethyleneimine-barium alginate to perform repeated oxidative ring expansion of penicillin G to DAOG. Product formation rate was higher with free than... 

Success with resting cells acting on penicillin G allowed Cho et al. [14] to demonstrate the ring expansion of penicillin G. By increasing the concentration of the substrate (penicillin G), cosubstrate (a-ketoglutarate), the chief cofactor, (Fe2+), and cells, the conversion of penicillin G to DAOG was improved. Using such improved conditions, activity with cell-free extracts was also demonstrated on 14 other penicillins. 

Immobilized cells of S. clavuligerus NP1, entrapped on a polymeric matrix, were able to perform oxidative ring expansion of penicillin G into DAOG. Cells entrapped in polyethyleneimine barium alginate (1.5%) were able to sustain activity for at least four 2-hr cycles, whereas free resting cells were inactive after the second cycle. 

JE Baldwin, RM Adlington, JB Coates, MJC Crabbe, JW Keeping, GC Knight, T Nomoto, CJ Schofield, H-H Ting. Enzymatic ring expansion of penicillins to cephalosporins side chain specificity. J Chem Soc Chem Commun 374-375,1987. 

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