Ring expansion, of penicillin

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. 

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... 

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). 

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. 

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. 

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. 

M Yoshida, T Konomi, M Kohsaka, JE Baldwin, S Herchen, P Singh, NA Hunt, AL Demain. Cell-free ring expansion of penicillin N to deacetoxycephalosporin C by Cephalosporium acremonium C-19 and its mutants. Proc Natl Acad Sci (USA) 75 6253-6257, 1978. 

Y Sawada, NA Hunt, AL Demain. Further studies on microbiological ring-expansion of penicillin N. J Antibiot 32 1303-1310, 1979. 

Some examples of ring expansion of penicillins to homopenicillins are depicted in Scheme 18 they can be either /3,7-unsaturated 32 <2004CC2332>, cr,/3-unsaturated 33 <1983TL3419, 20070BC160>, or saturated 34 <1988CC110> bicyclic compounds. 

Scheme 18 Ring expansion of penicillins to homopenicillins (three examples).

The A -double bond makes a considerable contribution to the bioactivity of cephems. Further, cephams having no double-bond exhibit no activity. These compounds can be obtained by catalytic hydrogenation [74], or as intermediates during the ring expansion of penicillin sulphox-ides. The ratio of (51) and (20a) and the nature of the group depend on the conditions [12-14, 75-77]. 

Ring expansion of the penicillins to the cephalosporins has been examined. Following the elucidation of the structure of penicillin N and cephalosporin C, small amounts of isopenidllin N with an L-aminoadipoyl side-chain and de-acetoxycephalosporin C were found in Cephalosporium acremonium. Cell-free systems were obtained from the fungus which catalysed the ring expansion of penicillin N to deacetoxycephalosporin C (3.19) and the hydroxylation of this. A radical mechanism has been proposed in which the 3(pro-R)-methyl group of valine (marked in 3.17) becomes the ring methylene (marked in 3.19) of cephalosporin C. 

Figure 12.2-8. Production of 7-ADCA has undergone remarkable changes. In the early days (left-hand side), chemical ring expansion of penicillin C resulted in the formation of the cephalosporin nucleus. The phenylacetyl moiety was then removed chemically. Later on, this last step was replaced by a biocatalytic step using penicillin amidase (middle). On the right hand side, a completely new route is presented. Dicarboxyl-7-ADCA is obtained directly by fermentation. A dicarboxyl amidase is used to remove the dicarboxyl group.

Ring expansion of penicillin N into deacetoxy cephalosporin C... 

Ring expansion of penicillin G into phenylacetyl-7-aminodeacetoxy-cephalosporanic acid ... 

A recent enantioselective synthesis of homopenicillin took advantage of a new method for the ring expansion of penicillin [79]. Irradiation of the P-ketosulphoxonium ylide 124 resulted in the smooth formation of y-lactam 127. The reaction probably involved the formation of an acylcarbene 125 which undergoes Wolff rearrangement to the ketene intermediate 126. Intramolecular nucleophilic attack formed the new five-membered ring (Scheme 43). Homo-... 

M., Baldwin, J.E. et al. (1978) Cell-free ring expansion of penicillin... 

The ring expansion of penicillin N to desacetoxycephalosporin is interesting as well. In spite of intensive efforts, this biosynthesis is stiU not completely clear. On the basis of substrate studies and kinetic investigations, it is assumed, that the chemistry of isopenicUlin N-synthase and expandase are alike and that the ring expansion also proceeds via a ferryl-oxo-intermediate, [41] and that a radical mechanism is involved as well. [42]... 

Figure 4.37 Proposed mechanisms of DAOCS/DACS catalysis of the ring expansion of penicillin N to DAOC.

Similarly, deacetoxy-Zdeacetylcephalosporin synthase C (DOAC/DAC synthase) catalyzes enzymatic ring expansion of penicillin N to deacetoxy- and deacetyl-cephalosporin C as shown in eq. (21) in the presence of Fe, a-ketoglutaric acid. The enzyme has been purified from stereptomyces clavuligerus, Mr 36 kDa [346, 347] and recombinant streptomyces clavulgerus [348]. A mechanism involving iron(IV) oxene formed from ferrous iron has been proposed [349,350]. 

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