The mechanism of the enzymatic ring expansion of penicillin N to deacetoxycephalosporin C

• Main Author: Crouch, Nicholas
• Other Authors: Baldwin, Jack E.
• Published: University of Oxford 1988
• Subjects: 615.1; Penicillin : Ring formation (Chemistry) : Cephalosporins
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233476

The order of events in the Deacetoxycephalosporin C/Deacetylcephalosporrn C Synthetase (DAOC/DAC Synthetase) catalysed ring expansion of penicillin N to deacetoxycephalosporin C has been investigated by the use of labelled/unlabelled penicillin N mixed competitive kinetic isotope effect experiments, in which the labelled penicillin N substrates were either labelled in the pro <strong>R</strong>- and pro <strong>S</strong>-methyl groups or at C-3. In addition, to assisting in the determination of the position of the first irreversible event in this reaction, deuteration at C-3 gave rise to a bifurcation of the natural biosynthetic pathway which led to enhanced production of the shunt metabolite, (2<strong>R</strong>,3<strong>S</strong>,6<strong>R</strong>,7<strong>R</strong>)-l-aza-3- methyl-3-hydroxy-7-[(5<strong>R</strong>)-5-amino-5-carboxy-pentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylate. The biosynthetic precursor to the 3<strong>S</strong>-hydroxycepham shunt metabolite has been investigated and the origin of the 3<strong>S</strong>-hydroxyl oxygen atom has been determined by the use of labelling studies with ¹⁸O₂ and shown to be derived from molecular oxygen. ¹³C-labelling studies are described which indicate that the ring expansion process is stereospecific to within the limits of the detection system employed. These experiments confirm earlier investigations but, in addition to improving upon the assessment of the degree of stereospecificity, have shown that the 3<strong>S</strong>- hydroxycepham shunt metabolite is produced with the same stereospecificity as that observed for the usual biosynthetic products, DAOC and DAC. Chapter 5 describes an investigation of the anomalous C-2 deuterium exchange detected in DAOC produced by incubation of di-(²H₃-methyl)-penicillin N with DAOC/DAC synthetase. The preliminary results from this study indicate that initially exchange occurs stereospecifically with the pro <strong>R</strong> C-2 deuterium atom being replaced by a hydrogen atom. The origins of the unusual tripeptides <strong>L</strong>-α-aminoadipyl-<strong>L</strong>-serinyl-<strong>D</strong>-valine (<strong>L</strong>,<strong>L</strong>,<strong>D</strong>-ASV), α-aminoadipyl-serinyl-isodehydrovaline (ASdV) and α-aminoadipyl-cysteinyl- β-hydroxyvaline (AC-[β-OH]-V) isolated from Penicillium chrysogenum and Cephalosporium acremonium, have been examined by the use of variously ¹³C-labelled <strong>L</strong>,<strong>L</strong>,<strong>D</strong>-α-aminoadipyl-cysteinyl-valine (<strong>L</strong>,<strong>L</strong>,<strong>D</strong>-ACV) and <strong>D</strong>,<strong>L</strong>,<strong>D</strong>-α-aminoadipyl- cysteinyl-valine (<strong>D</strong>,<strong>L</strong>,<strong>D</strong>-ACV) tripeptide isotopomers. The initial results obtained from this investigation may be considered as circumstantial evidence that ASdV is formed by the action of IPNS upon <strong>L</strong>,<strong>L</strong>,<strong>D</strong>-ACV. Finally, various substrate analogues have been prepared and evaluated as substrates for the ring expansion and hydroxylation activities of the bifunctional DAOC/DAC synthetase enzyme.