John J. Usher

Penicillin biosynthesis: active site mapping with aminoadipoylcysteinylvaline variants

A series of structural variants on the aminoadipoly moiety of the natural precursor of penicillins, δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine, have been synthesised and their effectiveness as substrates for the enzyme isopenicillin N synthetase has been determined.

Synthesis of δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine and some carbon-13 and nitrogen-15 labelled isotopomers

δ-(L-α-Aminoadipoyl)-L-cysteinyl-D-valine (7a), δ-(L-α-aminoadipoyl)-L-[3-13C]cysteinyl-D-valine (7b), δ-(L-α-aminoadipoyl)-L-[3-13C]cysteinyl-D-[3-13C]valine (7c), and δ-(L-α-aminoadipoyl)-L-[3-13C]cysteinyl-D-[3-13C, 15N]valine (7d) were prepared by conventional means using benzyl-based protective groups.

Penicillin biosynthesis. On the stereochemistry of carbon–sulphur bond formation with modified substrates

Enzymatic conversion of the two modified substrates (S-α-amino-δ-adipyl)-S-cysteinyl-(2R,3R)-3-2H-α-aminobutyrate (2c) and (S-α-amino-δ-adipyl)-S-cysteinyl-(2R,3S)-3-2H-α-aminobutyrate (2d) by the enzyme isopenicillin N synthetase gave from both precursors the same penam product, namely (2S)-2-deuterio-norisopenicillin N, indicating that this enzyme is capable of forming carbon–sulphur bonds by retention and also inversion pathways respectively.

The synthesis and reactions of a monocyclic β-lactam tripeptide, 1-[(1R)-carboxy-2-methylpropyl]- (3R -[(5S-5-amino- 5-carboxypentanamido]-(4R)-mercaptoazetidin-2-one, a putative intermediate in penicillin biosynthesis

Abstract The disulphide corresponding to the above thiol has been synthesised, but all attempts to reduce this substance to the thiol were unsuccessful, although an alternative procedure via a thiomercury intermediate, enabled the thiol to be generated in situ , the properties of this thiol, however, are not in accord with those previously described for a putative free intermediate in penicillin biosynthesis. 1

Direct 1H n.m.r. observation of the cell-free conversion of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine and δ-(L-α-aminoadipyl)-L-cysteinyl-D-(–)-isoleucine into penicillins

The cell-free conversion of δ-(L-aminoadipyl)-L-cysteinyl-D-(–)-isoleucine (lb) into a penicillin (2b) was observed directly by 1H n.m.r. spectroscopy and the thiazolidine ring formation was shown to occur with retention of stereochemistry at C-3 of the isoleucinyl residue by 1H nuclear Overhauser enhancement studies of the product penicillin in the deproteinated incubation mixture.

Direct n.m.r. observation of cell-free conversion of (L-α-amino-δ-adipyl)-L-cysteinyl-D-valine into isopenicillin N

Carbon-13 n.m.r. spectroscopy has been used to observe the efficient conversion of (L-α-amino-δ-adipyl)-L-cysteinyl-D-valine into isopenicillin N in cell-free extracts of Cephalosporium acremonium.

Penicillin biosynthesis: conversion of deuteriated (L-α-amino-δ-adipyl)-L-cysteinyl-D-valine into isopenicillin N by a cell-free extract of Cephalosporium acremonium

The tripeptide (L-α-amino-δ-adipyl)-L-cysteinyl-D-[22H, Me2–2H6]valine was converted into isopenicillin N by a cell-free extract ofCephalosporium acremonium;; complete retention of all deutrons was confirmed by 2H n.m.r. spectroscopy

On the possible role of the 3-methylene isomer of deacetoxycephalosporin C in the biosynthesis of cephalosporins

The 3-methylene isomer of [7α-3H]deacetoxy-cephalosporin C was incubated with a cell-free extract capable of converting penicillin N into deacetoxycephalo-sporin C; although there was no formation of deacetoxy-cephalosporin C from the 3-methylene isomer this compound was a powerful inhibitor of the penicillin N into cephalosporin bioconversion.

Conversion of 3-exomethylene cephalosporin C into deacetyl cephalosporin C in a cell-free extract from Cephalosporium acremonium(CW-19)

In a cell-free extract of the producing strain C. acremonium(CW-19)3-exomethylene cephalosporin C was transformed into a strongly antibiotic substance, which was identified as deacetyl cephalosporin C.