Studies on the biosynthesis of clavulanic acid. Part 5. Absolute stereochemistry of proclavaminic acid, the monocyclic biosynthetic precursor of clavulanic acid

Abstract

Proclavaminic acid (1) was synthesized by a route which indicated its constitution to be (2S,3R)-5-amino-3-hydroxy-2-(2-oxoazetidin-1-yl)valeric acid. The spectroscopic properties of the synthetic material were identical with those of natural proclavaminic acid, and, like the natural product, it was converted into clavaminic acid (2) by clavaminic acid synthase. An efficient synthesis of 3-hydroxyornithine derivatives was devised which allowed the separation of diastereoisomers and the resolution of a threo compound by the acylase [EC 3.5.1.11] from Escherichia coli. The β-lactam ring was subsequently elaborated by Michael addition of a protected 3-hydroxyornithine to acrylic acid followed by ring closure using triphenylphosphine/di-2-pyridyl disulphide. Model reactions were carried out with enantiomerically pure threonine derivatives to confirm that the formation of the β-lactam moiety did not impair the integrity of the α- and β-chiral centres and that the enzymatic deacylation reaction was capable of resolving the α-centre of an α-amino-β-hydroxy acid. The enantiomeric purity of intermediates was determined using HPLC, ¹H NMR spectroscopy utilising the chiral solvating reagents (R)- and (S)-1-(9-anthryl)-2,2,2-trifluoroethanol, and chiral GLC techniques.