Analytical and preparative separation of the diastereomers of l-buthionine ( SR)-sulfoximine, a potent inhibitor of glutathione biosynthesis

Abstract

Buthionine sulfoximine inhibits γ-glutamylcysteine synthetase, the enzyme catalyzing the first reaction of glutathione (GSH) biosynthesis. GSH synthesis is blocked in animals or cultured cells exposed to buthionine sulfoximine, and GSH is substantially depleted in cells or tissues with moderate to high rates of GSH utilization. Studies reported to date have used dl-buthionine ( SR)-sulfoximine or l-buthionine ( SR)-sulfoximine, mixtures of four and two isomers, respectively. The present report describes a chiral solvent HPLC procedure for the analytical separation of the diastereomers of l-buthionine ( SR)-sulfoximine and the separation of those isomers from the unresolved diastereomers of l-buthionine ( SR)-sulfoximine. l-Buthionine ( R)-sulfoximine was isolated preparatively by repeated crystallization of l-buthionine ( SR)-sulfoximine from water; l-buthionine ( S)-sulfoximine was obtained by crystallization as the trifluoroacetate salt in ethanol/hexane mixtures. The absolute configuration, bond lengths and angles of l-buthionine ( R)-sulfoximine were determined by X-ray diffraction. In vitro studies demonstrate that l-buthionine ( R)-sulfoximine is a relatively weak inhibitor of rat kidney γ-glytamylcysteine synthetase; binding is competitive with l-glutamate. l-Buthionine ( S)-sulfoximine is a tight-binding, mechanism-based inhibitor of the enzyme. Since l-buthionine sulfoximine is initially bound as a transition-state analogue, identification of the inhibitory diastereomer elucidates the steric relationships among ATP, glutamate, and cysteine within the active site. When administered to mice, l-buthionine ( S)-sulfoximine (0.2 mmol/kg) was as effective as l-buthionine ( SR)-sulfoximine (0.4 mmol/kg) in causing GSH depletion in liver, kidney, and pancreas. l-Buthionine ( R)-sulfoximine (0.2 mmol/kg) did not cause significant GSH depletion in liver or pancreas. The l-( R)-diastereomer caused a modest GSH depletion in kidney that is tentatively attributed to interference with γ-glutamylcyst(e)ine transport.