Synthesis and Reaction of Potential Alternate Substrates and Mechanism-Based Inhibitors of Clavaminate Synthase

Clavaminate synthase is an FeII/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In the presence of dioxygen it catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process. Samples of...

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Bibliographic Details
Published in:	Journal of natural products (Washington, D.C.) Vol. 56; no. 8; pp. 1373 - 1396
Main Authors:	Iwata-Reuyl, Dirk, Basak, Amit, Silverman, Lisa S, Engle, Christopher A, Townsend, Craig A
Format:	Journal Article
Language:	English
Published:	Washington, DC American Chemical Society 01-08-1993 Glendale, AZ American Society of Pharmacognosy
Subjects:	Analytical, structural and metabolic biochemistry Aza Compounds - chemistry Biological and medical sciences Clavulanic Acids - chemical synthesis Clavulanic Acids - chemistry Cyclization Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Hydroxylation Indicators and Reagents Mixed Function Oxygenases - antagonists & inhibitors Mixed Function Oxygenases - chemistry Molecular Conformation Oxidoreductases Prodrugs - chemistry Stereoisomerism Structure-Activity Relationship Substrate Specificity Trimethylsilyl Compounds - chemistry Enzyme Clavulanic acid Substrate specificity Enzyme inhibitor Biosynthesis β-Lactamase Mechanism of action
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Summary:	Clavaminate synthase is an FeII/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In the presence of dioxygen it catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process. Samples of (4'R)- and (4'S)-D,L-[4'-2H]proclavaminic acid have been prepared and used to demonstrate that oxazolidine ring formation occurs with retention of configuration. The stereochemical course of oxygen insertion from substrate that takes place in this oxidative cyclization is the same as that observed from molecular oxygen in several hydroxylation reactions catalyzed by other FeII/alpha-ketoglutarate-dependent enzymes. The ferryl (FeIV = O) species thought to be transiently involved in each of these processes was investigated in the present work with clavaminate synthase and three structural analogues of proclavaminic acid bearing vinyl or ethynyl groups at C-4' or a cyclopropyl at C-4. In the synthesis of the former two derivatives and proclavaminic acid stereoselectively labeled with deuterium at C-4', introduction of the unsaturated substituents in a stereochemically defined manner at C-4' relied upon ready access to (4R)-4-thiophenyl-2-azetidinone. Trimethylsilyl substitution could be easily achieved at C-3 of the optically pure starting material to give the readily separable cis and trans diastereomers. In radical chain reactions in which the thiophenyl was replaced by deuterium or in anionic reactions in which the thiophenyl was eliminated as its sulfone and replaced by addition of carbanions, the steric bulk of the trimethylsilyl group at C-3 governed the approach of incoming reagents to give the trans product. The enzymatic fate, however, of these derivatives was disappointing, yielding neither detectable reaction nor hoped-for inactivation of clavaminate synthase. Finally, as mixed competitive/noncompetitive inhibitors of catalysis, they gave unexceptional inhibition constants in the range 2-10 mM.
Bibliography:	ark:/67375/TPS-TBQWC6R0-S istex:2C20BDA77F85AE5011CB6691232CEEE6C7A531BC ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0163-3864 1520-6025
DOI:	10.1021/np50098a022