SYNTHESIS AND REACTION OF POTENTIAL ALTERNATE SUBSTRATES AND MECHANISM-BASED INHIBITORS OF CLAVAMINATE SYNTHASE

Clavaminate synthase is an Fe(II)/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulani c acid. In the presence of dioxygen it catalyzes the oxidative cycliza tion/desaturation of proclavaminic acid to clavaminic acid in a two-st ep process. Samples of (4'R)- and (4'S)-D,L-(4'-H-2]proclavaminic acid have been prepared and used to demonstrate that oxazolidine ring form ation occurs with retention of configuration. The stereochemical cours e of oxygen insertion from substrate that takes place in this oxidativ e cyclization is the same as that observed from molecular oxygen in se veral hydroxylation reactions catalyzed by other Fe(II)/alpha-ketoglut arate-dependent enzymes. The ferryl (Fe(IV)=O) species thought to be t ransiently 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 cy clopropyl at C-4. In the synthesis of the former two derivatives and p roclavaminic acid stereoselectively labeled with deuterium at C-4', in troduction of the unsaturated substituents in a stereochemically defin ed manner at C-4' relied upon ready access to (4R)-4-thiophenyl-2-azet idinone. Trimethylsilyl substitution could be easily achieved at C-3 o f the optically pure starting material to give the readily separable c is and trans diastereomers. In radical chain reactions in which the th iophenyl was replaced by deuterium or in anionic reactions in which th e thiophenyl was eliminated as its sulfone and replaced by addition of carbanions, the steric bulk of the trimethylsilyl group at C-3 govern ed the approach of incoming reagents to give the trans product. The en zymatic fate, however, of these derivatives was disappointing, yieldin g neither detectable reaction nor hoped-for inactivation of clavaminat e synthase. Finally, as mixed competitive/noncompetitive inhibitors of catalysis, they gave unexceptional inhibition constants in the range 2-10 mM.