An approach to trapping .gamma.-glutamyl radical intermediates proposed for vitamin K dependent carboxylase: .alpha.,.beta.-methyleneglutamic acid

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Abstract
The vitamin K dependent carboxylase activities the glutamyl .gamma.-CH of substrate peptides for carboxylation by producing a .gamma.-glutamyl free radical, a .gamma.-glutamyl carbanion, or through a concerted carboxylation. We propose to intercept the putative .gamma.-glutamyl free radical by the intramolecular rearrangement of a substrate containing the .alpha.,.beta.-cyclopropane analogue of glutamic acid. The rearrangement of cyclopropylcarbinyl radicals into 2-butenyl radicals is rapid, exothermic, and considered diagnostic of free-radical formation. 1-Amino-2-(carboxymethyl)cyclopropane-1-carboxylate, the .beta.-cycloproprane analogue of glutamic acid, was synthesized starting from diethyl .alpha.-ketoglutarate. The .alpha.-keto ester was first treated with benzonitrile in sulfuric acid, to yield diethyl .alpha.,.alpha.-dibenzamidoglutarate. The .alpha.,.alpha.-dibenzamido acid was cleaved to produce the .alpha.,.beta.-dehydroamino acid acid and benzamide on treatment with p-toluenesulfonic acid in hot benzene. Diazomethane addition to the dehydroamino acid resulted in cycloaddition of diazomethane and production of the pyrazoline, which upon irradiation lost n2 to give the protected cyclopropane-containing amino acid analogue. Acidic hydrolysis of the N-benzoyl-.alpha.,.beta.-methyleneglutamate diethyl ester resulted in the production of the unprotected amino acid, .alpha.,.beta.-methyleneglutamic acid, in high yield. A single dehydroamino acid and a single methyleneglutamic acid isomer were produced in this synthesis; both are identified as the Z isomer, the former by NMR using the nuclear Overhauser effect and the latter through X-ray crystallographic analysis of N-benzyl-.alpha.,.beta.-methyleneglutamate diethyl ester. Saponification of a N-protected methyleneglutamic acid dialkyl ester using limiting alkali was shown to selectively yield the .alpha.-alkyl ester .gamma.-acid. The reaction was used to produce .alpha.,.beta.-cycloproprane-containing analogues of the carboxylase substrates N-t-Boc-L-glutamic acid .alpha.-benzyl ester and N-benzoly-L-glutamic acid .alpha.-ethyl ester. The cyclpropane-containing analogues were tested and found to be neither substrates for nor inhibitors of the rat liver microsomal vitamin K dependent carboxylase. The inability of the enzyme to recognize these substrate analogues is attributed to the .alpha.-alkyl substitution, which apparently abolishes substrate binding.