An Approach to Trapping γ-Glutamyl Radical Intermediates Proposed for Vitamin K Dependent Carboxylase: α,β-Methyleneglutamic Acid

Article abstract of DOI:10.1021/jm00164a056

The vitamin K dependent carboxylase activates the glutamyl γ-CH of substrate peptides for carboxylation by producing a γ-glutamyl free radical, a γ-glutamyl carbanion, or through a concerted carboxylation.We propose to intercept the putative γ-glutamyl free radical by the intramolecular rearrangement of a substrate containing the α,β-cyclopropane analogue of glutamic acid.The rearrangement of cyclopropylcarbinyl radicals into 2-butenyl radicals is rapid, exothermic, and considered diagnostis of free-radical formation.1-Amino-2-(carboxymethyl)cyclopropane-1-carboxylate, the β-cyclopropane analogue of glutamic acid, was synthesized starting from diethyl α-ketoglutarate.The α-keto ester was first treated with benzonitrile in sulfuric acid, to yield diethyl α,α-dibenzamidoglutarate.The α,α-dibenzamido acid was cleaved to produce the α,β-dehydroamino 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.Acid hydrolysis of the N-benzoyl-α,β-methyleneglutamate diethyl ester resulted in the production of the unprotected amino acid, α,β-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-benzoyl-α,β-methyleneglutamate diethyl ester.Saponification of a N-protectedmethyleneglutamic acid dialkyl ester using limiting alkali was shown to selectively yield the α-alkyl ester γ-acid.The reaction was used to produce α,β-cyclopropane-containing analogues of the carboxylase substrates N-t-Boc-L-glutamic acid α-benzyl ester and N-benzoyl-L-glutamic acid α-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 α-alkyl substitution, which apparently abolishes substrate binding.