Synthesis of L-glutamic acid stereospecifically labelled at C-4 with tritium: stereochemistry of tritium release catalyzed by the vitamin K-dependent carboxylase in the absence of carboxylation

Abstract

[4-³H₁]-L-Glutamic acids have been synthesized by reduction of (2S,4S)- and (2S,4R)-N-benzyloxy-carbonyl-4-halogenoglutamic acid dimethyl esters with [³H₄]sodium borohydride in dimethylformamide. The degree of stereospecific labelling differed according to the diastereoisomeric configuration and the nature of the halogen substituent. Only the (2S,4R)-[4-³H₁]-acid could be satisfactorily obtained by this method. An enzymatic method, using an isocitrate lyase–isocitrate dehydrogenase–glutamate dehydrogenase system, was successfully used to obtain (2S)-[2-³H₁]succinate and to convert it into (2S,4S)-[4-³H₁]glutamic acid. The introduction of the labelled glutamic acids into a peptide previously shown to be a substrate of the rat liver vitamin K-dependent carboxylase allowed us to demonstrate that the hydrogen exchange catalyzed by this preparation in the absence of CO₂ proceeds by a stereospecific abstraction of the same 4-pro-S-hydrogen atom of the glutamyl residue which is eliminated in the carboxylation reaction. A 4-methyl (threo) L-glutamic acid-containing peptide exerts the same competitive inhibition on the exchange and on the carboxylation reaction. These results ensure that the previously demonstrated hydrogen exchange is part of the carboxylation reaction.