Synthesis of Mono- and Di-Deuterated (2S, 3S)-3-Methylaspartic Acids to Facilitate Measurement of Intrinsic Kinetic Isotope Effects in Enzymes.

Kinetic isotope effects provide a powerful method to investigate the mechanisms of enzyme-catalyzed reactions, but often other slow steps in the reaction such as substrate binding or product release suppress the isotopically sensitive step. For reactions at methyl groups, this limitation may be overcome by measuring the isotope effect by an intra-molecular competition experiment. This requires the synthesis of substrates containing regio-specifically mono- or dideuterated methyl groups. To facilitate mechanistic investigations of the adenosylcobalamin-dependent enzyme, glutamate mutase we have developed a synthesis of mono- and di-deuterated (2S, 3S)-3-methylaspartic acids. Key intermediates are the correspondingly labeled mesaconic acids and their dimethyl esters that potentially provide starting materials for a variety of isotopically labeled molecules.