Synthesis and configurational assay of asymmetric methyl groups.

Abstract

By chemical synthesis starting from phenylacetylene and utilizing stereospecific reactions, two specimens of acetic acid were prepared in which the following conditions were closely approached: (a) some of the methyl groups consisted of one hydrogen, one deuterium, and one tritium atom attached to carbon; (b) all such methyl groups in one specimen had the R configuration, and all such methyl groups in the other specimen had the S configuration, and the correct absolute configuration was assignable to each specimen from the method of synthesis used; (c) all methyl groups containing a tritium atom also contained a deuterium atom. Each specimen of acetic acid was converted enzymically into acetyl‐coenzyme A and (in the same incubation) this product was condensed with glyoxylic acid on malate synthase to form two specimens of S‐malic acid. Each specimen of malic acid was incubated with fumarase until the tritium content of the organic acids (fumarate + malate) had ceased to fall. Malic acid synthesized from R‐acetic acid retained 69% of its carbon‐bound tritium (in fumarate + malate) on incubation with fumarase. Malic acid synthesised from S‐acetic acid retained 31% of its carbon‐bound tritium. The enzymic assay described in paragraphs 2–4 constitutes a method for determining the absolute configuration of asymmetric methyl groups, provided that a substantial proportion of the tritium in the specimen examined is present in such groups. The method thus established can be used to study the mechanism of enzymic reactions in which a methylene group is converted stereospecifically into a methyl group. With one logical proviso–that it is necessary to known whether protium or deuterium is preferentially removed by the enzyme from methyl groups also containing tritium–the method is also applicable to enzymic reactions in which a methyl group is converted stereospecifically into a methylene group. Subject to the above proviso, it is indicated that on malate synthase the removal of hydrogen and the attachment of the glyoxylate residue occurs with inversion of configuration.