Stereospecific exchange of a β-methylene proton in phenylalanine preceding biosynthetic incorporation into gliotoxin

Abstract

Feeding experiments with the (3′R)- and (3′S)-forms of [3′-²H]- and [3′-³H]-phenylalanine show that biosynthesis of gliotoxin in Trichoderma viride takes place with loss of the pro-R- and retention of the pro-S-proton from the methylene group of the amino-acid precursor. Gliotoxin derived from [3′,3′-²H₂]phenylalanine is mainly monodeuteriated but contains some dideuterio-species; hydrogen (deuterium) loss is not therefore obligatory in gliotoxin biosynthesis. N.m.r. examination of the monodeuteriogliotoxin indicates an S-configuration for the new, deuterium-bearing chiral centre corresponding to incorporation of the original 3′-pro-S-deuterium with retention of configuration. Incorporation of [3′-³H]phenylalanine into mycelial protein also involves extensive loss of 3′-pro-R-tritium and retention of 3′-pro-S-tritium. Hydrolysis of the labelled protein gives [3′-³H]phenylalanine which is shown, by degradation with phenylalanine ammonia-lyase, to have predominantly the 3′S-configuration. Protein-phenylalanine and gliotoxin derived from [2′-³H]-phenylalanine contain minimal amounts of tritium. We conclude that, in T. viride, phenylalanine undergoes an unusual exchange reaction involving replacement of the pro-R-methylene proton by an external proton with overall retention of configuration. Exchange proceeds substantially faster than incorporation of the amino-acid into protein or gliotoxin but is not an obligatory preliminary step for either process.