Studies on the Properties of (—)-2-α-Hydroxyethyl-Thiamine Pyrophosphate (“Active Acetaldehyde”)

Abstract

An improved enzymatic preparation of (—)-HETPP from TPP with pig heart pyruvate dehydrogenase is described, yielding an amorphous product of satisfactory elementary analysis and constant optical rotation. The optical absorption spectrum of HETPP was recorded at various pH values and briefly explained and compared with that of TPP. The proton magnetic resonance spectrum of HETPP in deuterium oxide, as compared with those of TPP, 2-α-hydroxyethyl-4methyl-5-β-hydroxyethyl-thiazole, and 2-hydroxymethyl-3,4-dimethyl-thiazolium iodide, afforded additional and more accurate proof for the structure of HETPP. The possibility of hydrogen exchange in the α-position of the 2-side chain of a model compound, 2-hydroxymethyl-3,4dimethyl-5-β-hydroxyethyl-thiazolium iodide, and of HETPP itself was checked under varying conditions. No proton exchange was found in the absence of enzyme at pH values below 8.6. HETPP, ³H-labelled in the 2-side chain, was prepared and treated with apo-pyruvate decarboxylase and Mg++. Unconverted HETPP from the reaction mixtures was found to have the specific radioactivity of the starting material. The experimental results are explained in terms of the 2-α-carbanionof HETPP being the real “TPP-activated acetaldehyde” rather than HETPP itself as was hitherto believed.