Kinetics and thermodynamics of the structural transformations of thiamine in basic aqueous media. Part 3. Interpretation of the lability of the 2-proton via an intramolecular σ-adduct

Abstract

The structural transformations of thiamine into its yellow form J^– in very basic media (pH > 11) have been investigated by pH stopped-flow jump and temperature-jump techniques. This transformation via the deprotonation of the thiazolium 2-position to give the resonance-stabilized carbene A_–H, hitherto considered as the biocatalyst of vitamin B1 metabolic activity, is questioned, since deprotonation of the thiamine 2-position is unlikely for pK < 15. Formation of the J^– species occurs through the σ-adduct D resulting from intramolecular NH₂ base-promoted nucleophilic addition at the thiazolium 2-position. This ring formation occurs with a second-order rate constant k₁₄of 99 l mol^–1 s^–1. σ-Adduct D generates J^– by a base-promoted reaction: k₄₅ 5.15 × 10⁶ l mol^–1 S^–1. The deuterium isotope effect k_45(H2O)/k_45(D2O) 2.05 and ΔpK₄ 0.75 for this D ⇄ J^– reaction imply the involvement of several proton transfers. It is surmised that this D ⇄ J^– transformation takes place through the deprotonation of the 2-position of D into a carbanion intermediate D^– which by prototropic ring-opening yields J^–. This hypothesis accounts for the half-life of the 2-proton, known from n.m.r. measurements in neutral aqueous media. This tentative mechanism answers a long standing question on the role played by the amino pyrimidine moiety in the biocatalytic activity of thiamine. The existence of such a carbanion D^– species as a candidate among the catalysts which monitor the metabolic activity of vitamin B1 is, therefore, proposed with caution.