Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate

Abstract

The synthesis of 2-acetylthiamin pyrophosphate (acetyl-TPP) is described. The synthesis of this compound is accomplished at 23 degrees C by the oxidation of 2-(1-hydroxyethyl)thiamin pyrophosphate using aqueous chromic acid as the oxidizing agent under conditions where Cr(III) coordination to the pyrophosphoryl moiety and hydrolysis of both the pyrophosphate and acetyl moieties were prevented. Although the chemical properties exhibited by acetyl-TPP are similar to those of the 2-acetyl-3,4-dimethylthiazolium ion examined by Lienhard [Lienhard, G.E. (1966) J. Am. Chem. Soc. 88, 5642-5649], significant differences exist because of the pyrimidine ring in acetyl-TPP. Characterization of acetyl-TPP by ultraviolet spectroscopy, 1H NMR, 13C NMR, and 31P NMR provided evidence that the compound in aqueous solution exists as an equilibrium mixture of keto, hydrate, and intramolecular carbinolamine forms. The equilibria for the hydration and carbinolamine formation reactions at pD 1.3 as determined by 1H NMR are strongly dependent on the temperature, showing an increase in the hydrate and carbinolamine forms at the expense of the keto form with decreasing temperature. The concentration of keto form also decreases with increasing pH. Acetyl-TPP is stable in aqueous acid but rapidly deacetylates at higher pH to form acetate and thiamin pyrophosphate. Trapping of the acetyl moiety in aqueous solution occurs efficiently with 1.0 M hydroxylamine at pH 5.5-6.5 to form acetohydroxamic acid and to a much smaller extent with 1.0 M 2-mercaptoethanol at pH 4.0 and 5.0 to form thio ester. Transfer of the acetyl group to 0.5 M dihydrolipoic acid at pH 5.0 and 1.0 M phosphate dianion at pH 7.0 is not observed to any significant extent in water. The kinetic and thermodynamic reactivity of acetyl-TPP with water and other nucleophiles is compatible with a hypothetical role for acyl-TPPs as enzymatic acyl-transfer intermediates.

Observation of an Acryloyl−Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

“…17 Its lowest energy absorption band in acetonitrile (λ max = 316 nm, ε M = 7,150 M −1 cm −1 ) was fully within expectations. Addition of trifluoroacetic acid (TFA) allowed measurement of the transient protonated species, blue-shifted in accord with the literature (λ max = 312 nm), 12,15 before rapid conversion to the TFA addition product 12.…”

“…A careful study by Gruys et al of a related system, acetyl-ThDP (18, Scheme 2) showed that, while imine formation could not be detected, cyclization to the carbinolamine (19, Scheme 2) was clearly evident. 12 To examine the UV-spectroscopic behavior of such a possible conjugated π-system, a second model, N-(1-(4-methylthiazol-2-yl)allylidene)benzenamine (17, Scheme 2) was constructed. Once again 4-methylthiazole (9) Charge-transfer (CT) phenomena are often invoked to explain bathochromic shifts seen in protein chromophores, and, indeed, they have been proposed for other ThDP-dependent enzymes.…”

“…4-Methylthiazole (9) was lithiated at −78 °C with n-BuLi and reacted with acrolein. 12 The sensitive allylic alcohol 10 could be oxidized to 11 in 87% yield with the Dess-Martin periodinane. 17 Its lowest energy absorption band in acetonitrile (λ max = 316 nm, ε M = 7,150 M −1 cm −1 ) was fully within expectations.…”

Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum. Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

“…The net rate constant of the phosphorolytic cleavage of AcThDP (k′ 6 3 , and k′ 4 ). Because all of these rate constants have been already determined, the calculation of k′ 6 is straightforward.…”

“…It should be noted that AcThDP is in equilibrium with its internal hydrate and a carbinolamine form under the chosen conditions (3) and that all three species occur in comparable fractions. It is therefore challenging to observe AcThDP in only small amounts being present at steady state because the analytical signal splits off further.…”