Abstract
Abstract The bell-shaped pH rate profile for the reaction of tetrahydrofolic acid with formaldehyde to form 5,10-methylene tetrahydrofolic acid has been shown to result from a change in rate-determining step with changing acidity. In alkaline solution, the rate-determining step is the acid-catalyzed dehydration of hydroxymethyl tetrahydrofolic acid and its conjugate base. This step is subject to general acid catalysis with a Bronsted slope, α, of approximately 0.75. In acid solution, attack of tetrahydrofolic acid on formaldehyde is rate-determining. This step is subject to catalysis by general acids, including the solvated proton, with a Bronsted slope of 0.20. At pH values above 4, the reaction is inhibited by thiols, which combine with formaldehyde in a base-catalyzed reaction to form hemithioacetals. Depending on the reaction conditions, this inhibition can result from a lowering of the equilibrium concentration of formaldehyde or from a trapping by thiol anion of unhydrated formaldehyde, as it is formed from formaldehyde hydrate. The equilibrium constant for hemithioacetal formation from formaldehyde and mercaptoethanol is approximately 620 m-1. Secondary amines such as morpholine and imidazole catalyze the reaction of tetrahydrofolic acid with formaldehyde by nucleophilic catalysis by a pathway which must involve the intermediate formation of cationic imines, [see PDF for structure]
Highlights
5.60 and 7.00, which indicates that the reaction is first order in respect to formaldehyde
If the reaction is carried out in the absence of mercaptoethanol and the THF is protected from oxidation by argon and ethylenediaminetetraacetic acid, the observed rate constants above pH 4 are considerably larger and the maximum and inflection points of the pH rate profile occur at higher pH values
Conditions, the THF competes with thiol for the limited amount of unhydrated formaldehyde and, since the reactive thiol is present in much larger concentrations than that of THF, the THF reaction is inhibited
Summary
The concentration of mercaptoethanol in the THF sample from Nutritional Biochemicals, which was used in some of the early experiments, was not sufficient to interfere with the kinetic results after dilution I:5000 in the reaction mixtures. There was no significant difference in the kinetic results obtained with the different THF preparations. Reagent grade formaldehyde was obtained from Fisher in 36.6 to 37.2 s solutions which contained 10 to 11 y0 methanol. This solution was diluted to a 1.0 M stock solution and was further diluted to 0.025 M every 1 to 2 days. Reagent grade formic acid, substitut,ed acetic acids, 2-mercaptoethanol, ethanethiol, mercaptoacetic acid, and inorganic salts were used without further purificat,ion
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