Tritium-Labeled Compounds VII. Isotope Effects in the Oxidation of d-Mannitols-C(14) and d-Mannitols-t to d-Fructoses

d-Mannitols, labeled either with carbon-14 at C1, C2, or C3, or with tritium attached to C1, C2, or C3, were prepared. After oxidation by Acetobacter suboxydans, the distribution of radioactivity in each of the resulting labeled d-fructoses was determined. Labeled d-mannitol is unique among the hexitols in that it may be oxidized by A. suboxydans in either the labeled or the unlabeled part of the molecule. Except in the oxidation of d-mannitol-2-t, the competing reactions result in the formation of a mixture of d-fructoses, each having radioactivity in one of two different positions. Hence, the isotope effect, k*/k, (where k* and k are, respectively, the rate constants for oxidation in the labeled and in the unlabeled part of the labeled d-mannitol molecule) is the ratio of the activities at the two positions of the product, d-fructose. The following isotope effects were found for the bacterial oxidation of labeled d-mannitols: (1) for d-mannitol-2-C(14), k*/k=0.93; (2) for d-mannitol-2-t, k*/k=0.23; and (3) for d-mannitol-3-t, k*/k=0.70. For d-mannitols labeled at other positions, no isotope effect was detected, since k*/k was unity. The large isotope-effect for d-mannitol-2-t is indicative of rupture of the C2–H bond in the rate-determining process. It is suggested that the secondary isotope-effect for tritium at C3 indicates hyperconjugation of the C3 hydrogen atom in the activated enzyme—substrate complex; the lack of such effect for tritium at C1 may be due to unfavorable steric conditions for hyperconjugation of the C1 hydrogen atoms in the complex. The following substances were prepared and their isotopic distributions determined: d-fructose-1,6-C(14) and d-fructose-1,6-t (from 1-labeled d-mannitols); d-fructose-2,5-C(14) and d-fructose-5-t (from 2-labeled d-mannitols); and d-fructose-3,4-C(14) and d-fructose-3,4-t (from 3-labeled d-mannitols). A procedure, employing d-fructose-1,6-C(14) as an internal standard, was devised for the analysis of d-fructose-3,4-t.