Tritium - Labeled Compounds VII . Oxidation of D - Fructoses 1 D - Mannitols - C 14 Isotope Effects in the and D - Mannitols - t to

n -Ma nni tols, la beled eit her wi t h carbo n-14 at Cl , C2, or C3, o r wi t h tri t ium a t tached to Cl , C2, or C3, were prepared . After oxidatio n by Acetobacter suboxydans, t he dist ribu tion of r adioactivity in each of t he r esul t ing labeled n-fructoses wa deter mined . La beled nmanni tol is uni que a mong t he hexito ls in t ha t i t may be ox idi zed by A . suboxydans in eit her t he labeled or t he unla beled par t of t he molecule. Excep t in t he oxidat ion of n-manni tol-2-t, t he co mpet ing reactio ns r esult in t he formation o f a mi xt ure of D-fr uctoses, ea ch hav ing radioact ivity in one of t wo d ifferent posit ions. H cnce, t he iso tope effect, k */k, (where k * a nd k are, r espectively, t he rate co ns tan ts for oxidation ill t he labeled and in t he unla beled part of t he la beled D-ma nnitol molecule) is t he rat io of t he act ivit ies at the t wo posit ions of t he produ ct, n-fructose. The followin g isoto pe effects were fou nd for t he bacterial oxida t ion of la beled D-mannito ls : (1 ) for n-ma n ni tol-2-C14, k * / k = 0.93 ; (2) for n-mannito l-2-t, Ie * /k = 0.23; and (3) for D-manni to l-3-t, k*/k = 0.70 . For n-m a nni to ls la beled at other posit io ns, no isotope effect was detected, s ince k*/k was uni t y . The large isotope-effect fo r n-ma nn itol-2-t is in dicat ive of rupt ure of t he C2H bo nd in t he ra t e-d etermin in g process. It is s ugges ted t hat t he secondary isoto pe-effect for t ri t iu m a t C3 indi cat es hyperconjugat ion of t he C3 hydrogen ato m in t he activa ted enzymesubstrate co mplex ; t he lack of such effect fo r t ri t iu m at Cl may be du e to unfavora ble s teric condi t ions fo r hypercon juga t io n of t he Cl h ydrogen a toms in the co mplex. The fo llowing su bst a nces wer e p repared a nd t heir isoto pic dist ribu t ions dete rmin ed : n-fr uctose-l ,6-C" a nd n-fructose-l ,6-t (from I-la beled n-m a nnito ls); n-fru ctose-2,5-C14 a nd n-fru ctose-5-t (from 2-labeled n-manni tols); and n-fr uctose-3,4-C14 and n-fru ctose-3,4 -t (fro m 3-1abeled D-manni tols). A procedure, employ ing n-fru ctose-J ,6-C14 as a n int ern al standard , was devised for t he analysis of D-fru ctose-3,4-t.