THE MECHANISM OF ACTION OF VITAMIN-B12

A novel rearrangement reaction is introduced as a model for the rearrangement of methylitaconic acid (III) to .alpha.-methyleneglutaric acid (IV), 1 of 3 enzyme catalyzed, coenzyme B12-dependent, C-skeleton rearrangements whose mechanism was a source of puzzlement for many years. The key feature of the new model is the direct attachment of the substrate, methylitaconic acid, to the Co atom of vitamin B12. This was accomplished by reacting butadiene-2,3-decarboxylic acid with hydrobromic acid generating bromomethylitaconic acid (VIII). Use of 2 moles of hydrobromic acid yielded bis-2,3-(bromomethyl)succinic acid (IX). Reaction of the monobromide VIII with vitamin B12 did not yield the desired C.sbd.Co bonded adduct. The lactone .alpha.-methylene-.gamma.-butyrolactone-.beta.-carboxylic acid (X) was formed. The ester, dimethyl bromomethylitaconate (XIa), was reacted with vitamin B12 and yielded the C.sbd.Co bonded adduct XIIa. Bis-trimethylsilyl bromomethylitaconate did not yield an adduct when reacted with vitamin B12, but bis-tetrahydropyranyl brmomethylitaconate (XIb) did yield the adduct XIIb. The ester cobalamin XIIb undergoes spontaneous decomposition at room temperature, in aqueous solution, at pH 8 and in the dark.sbd.biochemically ideal circumstances.sbd.yielding a mixture of: butadiene-2,3-decarboxylic acid (VII), methylitaconic acid (III) and .alpha.-methyleneglutaric acid (IV). The presence of the latter indicates that a skeletal change has taken place in a way which mimics the enyzmatic reaction. This is the first enzymic model in this C-skeleton rearrangement series. The methyl ester cobalamin XIIa was stable in the dark but did decompose on irradiation with a sunlamp to butadiene-2,3-decarboxylic acid (VII) and methylitaconic acid (III). No .alpha.-methyleneglutaric acid IV was observed in the latter reaction. Authentic methylitaconic acid (III) was prepared by alkylation of triethyl prop-2-ene-1,1,2-tricarboxylate (XIII) with methyl iodide followed by hydrolysis and decarboxylation. The lactone X and lactone .alpha.-methyl-.gamma.-butyrolactone-.beta.-carboxylic acid (XVI) were prepared by condensing the triester XIII with formaldehyde, hydrolyzing the lactone diester XV to the lactone X and hydrogenating to the saturated lactone XVI.