OCTOBER 1963

The Reaction of CobaJamins with ThioIs: an Alternative Synthesis of AlkyI-cobamide Coenzyme Analogues

By D. H. DOLPHIN and A. W. JOHNSON (DEPARTMENT OF CHEMISTRY, UNIVERSITY OF NOTTINGHAM)

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STUDIES of the biosynthesis of the vitamin Bu markedly faster if the hydroxocobalamin and sodium coenzyme1,2 (I; R 5'-deoxyadenosyl) from hydrogen sulphide solutions are allowed to react to hydroxocobalamin (vitamin Bub) (I; R H 20) the brown stage before addition of methyl iodide. have established that the 5'-deoxyadenosyl group is The brown intermediate can also be prepared derived from adenosine triphosphate (ATP) and that directly from vitamin Bur by adding a degassed necessary co-factors for the reaction are a thiol (i.e., oxygen-free) aqueous solution of sodium sul-(glutathione and 2-mercaptoethanol were exempli- phide, but the product is not sufficiently stable to be fled), reduced flavin (FADH2), and manganese ions. isolated in the solid state. Vitamin Bur does not The in vivo reaction therefore does not parallel the react with methyl iodide in the absence of sulphide in vitro partial synthesis of the coenzyme from ions, and it is probable that the reactive brown inter-hydroxocobalamin,3,4 which involves a preliminary mediates are complexes (III) of bivalent cobalt. This reduction to the so-called vitamin Bus, a cobalt implies a final oxidation step, although it has been hydride.3- 6

We have examined the reaction of hydroxoco-balamin with a variety of thiols including glutathione, 2-mercaptoethanol, thioglycollic (mercaptoacetic) acid, cysteine, homocysteine, ethanethiol, toluene-w-thiol, and sodium hydrogen sulphide (present in aqueous solutions of sodium sulphide), and in all cases the initial colour change is from red to violet. When methyl iodide is added to the mixture of aqueous hydroxocobalamin and anyone of the above thiols with exclusion of light, methylco balamin is formed (identified by chromatography and spectra3). In the case of sodium hydrogen sulphide, the yield of methylcobalamin is very high (ca. 90%) and the product is easily isolated in the crystalline form. This reaction has been examined in detail and it has been shown not to be a simple displacement reaction of type (A).

OH 2 -SR Me -} -} I (A) Colli -~ COlli COlli

([I) In fact the reactive intermediate in the formation

of methylcobalamin was not the violet product, which is believed to be the species (II), but a second-ary brown compound formed by further reduction of (II) by sulphide. The spectrum of the brown com-pound is similar to that of vitamin B12r,7 a reduction product of vitamin Bu containing bivalent cobalt 8 (see, however, Hill et aI.6). Solutions of the brown compound react very rapidly with methyl iodide to yield methylcobalamin, and the overall reaction is

observed that the rate of reaction of hydroxoco-balamin, sodium sulphide, and methyl iodide to form methylcobalamin is faster in the absence of oxygen (10-6 mm.) than the similar reaction with oxygen present. The precise nature of the oxidation step has not been elucidated. The cycle is reversed when the final solution is irradiated, as methylcobalamin is rapidly photolysed to re-form hydroxocobalamin.

OH2 -SR fMe 1 Me 1 NaHS 1 Mel l I I Colli COII __ ~ Collj __ ~Colli

"" (III) / hv

1 Brady, Castanera, and Barker, J. Bioi. Chem., 1962,237,2325. 2 Weissbach, Redfield, and Peterkovsky, J. Bioi. Chem., 1962,237,3217. 3 Smith, Mervyn, Johnson, and Shaw, Nature, 1962, 194, 1175; J. Chem. Soc., 1963 in the press. 4 Bernhauser, Muller, and Muller, Biochem. z., 1962,336, 102,299. '

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The displacement of the sulphur ligand by an alkyl group is more susceptible to steric hindrance than the corresponding hydride displacement, and the reaction with ethyl iodide is slower than that with methyl iodide, although the crystalline ethyl-cobalamin was obtained without difficulty. How-ever, in this case the formation of the brown inter-mediate with its characteristic spectrum could be clearly observed. The following alkylating agents were also employed successfully in the reaction:

PROCEEDINGS

methyl toluene-p-sulphonate, l-chloro-, 1-bromo-, and l.iodo-propane, 1-iodobutane, chloroacetic acid, ,B-chloropropionic acid, and acetyl chloride.

The displacement reactions involving cobalt-suJphur ligands appear to be analogous to the bio-logical syntheses of the coenzymes, but the exact parallel of the biosynthesis, i.e., the use of ATP as the alk.ylating agent, has not yet been achieved in vitro. (Received, July 9th, 1963.)