OCTOBER

1963

311

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)

of the biosynthesis of the vitamin Bu

coenzyme1,2 (I; R
5'-deoxyadenosyl) from
hydroxocobalamin (vitamin Bub) (I; R
H 20)
have established that the 5'-deoxyadenosyl group is
derived from adenosine triphosphate (ATP) and that
necessary co-factors for the reaction are a thiol
(glutathione and 2-mercaptoethanol were exemplifled), reduced flavin (FADH 2), and manganese ions.
The in vivo reaction therefore does not parallel the
in vitro partial synthesis of the coenzyme from
hydroxocobalamin,3,4 which involves a preliminary
reduction to the so-called vitamin Bus, a cobalt
hydride. 3- 6
We have examined the reaction of hydroxocobalamin with a variety of thiols including glutathione,
2-mercaptoethanol, thioglycollic (mercaptoacetic)
acid, cysteine, homocysteine, ethanethiol, toluenew-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).

STUDIES

OH 2
(A)

-}

-SR

-}

Colli -~ COlli

Me

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 secondary brown compound formed by further reduction
of (II) by sulphide. The spectrum of the brown compound is similar to that of vitamin B 12r,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

markedly faster if the hydroxocobalamin and sodium

hydrogen sulphide solutions are allowed to react to
the brown stage before addition of methyl iodide.
The brown intermediate can also be prepared
directly from vitamin Bur by adding a degassed
(i.e., oxygen-free) aqueous solution of sodium sulphide, but the product is not sufficiently stable to be
isolated in the solid state. Vitamin Bur does not
react with methyl iodide in the absence of sulphide
ions, and it is probable that the reactive brown intermediates are complexes (III) of bivalent cobalt. This
implies a final oxidation step, although it has been

observed that the rate of reaction of hydroxocobalamin, 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.
-SR

OH 2

Colli

""

NaHS

fMe

Mel

COII _ _ ~

(III)

lI

Me

Collj _ _ ~Colli

hv

Brady, Castanera, and Barker, J. Bioi. Chem., 1962,237,2325.

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.
'
<; Smith and Mervyn, Biochem. J., 1962, 86, 2p.
6 Hill, Pratt, and Williams, J. Theor. Bioi., 1962, 3, 423.
7 Diehl and Mucie, Iowa State Call. J. Sci., 1952,26, 555; Jaselkis and Diehl, J. Amer. Chem. Soc., 1954,76,4345;
Beaven and Johnson, Nature, 1955, 176, 1264.
8 Hogenkamp, Barker. and Mason, Arch. Biochem. Biophys., 1963,100,353.
1

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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 ethylcobalamin was obtained without difficulty. However, in this case the formation of the brown intermediate 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 cobaltsuJphur ligands appear to be analogous to the biological 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.)