There are approximately fifteen enzyme reactions known which require one of the two coenzyme forms of vitamin B12 to function. Two of these enzymes, methionine synthase and methylmalonyl-coenzyme A mutase, occur in humans (Dolphin, D. (ed.) B12; John Wiley & Sons, Inc.: New York, USA, 1982; Banerjee, R. (ed.) Chemistry and Biochemistry of B12; John Wiley & Sons, Inc.: New York, USA, 1999). The B12-dependent enzyme reactions play a vital role in maintaining healthy nerve and red blood cells and are required for the synthesis of DNA. Formula (I) depicts the structure of vitamin B12 (X=CN−, cyanocobalamin) and its derivatives, commonly referred to as the cobalamins. The α (or lower) axial site is occupied by an intramolecularly-bound 5,6-dimethylbenzimidazole, and the β (or upper) axial site can be occupied by a variety of ligands. The cobalamins belong to a family of compounds known as the corrinoids, which differ from one another in the specific nucleotide occupying the α axial site of the cobalt corrin complex.
    (I) X=CN− cyanocobalamin (vitamin B12)    (II) X=H2O/OH− aquocobalamin/hydroxycobalamin    (III) X=CH3 methylcobalamin    (IV) X=5′-deoxyadenosyl (adenosylcobalamin, coenzyme B12)    (V) X=glutathione (glutathionylcobalamin)
Three forms of vitamin B12 have long been recognised to occur in biology, aquocobalamin/hydroxycobalamin (II), methylcobalamin (CH3Cbl) (III) and adenosylcobalamin (AdoCbl) (IV) (Golding, B. T. Chem. Brit. 1990, 950). The latter two forms play a crucial role in the B12-dependent enzyme reactions, and are frequently referred to as the B12 coenzymes. Severe B12-deficiency may lead to megoblastic anaemia and/or neurological impairment. Insufficient vitamin B12 as a result of faulty absorption frequently manifests as pernicious anaemia. B12-related conditions arising from malabsorption can be easily (and reversibly) treated by administering vitamin B12 (I) or its hydroxycobalamin derivative (II), either orally or by injection into muscle tissue.
Recently, other therapeutic applications have been identified. McCaddon and co-workers have proposed that glutathionylcobalamin (GluSCbl) (V) may be an effective therapeutic for the treatment of Alzheimer's disease (AD) and other neurological diseases (McCaddon, A.; Regland, Bjorn; Hudson, P.; Davies, G. Neurol. 2002, 58, 1395–1399). It is now generally accepted that “oxidative stress” is an important neurodegenerative element in AD and several other neurological diseases. Glutathionylcobalamin is a naturally occurring intracellular form of cobalamin and is more readily absorbed and retained longer than cyanocobalamin. It has been proposed that, in vivo, GluSCbl is an intermediate in the conversion of biologically inactive cyanocobalamin to the active coenzyme forms adenosylcobalamin (IV) and methylcobalamin (III). The reducing agent glutathione (GluSH) is required for the formation of GluSCbl, and is likely to be present in lower levels in AD patients compared with healthy individuals as a result of oxidative stress. Thus, GluSCbl has the potential to offer a valuable source of cobalamin in therapeutic applications requiring administration of a vitamin B12 derivative.
GB 945722 (1964 to Merck & Co., Inc.) describes a method of preparing GluSCbl by reacting a 1:1 ratio hydroxocobalamin (hydroxycobalamin or aquocobalamin) and glutathione in water and precipitating the resulting complex to give a product of at least 95% purity. However, the technical sophistication available today for determining product purity was not available at the time of this earlier work and subsequent repeated attempts of this work have identified that the resulting product is in fact of only 60–70% purity (see Reference Examples A and B).
More recently, other methods for preparing GluSCbl in high purity have been reported (Pezacka, E., et al, Biochem. Biophys. Res. Commun., 1990, 169, 443; Brown K., et al, Biochem., 1993, 32, 8421; Brasch N., et al, Inorg. Chem., 1999, 76, 197) using a large excess of GluSH (5–12×), however, an additional chromatographic step is required to provide a product of 98% purity.
Thus, there exists a need for methods of preparing GluSCbl of an acceptable purity level which do not require the cost and effort of a chromatographic purification step, which also can be performed in the presence of air, and which presents a method leading to a commercially viable process for the synthesis of glutathionylcobalamin.