Vitamin B12 and α-Ribonucleosides

Various nucleoside/carbohydrate bearing species found in nature are important biomolecules. Nucleosides/carbohydrates play a vital role in biological systems and control a number of reactions in living cells. Naturally occurring ribonucleosides are almost always in the β-anomeric configuration with the exception of the vitamin B12 family (Fig. 1), in which an α-ribonucleoside is included in the structure as the lower axial ligand.1,2 About a dozen enzymes, including the B12 dependent ribonucleoside triphosphate reductase,3 ethanolamine amamonia lyase,4 glutamate mutase5 and methylmalonyl CoA mutase require a B12 cofactor. The cofactor for these reactions, 5- deoxyadenosylcobalamin (AdoCbl, coenzyme B12, Fig 2), plays an important role by releasing a reactive species, 5-deoxyadenosyl radical, upon enzyme-induced cleavage of the carbon-cobalt bond. The mechanism of this enzymatic “activation” of the co-enzyme, in which homolysis of C-Co bond catalyzed by some 109 to 1014-fold remains unknown. Figure 1 Vitamin B12 derivatives with altered lower axial ligands are of interest as probes of the mechanism of action of coenzyme B12. In order to determine what role, if any, the lower axial ligand plays in the enzymatic activation of coenzyme B12, it is necessary ... Fig. 2 Vitamin B12 derivatives are octahedral coordination complexes of Co(III) with a macrocyclic tetradentate equatorial ligand (the corrin ring), a lower axial 5,6 dimethylbenzimidazole (DMBI) ligand coordinated to cobalt through the imidazole (N-3) atom, and attached to a corrin ring side chain as an unusual α-ribonucleoside (to form the “nucleotide loop”), and various upper axial ligands (cyanide = vitamin B12, CH3 = methylcobalamin, 5-deoxyadenosyl = AdoCbl). The Co-C linkage in AdoCbl was the first metal-carbon bond described in biological systems and is the key to the enzymatic activity of the B12 coenzyme. This report addresses recent advances in the synthesis of α- ribonucleosides, for which little prior literature exists, and which permits modification of the lower axial ligand of B12 for mechanistic studies. 5,6-Dimethyl-α-d-ribofuranosylbenzimidazole is one of the few naturally occurring α-ribonucleosides that has attracted significant attention due to its possible role the activation of co-enzyme B12.1 A number of nucleoside and oligonucleotide analogues have been used in medicinal applications due to their diverse biological effects human beings,16 but the majority of these compounds are analogues of β-nucleosides. β-Nucleosides are common in nature and can be prepared easily by established glycosylation methods. On the other hand, the use of α-nucleosides and their analogues is limited due to limited availability of synthetic routes to these anomers. Thus, only a limited number of α-nucleosides are known for their biological activity in human beings. α-2-Deoxythioguanosine has shown promising antitumer activity and been found to be less toxic compared to its β-counterparts.17 As α-ribonucleosides have not attracted much synthetic attention, most α-ribonucleosides arise as by-products of glycosylation reactions intended for the synthesis of β-ribonucleosides. Although some general glycosylation reactions produces up to 40–50% of the α-anomer,15 it is often difficult to separate the α-anomer from its β-counterpart by column chromatography or crystallization, due to their similar Rf values and chemical nature. Since indoles and indolines are not incorporated in to cobalamin structure when the media of B12 overproducing bacteria are supplemented with them, it is necessary to prepare the α-nucleosides or nucleotides of indole, and their substituted derivatives for semisynthesis of B3-deaza derivatives of vitamin B12. The replacement of the 5,6-dimethybenzimidazole nucleotide18 with a 5,6-dimethyindole nucleotide loop will produce an exact replica of the cobalamin without a coordinating nitrogen and serve as a test of the role, if any, of axial ligand coordination in the enzymatic activation of AdoCbl. Vitamin B12 has been widely used for targeting cancer cells and for incorporating various bioactive molecules.19 Many bioconjugates of vitamin B12 have been prepared and used as receptor modulating agents for treating cancer. Neoplastic disorders such as leukemia, sarcoma, myeloma, carcinoma, neuroma, melanoma, cancers of the lung, liver, breast, colon, cervix and prostate can be treated by such receptor modulating agents.20 B12 is a large water soluble biomolecule and structural alterations are possible without affecting the affinity to different transporter and receptor proteins. Recent uses of cobalamin and its conjugates as delivery vechicles for cytotoxic anticancer drugs and fluorescent tumor imaging agents for malignant cells have given hope for new therapeutics for the treatment of these deadly diseases. Vitamin B12 can be easily conjugated to anticancer and radiotracer drugs for transport to specific sites. Grissom and co-workers have attached a number of cytotoxic drugs to cobalamin as upper axial ligands or to the 5′ carbon of the ribose sugar (Fig 3).19 (b–e) Such B12-drug conjugates are nontoxic until they reach the target site. Other bioconjugates of B12 modified at the 5′-sugar carbon have been reported, and have affinity for intrinsic factor similar to that of CNCbl-e-monocarboxylate. The 5′-hydroxyl group has been activated with 1,1-carbonyldiimidazole, 1,1-carbonyldi (1,2,4-triazole) or di(1-benzotriazolyl)carbonate and then added to aminoalkanes, diaminoalkanes or alkanedihydrazides (Fig 4) to produce CNCbl analogues. Fig. 3 Fig. 4 Cobalamins and their derivatives have been used as efficient catalysts.21 The availability of the different oxidation states of cobalt in B12 results in the formation of Co-alkyl derivatives and also in the lability of the Co-C bond, which is easily cleaved thermally or photochemically. The reducing properties of the B12 have been widely explored in reductive dehalogenations.22 Polychlorinated ethylenes are among the most commonly found volatile organics in ambient ground water in the United States. B12 is involved in both the enzymetic and non-enzymetic dehalogenation reactions.22 Several microorganisms have been isolated recently that can convert perchloroethylene (PCE) into trichloroethene (TCE), and subsequently TCE into cis-1,2-dichloroethene. The enzyme responsible for this dechlorination reaction uses a vitamin B12 derivative as its active cofactor. While the mechanisms involved in reductive dehalogenation of TCE are still under investigation, it is clear that the availability of reduced oxidation states is crucial and the reduction pathway goes through cis-DCE formation.23