Pseudocoenzyme B12 and Adenosyl-Factor A: Electrochemical Synthesis and Spectroscopic Analysis of Two Natural B12 Coenzymes with Predominantly ‘Base-off' Constitution

Abstract

Pseudocoenzyme B12 (=Coβ-(5′-deoxy-5′-adenosyl)-(adenin-7-yl)cobamide; 1) and adenosyl-factor A (=Coβ-(5′-deoxy-5′-adenosyl)-(2-methyladenin-7-yl)cobamide; 3) are two natural analogues of coenzyme B12 (=adenosylcobalamin-Coβ-(5′-deoxy-5′-adenosyl)-(5,6-dimethyl-1H-benzimidazolyl)cobamide; 2), where the Co-coordinating 5,6-dimethyl-1H-benzimidazole nucleotide base of 2 is replaced by the purine bases adenine and 2-methyladenine. In contrast to 2, which exists solely in the ‘base-on' form, UV/VIS spectroscopy qualitatively indicates ‘base-off' constitution for 1 and 3 in aqueous solution. (cf. the established ‘base-off' form as unexpected binding mode of B12 cofactors in several B12-dependent enzymes, such as in methionine synthase from Escherichia coli and in glutamate mutase from Clostridium cochlearium). In the present work, pseudocoenzyme B12 (1) was synthesized in 85% yield by alkylation with 5′-O-tosyladenosine of (adenin-7-yl)cob(I)amide, which was produced electrochemically from pseudovitamin B12 (Coβ-cyano-(adenin-7-yl)cobamide). Likewise, adenosyl-factor A (3) was prepared in ca. 70% yield from factor A (=Coβ-cyano-(2-methyladenin-7-yl)cobamide; 5). All the spectroscopic properties of 1 and 3 in aqueous solution indicated that these two Coβ-(5′-deoxy-5′-adenosyl)-(adenin-7-yl)cobamides exist predominantly in a ‘base-off' constitution, with minor but significant contributions of the ‘base-on' form. From the UV/VIS spectra, the temperature-dependent equilibrium constants of the ‘base-off'/‘base-on' reconstitution reaction were determined as Kon (1)=0.30 and Kon (3)=0.48 at 25°, corresponding to a contribution of the ‘base-on' forms of 23% for 1 and of 32% for 3.