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

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 K_on ( 1 )=0.30 and K_on ( 3 )=0.48 at 25°, corresponding to a contribution of the ‘base‐on' forms of 23% for 1 and of 32% for 3 .