Pseudocoenzyme B 12 ( Cob-(5'-deoxy-5'-adenosyl)-(adenin-7-yl)cobamide; 1) and adenosyl-factor A ( Cob-(5'-deoxy-5'-adenosyl)-(2-methyladenin-7-yl)cobamide; 3) are two natural analogues of coenzyme B 12 ( adenosylcobalamin-Cob-(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 B 12 cofactors in several B 12 -dependent enzymes, such as in methionine synthase from Escherichia coli and in glutamate mutase from Clostridium cochlearium). In the present work, pseudocoenzyme B 12 (1) was synthesized in 85% yield by alkylation with 5'-O-tosyladenosine of (adenin-7yl)cob(I)amide, which was produced electrochemically from pseudovitamin B 12 (Cob-cyano-(adenin-7yl)cobamide). Likewise, adenosyl-factor A (3) was prepared in ca. 70% yield from factor A ( Cob-cyano-(2-methyladenin-7-yl)cobamide; 5). All the spectroscopic properties of 1 and 3 in aqueous solution indicated that these two Cob-(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 258, corresponding to a contribution of the -base-on× forms of 23% for 1 and of 32% for 3.