Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.