8‐Azidoadenine Analogs of NAD+ and FAD

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Abstract
The synthesis and purification of the 8-azidoadenine analogs of NAD+ (azido-NAD+) and FAD (azido-FAD) from 8-azidoadenosine 5''-phosphate and NMN+ or FMN, respectively, is described. The coenzyme analogs are characterized by absorption, NMR and circular dichroism spectra. The 2 latter methods indicate a folded structure of azido-NAD+ and azido-FAD. Upon irradiation at 300 nm in aqueous solution, a change of the UV absorption spectra of the coenzyme analogs indicates photolysis of the azido group. The coenzyme properties of azido-NAD+ are demonstrated with lactate, glutamate and alcohol dehydrogenase [from pig heart, bovine liver and horse liver, respectively] yielding 14, 154 and 60%, respectively, of the V observed with NAD+. Concomitantly, the Km values of the coenzyme analogs are 1.7, 3.5 and 3-fold higher than those of NAD+. Azido-FAD is a coenzyme of apo-glucose oxidase [from Penicillium notatum]. The recovery of activity is much slower in the presence of azido-FAD than with FAD. A final value of 66% of the activity with FAD is obtained. With apo-D-amino acid oxidase [from pig kidney], azido-FAD is completely inactive, although it is specifically bound to the enzyme.