A group of nicotinamide adenine dinucleotide (NAD⁺) analogs have been enzymatically synthesized and evaluated for their potential applications in photoaffinity labeling experiments for NAD⁺ dependent enzymes. The objective of molecular design for these analogs is to elucidate the three dimentional environment of active sites in NAD⁺ dependent enzymes. The functional group modification at the nicotinamide moiety is made possible by a simple yet unique enzymatic exchange reaction which avoid length organic synthesis in constructing such co- enzymes. Among the nine NAD⁺ analogs, two possess coenzyme activity, five were competitive inhibitors of LADH, and two were not inhibitors. Six of them were found covalently attached to the targeted enzyme upon irradiation. The potential application of these co-enyzmes in locating the active sites for NAD-utilizing enzymes has been clearly established through this study.