AN ¹²⁵I‐LABELED N⁶‐SUBSTITUTED AZIDO ANALOG OF NAD⁺ FOR THE PHOTOAFFINITY LABELING OF NAD⁺‐LINKED ENZYMES

Abstract ¹²⁵I‐N⁶‐(N‐[6‐N‐{5‐iodo‐4‐azidosalicyl}‐aminohexyl]‐aminocarbamoylmethyl)‐nicotinamide adenine dinucleotide (¹²⁵I‐N⁶‐I‐ASA‐AH‐NAD⁺) was synthesized by coupling N⁶‐([6‐aminohexyl]‐carbamoylmethyl)‐NAD⁺ with 4‐azidosalicylic acid N‐hydroxysuccinimide ester followed by radioiodination. The utility of ¹²⁵I‐N⁶‐I‐ASA‐AH‐NAD⁺ as an effective site‐directed photoprobe was demonstrated by the photolabeling of both glutamate dehydro‐genase and 15‐hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD⁺. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD⁺. These results indicate that ¹²⁵I‐N⁶‐I‐ASA‐AH‐NAD⁺ can be a specific photoprobe for NAD⁺‐linked enzymes.