p-Nitrophenyl 3-diazopyruvate and diazopyruvamides, a new family of photoactivatable cross-linking bioprobes

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Abstract
P-Nitrophenyl 3-diazopyruvate (DAPpNP) has been developed as a heterobifunctional cross-linking agent for synthesis of photoaffinity probes and photoactivatable cross-linking agents that are nucleophile specific. p-Nitrophenyl chloroglyoxylate is formed in high yield from oxalyl chloride and p-nitrophenol. Subsequent reaction with diazomethane produces DAPpNP in 50-60% overall yield. DAPpNP acylates primary and secondary amines to form 3-diazopyruvamides in high yields. 3-Diazopyruvamide derivatives have been formed from a wide variety of amines including aromatic amines, amino acids, and peptides. 3-Diazopyruvamides undergo photolysis and Wolff rearrangement at 300 nm to produce a ketene amide, which efficiently acylates nucleophilic species to form malonic acid amide derivatives. A family of photoactivatable 3-diazopyruvamide cross-linking agents was synthesized from amino acids. A cleavable, thiol-specific photoactivatable cross-linking agent was synthesized from cystamine. These reagents were caused to react with rabbit muscle aldolase to form mainly dimeric cross-linked species.