Synthesis and characterization of N-(4-azidophenylthio)phthalimide: A cleavable, photoactivable crosslinking reagent that reacts with sulfhydryl groups

The cleavable, photoaffinity label precursor, N-(4-azidophenylthio)phthalimide has been synthesized and purified. The recrystallized product was identified by infrared spectroscopy and nuclear magnetic resonance spectroscopy. The compound modifies free thiol groups to yield the corresponding S-4-azidophenylthio derivatives. In order to examine the biological applications of this compound, yeast iso-1-cytochrome c, containing a single free cysteine residue, was modified and characterized. The 102-S-(4-azidophenylthio)-iso-1-cytochrome c was found to contain 1 mol of label/mol of cytochrome c. The photoaffinity labeling of purified, detergent-solubilized yeast cytochrome c oxidase was examined. Photolysis products of crosslinking could be analyzed on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The crosslinked products were readily cleaved by dithiothreitol. The use of this compound as a sulfhydryl-specific photolabile, bifunctional crosslinking reagent is discussed.