Trifluoromethyldiazirinylphenyldiazenes: New hemoprotein active-site probes

The photoactivatable trifluoromethyldiazirinylphenyldiazene probes 1a and 2 a have been synthesized, and their utility in the mapping of hemoprotein ac tive sites has been validated with myoglobin (Mb). Reaction of the probes w ith Mb yields Fe-aryl adducts. Photolysis of these adducts unmasks a carben e that cross-links to active-site protein residues. Migration of the aryl g roup from the iron to a porphyrin nitrogen then attaches the porphyrin chro mophore to the labeled amino acid residue. Tryptic digestion of the labeled proteins followed by mass spectrometric analysis of the peptides has ident ified Leu-29, His-64, Ile-107, and Val-68 as active-site residues. Previous studies with an arylnitrene probe, which appears to only react with nucleo philic groups, identified His-64 as an active-site residue [Tschirret-Guth, R. A.; Medzihradszky, K. F.; Ortiz de Montellano, P. R. J. Am. Chem. Sec. 1998, 120, 7404-7410]. These studies have identified all but one of the act ive-site amino acids in contact with the probe. The present strategy not on ly labels active-site amino acids but also, because the probe is rigidly he ld in the active site, provides approximate locations for the labeled resid ues with respect to the heme iron atom. Validation of the Strategy with myo globin opens the way to use of the approach with hemoproteins of unknown ac tive-site structure.