Trifluoromethyldiazirinylphenyldiazenes: New Hemoprotein Active-Site Probes

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• 作者：Richard A. Tschirret-Guth ; Katalin F. Medzihradszky ; and Paul R. Ortiz de Montellano
• 刊名：Journal of the American Chemical Society
• 出版年：1999
• 出版时间：May 26, 1999
• 年：1999
• 卷：121
• 期：20
• 页码：4731 - 4737
• 全文大小：116K
• 年卷期：v.121,no.20(May 26, 1999)
• ISSN：1520-5126

文摘

The photoactivatable trifluoromethyldiazirinylphenyldiazene probes 1a and 2a have been synthesized,and their utility in the mapping of hemoprotein active sites has been validated with myoglobin (Mb). Reactionof the probes with Mb yields Fe-aryl adducts. Photolysis of these adducts unmasks a carbene that cross-linksto active-site protein residues. Migration of the aryl group from the iron to a porphyrin nitrogen then attachesthe porphyrin chromophore to the labeled amino acid residue. Tryptic digestion of the labeled proteins followedby mass spectrometric analysis of the peptides has identified 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 nucleophilic groups,identified His-64 as an active-site residue [Tschirret-Guth, R. A.; Medzihradszky, K. F.; Ortiz de Montellano,P. R. J. Am. Chem. Soc. 1998, 120, 7404-7410]. These studies have identified all but one of the active-siteamino acids in contact with the probe. The present strategy not only labels active-site amino acids but also,because the probe is rigidly held in the active site, provides approximate locations for the labeled residueswith respect to the heme iron atom. Validation of the strategy with myoglobin opens the way to use of theapproach with hemoproteins of unknown active-site structure.