Abstract
This review describes a new outcome of photoaffinity labeling methods for the analysis of functional sites within receptor molecules. Diazirine-based photoaffinity labeling was extensively investigated to improve azide-based conventional methods. First of all, a series of simple methods for modifying diazirines bearing an aromatic ring has been accomplished. This first versatile approach involving direct substitution on the aromatic ring of diazirines has been achieved by means of the aromatic thallation of alkoxyphenyldiazirines. Introduction of the thallium moiety was successfully followed by nitration, iodination, or palladiumcatalyzed carbonylation to give a family of substituted aryl diazirines useful for photolabeling. The methoxyphenyldiazirines were also found to be stable under certain demethylation conditions, and a tether to link diazirines with ligands or radioactive markers were readily introduced to the resulting phenols. These new methods of derivatization provide a practical approach to simplify the timeconsuming processes currently used for diazirine synthesis. Secondly, the diazirine-based photoaffinity labeling was systematically compared with a conventional aryl-azide method in the course of photoaffinity labeling of the eel sodium channels. A tetrodotoxin derivative carrying an aryl-diazirine was specifically photoincorporated to toxin-binding region within the sodium channel polypeptide. Labeled sites were successfully identified by probing protease-digested labeled fragments with several sequence-directed antipeptide-antibodies. The results suggest that the diazirine moiety on the toxin orients to the region between S5 and S6 in domain III and IV of the channel. The corresponding azide derivative gave no positive results probably due to their chemical limitations : their photogenerated intermediates are less reactive and the crosslinks induced between ligands and channels are less stable.
