Analysis of the Binding Sites of Opioid Receptor by Photoaffinity Labeling

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Research Purpose : Recent success in the construction of chimeric opioid receptors demonstrated the presence of several extra cellular regions which could be responsible for the distinct ligand-binding profiles of the each type of opioid receptors. However, it is generally impossible to exclude potential conformational changes which result in alterations of the ligand binding. The purpose of this research is the development and application of diazirine based photoprobes for the analysis of opioid binding sites.Results : A convenient synthesis of m- and p-CF_3-diazirinylbenzoic acid was developed. Using these photoreactive moieties, a novel pair of photoprobes which only differ in the carbene generating sites were synthesized. This structural feature of the probes are potentially useful to map the different sites within the ligand binding pocket. For avoiding the use of radioactive probes, a novel biotinylated photoreactive naltrexone analog was also developed. All new probes bearing diazirine bind reversibly with high affinity at mu-, delta-, and kappa- receptors. The photoaffinity labeling of recombinant delta-receptor protein was investigated with the biotinyl probe. The visualization of target molecule was successfully accomplished by the chemiluminescent detection of photochemically biotinylated proteins.