Evaluation of 3-azidiamantane as photoaffinity probe of cytochrome P450

3-azidiamantane (DIA-N-2) has been shown to be a photolabile carbene-genera ting probe interacting specifically with cytochrome P450 (P450) active cent re. To evaluate the modification of P450 by the probe, radiolabelled [9-H-3 ]-3-azidiamantane was prepared by reductive dehalogenation of its precursor , 3-oxo-9-bromodiamantane ethylene ketal. The synthesis was optimized as th e proper precursor and reaction conditions were concerned to produce 96% pu re product (overall yield 59%). An incorporation efficacy of the probe phot oactivated at 366 pm was examined with two different proteins, BSA and rat phenobarbital-inducible P450 2B1, both having hydrophobic binding sites. Un der photolysis the photoaffinity probe generated short-lived (> 90%) interm ediates binding immediately to the protein. The yield of photoactivated DIA -N2 incorporation was 12% and 11% for BSA and P450, respectively. The prese nce of reduced glutathione, a scavenger of reactive intermediates, did not affect the probe incorporation markedly. On the other hand, scavengers ente ring the P450 active centre, methanol and dithiothreitol, reduced the prote in labelling by 36% and 42%, respectively. Similarly, at DIA-N2, aminopyrin e (substrates), and metyrapone (inhibitor) 50 times molar excess over the p robe, prevented its binding by about 40%. In addition, when photoaffinity l abelling was carried out with microsomal preparation, the substrate with a high affinity for the P450 2B1, diamantane, (at 20 times molar excess to th e probe) caused 47% inhibition of the P450 covalent labelling. These result s, suggesting a high specificity of the probe binding, show that it can be applied as a photoaffinity probe for cytochrome P450 2B1 active centre stud ies.