title: Evaluation of 3-azidiamantane as photoaffinity probe of cytochrome P450.
abstract: 3-azidiamantane (DIA-N2) has been shown to be a photolabile carbene-generating probe interacting specifically with cytochrome P450 (P450) active centre. To evaluate the modification of P450 by the probe, radiolabelled [9-3H]-3-azidiamantane was prepared by reductive dehalogenation of its precursor, 3-oxo-9-bromodiamantane ethylene ketal. The synthesis was optimized as the proper precursor and reaction conditions were concerned to produce 96% pure product (overall yield 59%). An incorporation efficacy of the probe photoactivated at 366 nm was examined with two different proteins, BSA and rat phenobarbital-inducible P450 2B1, both having hydrophobic binding sites. Under photolysis the photoaffinity probe generated short-lived (> 90%) intermediates binding immediately to the protein. The yield of photoactivated DIA-N2 incorporation was 12% and 11% for BSA and P450, respectively. The presence of reduced glutathione, a scavenger of reactive intermediates, did not affect the probe incorporation markedly. On the other hand, scavengers entering the P450 active centre, methanol and dithiothreitol, reduced the protein labelling by 36% and 42%, respectively. Similarly, at DIA-N2, aminopyrine (substrates), and metyrapone (inhibitor) 50 times molar excess over the probe, prevented its binding by about 40%. In addition, when photoaffinity labelling was carried out with microsomal preparation, the substrate with a high affinity for the P450 2B1, diamantane, (at 20 times molar excess to the probe) caused 47% inhibition of the P450 covalent labelling. These results, suggesting a high specificity of the probe binding, show that it can be applied as a photoaffinity probe for cytochrome P450 2B1 active centre studies.
authors.affiliation: Department of Biochemistry, Charles University, Prague, Czech Republic. hodek@prfdec.natur.cuni.cz
authors.forename: P
authors.initials: P
authors.lastname: Hodek
authors.forename: S
authors.initials: S
authors.lastname: Smrcek
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008861:Microsomes / Q000187:drug effects; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D010782:Photolysis; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology; D014316:Tritium / Q000378:metabolism
chemical_list: C121999:3-azidiamantane; D019920:Photoaffinity Labels; D012997:Solvents; D014316:Tritium; D012710:Serum Albumin, Bovine; D003577:Cytochrome P-450 Enzyme System; D000218:Adamantane