Analogues of a novel class of template-competitive reverse transcriptase inhibitors (Li, K.; Lin, W.; Chong, K. H.; Moore, B. M.; Doughty, M. B. Bioorg. Med. Chem. 2002, 10, 507) were analyzed as photoprobes of HIV-1 reverse transcriptase (RT) heterodimer. The two photoprobes, 2-(4-azidophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 2 and the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 3, photodecomposed at 3500 A with half-lives of 4.0 and 2.5 min, respectively. Analysis of the photoproducts of 2m demonstrated that the etheno group is stable but the azido decomposes primarily to the 2-(S-[3H-diazepinon-4-yl]thio)-1,N(6)-etheno-dAMP. Photolysis of both 2 and 3 with RT resulted in a time-dependent loss of activity, with maximum inactivation of 83 and 60%, respectively. Both 2 and 3 showed concentration-dependent photoinactivation of RT in the concentration range from 0 to 100 microM, with EC(50)s of 20 and 25 microM and maximum inactivation of 80 and 60%, respectively. Both the time and concentration dependent photoinactivation were strongly protected by template-primer, but only poorly inhibited by even high concentrations of TTP. Radiolabeled analogues [beta,gamma-(32)P]-2 and [beta,gamma-(32)P]-3 photoincorporated into the p66 subunit, an incorporation also protected by template primer. Identification of the site of incorporation was problematic for both photoprobes, but evidence presented is consistent with labeling sites for the phenacyl side chains of both 2 and 3 in the template grip. Nevertheless, the photoinactivation and incorporation data are consistent with our earlier conclusions from the kinetic data that these inhibitors are specific for the free form of RT in competition with template/primer, and thus represent a novel class of inhibitors.