COMPARATIVE PHOTOAFFINITY-LABELING STUDY BETWEEN AZIDOPHENYL, DIFLUOROAZIDOPHENYL, AND TETRAFLUOROAZIDOPHENYL DERIVATIVES FOR THE GABA-GATED CHLORIDE CHANNELS

Syntheses of 2-aryl-substituted photoactivatable derivatives of 5-tert -butyl-1,3-dithiane and their oxidized bis-sulfone are described. The 4-azidoaryl and the 3,5-difluoro-4-azidoaryl groups were chosen as pho tosensitive moieties. Azidoaryl derivatives 3 and 13 were synthesized by diazotization and azidation of their corresponding arylamine precur sors. The o-difluoroazidophenyl derivatives 2 and 9 were synthesized b y transformation of the o-difluoro-substituted lithiophenyl into the c orresponding azido derivative. The reversible binding properties of th e photosensitive probes were established on bovine cortex P-2 membrane s by displacement of nyl-4-tert-butyl-2,6,7-trioxabicyclo[2.2.2]-octan e ([H-3]TBOB), a specific ligand for the channel blocker binding site. The 2-(3',5'-difluro-4'-azidophenyl)-5-tert-butyl- 1,3-dithianebis-su lfone, compound 2, exhibited the best K-i of about 11 nM, compared to K(i)s of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorop henyl analogue) and 3 (azidophenyl analogue). On irradiation, probe 2 (0.6 mu M) produced 18% irreversible loss of TBOB binding sites in bra in membranes while probe 3 did not produce any photoinactivation. The loss observed with 2 was fully protectable by TBOB, demonstrating the specificity of the photochemical inactivation by compound 2 for the co nvulsant site of the GABAA receptor. These results, when compared to t he photoaffinity labeling results obtained with the tetrafluorinated p robe 1 (25% selective irreversible photochemical inactivation), establ ish a hierarchy between fluorinated and nonfluorinated arylazido probe s and strengthen the potential of the newly described difluorinated pr obe 2, combining high affinity and good labeling efficacy.