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 photosensitive moieties. Azidoaryl derivatives 3 and 13 were synthesized by diazotization and azidation of their corresponding arylamine precursors. The o-difluoroazidophenyl derivatives 2 and 9 were synthesized by transformation of the o-difluoro-substituted lithiophenyl into the corresponding azido derivative. The reversible binding properties of the photosensitive probes were established on bovine cortex P2 membranes by displacement of [3H]-1-phenyl-4-tert-butyl- 2,6,7-trioxabicyclo[2.2.2]-octane ([3H]TBOB), a specific ligand for the channel blocker binding site. The 2-(3',5'-difluro-4'-azidophenyl)-5-tert-butyl- 1,3-dithianebis-sulfone, compound 2, exhibited the best Ki of about 11 nM, compared to Kis of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorophenyl analogue) and compared to Kis of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorophenyl analogue) and 3 (azidphenyl analogue). On irradiation, probe 2 (0.6 microM) produced 18% irreversible loss of TBOB binding sites in brain 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 convulsant site of the GABAA receptor. These results, when compared to the photoaffinity labeling results obtained with the tetrafluorinated probe 1 (25% selective irreversible photochemical inactivation), establish a hierarchy between fluorinated and nonfluorinated arylazido probes and strengthen the potential of the newly described difluorinated probe 2, combining high affinity and good labeling efficacy.