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.
Kapfer
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Laboratoire de Chimie Bio-organique, URA 1386 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, Illkirch, France.
Jacques
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Toubal
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Goeldner
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MP
D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000737:chemistry / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002417:Cattle; D002540:Cerebral Cortex / Q000648:ultrastructure; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology; D008566:Membranes / Q000502:physiology; D010777:Photochemistry; D018079:Receptors, GABA / Q000737:chemistry*; D013450:Sulfones / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology
C093049:2-(3',5'-difluoro-4'-azidophenyl)-5-tert-butyl-1,1,3,3-tetraoxide-1,3-dithiane; C093048:2-(4'-azidophenyl)-5-tert-butyl-1,1,3,3-tetraoxide-1,3-dithiane; D000345:Affinity Labels; D001386:Azides; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018118:Chloride Channels; D018079:Receptors, GABA; D013450:Sulfones; C085376:2-(4'-azidotetrafluorophenyl)-5-tert-butyl-1,3-dithiane-bis-sulfone; C070435:4-t-butyl-1-phenyl-2,6,7-trioxabicyclo(2.2.2)octane