Development of fluorescent pH indicator, fluorogenic protecting group, photoactivatable nucleophilic substitution (S[subscript N]1-type) reaction and photoactivatable fluorophores for biological applications
Nekongo, Emmanuel Etum
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In aqueous and alcohol solutions, colorless and non-fluorescent 9-aryl-9H-xanthen-9-ols equilibrate with brightly colored and fluorescent 9-arylxanthylium cations. The position of this equilibrium depends only on hydronium ion concentration and is not affected by general acids or other ions. The pH of the equivalence point can be adjusted by substitution in the xanthenol core. 3,6-dialkoxy-9-(o-tolyl)-9-xanthenol undergoes dehydroxylation to form corresponding cation at biologically relevant pH = 6.5. 9-Aryl-9H-xanthen-9-ol – based pH indicators permit following acidity changes by both fluorescent and absorption spectroscopies. Non-fluorescent 3,6-bis(benzyloxy)-9-aryl-9-xanthenol undergoes photo-dehydroxylation with simultaneous photo-Claisen rearrangement upon irradiation in aqueous methanol to give fluorescent isomeric products. Alcohol substrates are simply caged by refluxing the parent 3,6-bis(benzyloxy)-9-aryl-9-xanthenol with the desired alcohol in acetonitrile and catalytic amounts of acetic acid or using the alcohol as neat solvent for less expensive substrates. Formation of fluorescent fluorescein derivatives can be used to monitor substrate release. Photolabile protecting groups can also be used to weaken or totally eradicate the fluorescence of a fluorophore. Irradiation of the caged dye removes the protection thereby revealing the fluorophore. We have effectively used 3-(hydroxymethyl)napthalen-2-ol as a photoremovable protecting group for fluorescein. The caged fluorophore is weakly fluorescent. Photolysis of the caged dye releases the fluorophore with a fluorescent quantum yield = 0.75 -0.93. Novel light-triggered SN1 strategy for the formation of glycosidic bond has been developed. The reaction is initiated by the photochemical cleavage of the bond between anomeric carbon and aglycone containing 9-aryl-9-fluorenol. Irradiation of 9-aryl-9-fluorenol derivatives bearing the tetrahydropyranyl moiety in 1,1,1,3,3,3-hexafluoro-2-propanol and tetrahydrofuran in a Royonet reactor at 300 nm presumably resulted in a short-lived 9-aryl-9-fluorenyl cation. The carbocation intramolecularly attacks the ether oxygen. This intramolecular attack of the ether by the initially formed carbocation leads to heterolysis of the C-O bond and the formation of oxonium ion. The oxonium ion thus generated is trapped preferentially with added nucleophile such as 2-phenyl ethanol.