Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY.

摘要: A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the range 5-7 has been designed and synthesized by conjugatively linking BODIPY fluorophore at 3-position to pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) corresponding water-soluble sodium carboxylate, 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). fluorescence quantum yield Φ(f) of I very high (0.8-1.0) organic solvents tested. lifetime (ca. 4 ns) with varying polarity/polarizability (from cyclohexane acetonitrile) independent solvent a rate constant k(f) 2.4×10(8) s(-1). Probe readily loaded cytosol live cells, where its intensity remains nearly over extended time period. Water-soluble II exhibits two acid-base equilibria aqueous solution, characterized pK(a) values 6.0 12.6. value solution high: 0.6 cationic anionic forms ligand, 0.8 neutral imidazole. On protonation-deprotonation near-neutral range, UV/Vis absorption spectral shifts along isosbestic pseudo-isoemissive points are observed. This dual-excitation dual-emission emits intense green-yellow lower orange higher pH. influence ionic strength buffer concentration on absorbance steady-state also investigated. apparent equilibrium determined spectrophotometric fluorometric titration added salt concentration. In absence 5.20-7.45, dual exponential decays obtained decay τ(1)=4.3 ns τ(2)=3.3 form II. excited-state proton exchange becomes reversible addition phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, pH-dependent change times induced. Global compartmental analysis traces collected function was used recover deactivation constants excited (k(01)=2.4×10(8) s(-1)) (k(02)=3.0×10(8)