Fluorimetric studies of solutions of pyronin dyes: equilibrium constants in water and partition coefficients in organic-solvent—water systems

Abstract

The cytotoxic pyronin dyes provide simplified models for the xanthene squeleton of popular rhodamines. Kinetic analysis of fluorescence spectra of pyronin Y (PY) and pyronin B (PB) aqueous solutions allows us to study the reversible equilibria between R + zwitterions (λ Fluo, max=566 nm (PY), 570 nm (PB)) and colorless ROH xanthydrols (λ Fluo, max=463 nm (PY), 467 nm (PB)) formed through ion-pair reactions with nucleophile OH −. The rate and equilibrium constants of R + hydrolysis by OH − in water gives pK R+(S 0)= -2.6 for the two pyronins while fluorescent species are too short lived for any shift in the equilibrium to be detected in the S 1 state. Covalent ROH species are stable in basic solutions and poor ion solvating solvents; the reaction of R + with OH − is slow in neutral solutions. For further understanding and applications of pyronins on biomembranes, biphasic mixtures of an organic solvent (OS) with water (W) provide partition coefficients P and extraction percentages E of the different species in the OS phase. In the n-octanol-water (ratio 1:1) system, R + and ROH co-exist in both phases, resulting in P ion=1.8 (PY) and 2.6 (PB) at physiological pH with E ROH=0.09 (PY) and 0.16 (PB); most pyronin exists as R + in water owing to its pK R+ but the presence of the octanol phase shifts the equilibrium toward ROH. The ROH structure presents other features of interest because of its oxidization by air into RO xanthone in aprotic non-polar solvents. In the OS-W system (1:1) where OS is cyclohexane, the partitioning of pyronins between the OS and W phases is strongly one sided with R + remaining in water RO(λ Fluo, max=365–366 nm) completely trapped in the cyclohexane. The E percentages differ for PY and PB since E RO(PY)=0.8 but only 0.3 for PB. The implication of hydrophobic interactions and hydrogen-bonded (solvated) water in OS is also discussed.