Probing Hydrophobic Nanocavities in Chemical and Biological Systems with a Fluorescent Proton-Transfer Dye

Abstract

A new and sensitive molecular probe, namely, 2-(2'-hydroxyphenyl)-4-methyloxazole (HPMO), for exploring nanocavities in chemical and biological systems is presented. The incorporation of HPMO into hydrophobic cavities in aqueous medium involves rupture of its intermolecular hydrogen bond to water and formation of an intramolecular hydrogen bond in the sequestered molecule. Upon UV excitation (280 ± 330 nm) of this entity, a fast intramolecular proton-transfer reaction of the excited state produces a phototautomer, the fluorescence of which (l max 450 ± 470 nm) shows a largely Stokes-shifted band. Because of the existence of a twisting motion around the C2 À C1' bond of this phototautomer, the absorption and emission properties of the probe depend on the size of the host cavity. Experiments with cyclodextrins, calix [4]arene, micelles and the human protein serum albumin reveal that the emission of the sequestered phototautomer of HPMO is a simple and efficient tool for detecting and exploring the size of hydrophobic nanocavities.

Femtosecond Studies of Protein-Ligand Hydrophobic Binding and Dynamics: Human Serum Albumin

“…Second, the lifetimes change upon inclusion in CD or micelle, from a single-exponential decay in the free solvent to multiple-exponentials. This change in nonradiative decay reveals the heterogeneous local structure resulting from the anisotropic hydrophobic interactions between the probe and the surroundings (11,21,22). Third, the lifetimes in the HSA protein change differently from the blue side to the red side when compared with other environments studied (Fig.…”

“…HPMO is hydrophobic and barely dissolves in water. As a neutral species, its binding to HSA by the strong hydrophobic interactions is at site I, as discussed above in x-ray studies and by observation of enhanced fluorescence intensity upon complexation (11). Fig.…”

“…The rotational relaxation of HSA in water is on the time scale of tens of nanoseconds (11) and makes no contribution to the anisotropy of the ligand in the picosecond region. The high value of the initial anisotropy ¶ and the lack of any initial ultrafast decay indicate the strong hydrophobic attraction by molecular recognition, the hindrance of molecular ¶ The initial value of 0.33 is less than the ideal one (0.4) because the absorption and emission involve the different molecular structures of initial enol and final keto species.…”

“…[15][16][17]. The transformation of the initial enol form to the keto form results in a Stokes shift, as large as Ϸ10,000 cm Ϫ1 , observed in the steady-state emission spectrum (11,12).…”

“…The ligand HPMO was obtained as described (11). Heptakis-(2,6-di-O-methyl)-␤-cyclodextrin (dm-␤-CD), micelle of 1-O-octyl ␤-D-glucopyranoside (98%), HSA (99%), and all solvents (spectroscopy-grade) were purchased from Sigma and used as supplied.…”