Push-pull dioxaborine as fluorescent molecular rotor: far-red fluorogenic probe for ligand-receptor interactions

Abstract

Fluorescent solvatochromic dyes and molecular rotors increase their popularity as fluorogenic probes for background-free detection of biomolecules in cellulo in no-wash conditions. Here, we introduce a push-pull boron-containing (dioxaborine) dye that presents unique spectroscopic behavior combining solvatochromism and molecular rotor properties. Indeed, in organic solvents, it shows strong red shifts in the absorption and fluorescence spectra upon increase in solvent polarity, typical for push-pull dyes. On the other hand, in polar solvents, where it probably undergoes Twisted Intramolecular Charge Transfer (TICT), the dye displays strong dependence of its quantum yield on solvent viscosity, in accordance to Förster-Hoffmann equation. In comparison to solvatochromic and molecular rotor dyes, dioxaborine derivative shows exceptional extinction coefficient (120,000 M^-1 cm^-1), high fluorescence quantum yields and red/far-red operating spectral range. It also displays much higher photostability in apolar media as compared to Nile Red, a fluorogenic dye of similar color. Its reactive carboxy derivative has been successfully grafted to carbetocin, a ligand of the oxytocin G protein-coupled receptor. This conjugate exhibits >1000-fold turn on between apolar 1,4-dioxane and water. It targets specifically the oxytocin receptor at the cell surface, which enables receptor imaging with excellent signal-to-background ratio (>130). We believe that presented push-pull dioxaborine dye opens a new page in the development of fluorogenic probes for bioimaging applications.

Fig. 1

Structure and rotor properties of dioxaborine molecular rotors. (a) Structure of the molecular rotor DXB Red. (b) Fluorescence response of the molecular rotor DXB Red to the change of the environment from fluid (MeOH, black) to viscous (glycerol, red). (c) Structure of the carbetocin conjugate DXB-CBT.

Fig. 2

Normalized absorption (a) and fluorescence (b) spectra of DXB Red (1 μM) in the solvents of different polarity. Excitation wavelength was 540 nm.

Fig. 3

Photostability of DXB Red and of Nile Red in toluene. Excitation wavelength was 525 nm; emission was detected at 600 nm; illumination power density was ~1 mW cm-²; concentration of dyes was 200 nM.

Fig. 4

Solvatochromic and fluorogenic properties of DXB Red. (a) Position of emission maxima of DXB Red and Nile Red versus E_T(30) in aprotic solvents. The slope of the linear fits for DXB Red and Nile Red are 156 and 119 cm^-1, respectively, and r² value (goodness of fits) are 0.85 and 0.93, respectively. (b) Dependency of fluorescence quantum yield on solvent polarity. Red circle and blue triangle represent ethylene glycol and glycerol, respectively. (c) Fluorescence quantum yield as a function of water content in dioxane.

Fig. 5

(a) Fluorescence spectra of DXB Red in binary mixtures of methanol and glycerol of different ratio. (b) Correlation of the fluorescence quantum yield with the viscosity of the medium according to the equation LogΦ_F = C + xlogη (Förster-Hoffmann equation, where Φ_F is fluorescence quantum yield, η is solvent viscosity, x is dye-dependent constant and C is concentration and temperature dependent constant). The slope of the linear fits is 0.606, and r² value (goodness of fits) is 0.99.

Scheme 1

Synthesis of DXB-CBT.

Fig. 6

Fluorogenic behavior or DXB-CBT in solvents. Absorption (a) and fluorescence (b) spectra of DXB-CBT (1 µM) in water and 1,4-dioxane. Inset shows the zoomed fluorescence spectrum of DXB-CBT in water of negligibly low intensity. Excitation wavelength was 560 nm for both solvents.

Fig. 7

Confocal microscopy studies. Confocal images of OTR cells with 10 nM of DXB-CBT (a), 10 nM of DXB-CBT and 2 µM of CBT competitor (b), or 100 nM of DXB-CBT (c). Fluorogenic properties of DXB-CBT: average membrane and background fluorescence for all the images (d). DXB-CBT was incubated with cells 5 min before imaging. Laser excitation was done at 561 nm and the emission was collected at 575-750 nm interval.