Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores

Abstract

There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)thiophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes. Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly, both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and second harmonic generation, which are themselves environmentally sensitive. The combination of long wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for applications that require sensing or imaging deep inside tissues.

FIGURE 1

Absorption and emission spectra of 1d in different solvent environments. The emission curves in ethanol and PBS buffer have been scaled 10 and 100 times respectively. Here lipid vesicles prepared from soybean phosphatidylcholine are used to represent the environment of cell membranes. Excitation wavelength: 480 nm.

FIGURE 2

Normalized absorption and emission spectra of a series of dyes with different number of thienylene units, measured in vesicle suspensions containing 1 mg/mL soybean phosphatidylcholine in PBS buffer.

FIGURE 3

Normalized absorption and emission spectra of a series of dyes with different acceptors, measured in vesicle suspensions containing 1 mg/mL soybean phosphatidylcholine in PBS buffer.

FIGURE 4

Emission spectra of 1b and 1d in vesicle suspensions prepared from DOPC or 7:3 DPPC/cholesterol in PBS buffer. Lipid concentration = 0.017 mg/mL.

FIGURE 5

(A) SHG, (B) 2PF images of the same undifferentiated neuroblastoma cell; (C) SHG, (D) 2PF, and (E) merged images of differentiated neuroblastoma cells. The samples were stained with 5 μM 1d and excited at 1064 nm. A bandpass filter (615–665 nm) was used in 2PF imaging. SHG was collected in the transmitted light path at 532 nm.

SCHEME 1

A general route to amino(oligo)thiophene-based hemicyanines

SCHEME 2

Synthesis of a bridged hemicyanine

CHART 1

Structures of Indocyanine green (ICG), di-4-ANEPPS, and amino(oligo)thiophene-based hemicyanines (1–4).