Development and applications of a near-infrared dye–benzylguanine conjugate to specifically label SNAP-tagged proteins

Abstract

Near-infrared (NIR) fluorescent probes are advantageous over visible ones, for they can avoid the interference from the short-wavelength background emission in biological systems. However, there are a very limited number of NIR probes that can specifically label target proteins in living cells. In this work, a series of long-wavelength dyes (N-NIR, S-NIR, and K-NIR) analogous to the novel Changsha NIR family are synthesized conveniently through a new approach that is different from the previously reported one. These three dyes have similar conjugation structures but exhibit tunable photophysical properties. N-NIR and S-NIR have large extinction coefficients over 100 000, and high fluorescence quantum yields. Although NIR absorption and emission of K-NIR are inferior to the former two, it emits in a much longer wavelength region. And all the three dyes can easily pass through the cell membranes to obtain the high-resolution NIR fluorescence images. Furthermore, N-NIR is chosen as the NIR fluorophore to develop a protein-labeling reagent PYBG-D, since it demonstrates the highest fluorescence quantum yield of up to 0.4 (in methanol). PYBG-D is efficiently synthesized through Sonogashira coupling between bromo-substituted N-NIR and alkyne-substituted benzylguanine (PYBG). The conjugate PYBG-D proves to be a specific and efficient label for O⁶-alkylguanine-DNA alkyltransferase (SNAP-tag) that fused to target proteins in living cells, which contributes to high resolution NIR fluorescence images under a laser confocal microscope.