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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.

By connection of O(6)-benzylguanine (BG) to an "o-phenylenediamine-locked" rhodamine spirolactam responsive to nitric oxide (NO), a novel substrate (TMR-NO-BG) of genetically encoded SNAP-tag has been constructed. In living cells, labeling SNAP-tag fused proteins with TMR-NO-BG will in situ generate corresponding probe-protein conjugates (TMR-NO-SNAP) that not only inherit high NO sensitivity from the small-molecule parent but also guarantee the site-specificity to the designated subcellular compartments such as the mitochondrial inner membrane, nucleus, and cytoplasm. In two representative cellular processes, TMR-NO-BG demonstrates its applicability to monitor endogenous subcellular NO in the activated RAW264.7 cells stimulated by lipopolysaccharide and in the apoptotic COS-7 cells induced by etoposide.

We have developed two near-infrared fluorescent probes for pH, named pH-A and pH-B. The fluorescence quantum yields of pH-A and pH-B are 0.19 and 0.095 in methanol. And there is the neutral pKa value of pH-A (pKa = 7.2). What is the most important is that these probes can stain the cells, which can reflect the changes of pH in cells.

Genetically encoded technologies provide methods for specific labeling and imaging of proteins, which is essential for the understanding of their subcellular localization and function. Herein, we employ naphthalimide, an efficient two-photon fluorophore, to develop O6-benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP-tag. The three naphthalimide-BG derivatives, TNI-BG, QNI-BG and ONI-BG are conveniently synthesized through modular "click chemistry" with high yields. All of them show high labeling efficiency with SNAP-tag in solution (1~2 ×103 s-1 M-1) and in bacteria. Among them, ONI-BG shows high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP-tag in mammalian cells. The protein labeled naphthalimides exhibit high two-photon absorption cross section, which indicates the potential application in protein specific two-photon fluorescent imaging, such as two-photon fluorescent lifetime imaging and two-photon multi-color imaging. Therefore, ONI-BG is a versatile tool for tracking of subcellular proteins via multiple fluorescent techniques.