The long wavelength (far-red to NIR) analyte-responsive fluorescent probes are advantageous for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems. Thus, great interest in the development of new long wavelength analyte-responsive fluorescent probes has emerged in recent years. This review highlights the advances in the development of far-red to NIR fluorescent probes since 2000, and the probes are classified according to their organic dye platforms into various categories, including cyanines, rhodamine analogues, BODIPYs, squaraines, and other types (240 references).

Coumarins are a very large family of compounds containing the unique 2H-chromen-2-one motif, as it is known according to IUPAC nomenclature. Coumarin derivatives are widely found in nature, especially in plants and are constituents of several essential oils. Up to now, thousands of coumarin derivatives have been isolated from nature or produced by chemists. More recently, the coumarin platform has been widely adopted in the design of small-molecule fluorescent chemosensors because of its excellent biocompatibility, strong and stable fluorescence emission, and good structural flexibility. This scaffold has found wide applications in the development of fluorescent chemosensors in the fields of molecular recognition, molecular imaging, bioorganic chemistry, analytical chemistry, materials chemistry, as well as in the biology and medical science communities. This review focuses on the important progress of coumarin-based small-molecule fluorescent chemosensors during the period of 2012− 2018. This comprehensive and critical review may facilitate the development of more powerful fluorescent chemosensors for broad and exciting applications in the future.

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.

Fluorescence imaging has emerged as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution. Researchers have constructed a large number of synthetic intensity-based fluorescent probes for bio-imaging. However, intensity-based fluorescent probes have some limitations: variations in probe concentration, probe environment, and excitation intensity may influence the fluorescence intensity measurements. In principle, the use of ratiometric fluorescent probes can alleviate this shortcoming. Förster resonance energy transfer (FRET) is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. However, the development of synthetic FRET probes with favorable photophysical properties that are also suitable for biological imaging applications remains challenging. In this Account, we review the rational design and biological applications of synthetic FRET probes, focusing primarily on studies from our laboratory. To construct useful FRET probes, it is a pre-requisite to develop a FRET platform with favorable photophysical properties. The design criteria of a FRET platform include (1) well-resolved absorption spectra of the donor and acceptor, (2) well-separated emission spectra of the donor and acceptor, (3) donors and acceptors with comparable brightness, (4) rigid linkers, and (5) near-perfect efficiency in energy transfer. With an efficient FRET platform in hand, it is then necessary to modulate the donor-acceptor distance or spectral overlap integral in an analyte-dependent fashion for development of FRET probes. Herein, we emphasize our most recent progress on the development of FRET probes by spectral overlap integral, in particular by changing the molar absorption coefficient of the donor dyes such as rhodamine dyes, which undergo unique changes in the absorption profiles during the ring-opening and -closing processes. Although partial success has been obtained in design of first-generation rhodamine-based FRET probes via modulation of acceptor molar absorption coefficient, further improvements in terms of versatility, sensitivity, and synthetic accessibility are required. To address these issues with the first-generation rhodamine-based FRET probes, we have proposed a strategy for the design of second-generation probes. As a demonstration, we have developed FRET imaging probes for diverse targets including Cu²⁺, NO, HOCl, cysteine, and H₂O₂. This discussion of the methods for successfully designing synthetic FRET probes underscores the rational basis for further development of new FRET probes as a molecular toolbox for probing and manipulating a wide variety of biomolecules in living systems.