Hybrid Small-Molecule/Protein Fluorescent Probes.

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid probes are constructed by diverse site-specific chemical protein labeling approaches through chemical reactions to exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemical reactions. The hybrid small-molecule/protein fluorescent probes are employed for imaging protein trafficking, conformational changes, and bioanalytes surrounding proteins. In addition, fluorescent hybrid probes facilitate visualization of protein dynamics at the single-molecule level and the defined structure with super-resolution imaging. In this review, we discuss development and the bioimaging applications of fluorescent probes based on small-molecule/protein hybrids.

Recent advancements of fluorescent biosensors using semisynthetic probes.

Fluorescent biosensors are crucial experimental tools for live-cell imaging and the quantification of different biological analytes. Fluorescent protein (FP)-based biosensors are widely used for imaging applications in living systems. However, the use of FP-based biosensors is hindered by their large size, poor photostability, and laborious genetic manipulations required to improve their properties. Recently, semisynthetic fluorescent biosensors have been developed to address the limitations of FP-based biosensors using chemically modified fluorescent probes and self-labeling protein tag/peptide tags or DNA/RNA-based hybrid systems. Semisynthetic biosensors have unique advantages, as they can be easily modified using different probes. Moreover, the self-labeling protein tag, which labels synthetically developed ligands via covalent bonds, has immense potential for biosensor development. This review discusses the recent progress in different types of fluorescent biosensors for metabolites, protein aggregation and degradation, DNA methylation, endocytosis and exocytosis, membrane tension, and cellular viscosity. Here, we explain in detail the design strategy and working principle of these biosensors. The information presented will help the reader to create new biosensors using self-labeling protein tags for various applications.

An "OFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems.

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications.

The development of near-infrared (NIR) fluorescent probes over the past few decades has changed the way that biomolecules are imaged, and thus represents one of the most rapidly progressing areas of research. Presently, NIR fluorescent probes are routinely used to visualize and understand intracellular activities. The ability to penetrate tissues deeply, reduced photodamage to living organisms, and a high signal-to-noise ratio characterize NIR fluorescent probes as efficient next-generation tools for elucidating various biological events. The coupling of self-labeling protein tags with synthetic fluorescent probes is one of the most promising research areas in chemical biology. Indeed, at present, protein-labeling techniques are not only used to monitor the dynamics and localization of proteins but also play a more diverse role in imaging applications. For instance, one of the dominant technologies employed in the visualization of protein activity and regulation is based on protein tags and their associated NIR fluorescent probes. In this mini-review, we will discuss the development of several NIR fluorescent probes used for various protein-tag systems.

Development of an effective protein-labeling system based on smart fluorogenic probes.

Proteins are an important component of living systems and play a crucial role in various physiological functions. Fluorescence imaging of proteins is a powerful tool for monitoring protein dynamics. Fluorescent protein (FP)-based labeling methods are frequently used to monitor the movement and interaction of cellular proteins. However, alternative methods have also been developed that allow the use of synthetic fluorescent probes to target a protein of interest (POI). Synthetic fluorescent probes have various advantages over FP-based labeling methods. They are smaller in size than the fluorescent proteins, offer a wide variety of colors and have improved photochemical properties. There are various chemical recognition-based labeling techniques that can be used for labeling a POI with a synthetic probe. In this review, we focus on the development of protein-labeling systems, particularly the SNAP-tag, BL-tag, and PYP-tag systems, and understanding the fluorescence behavior of the fluorescently labeled target protein in these systems. We also discuss the smart fluorogenic probes for these protein-labeling systems and their applications. The fluorogenic protein labeling will be a useful tool to investigate complex biological phenomena in future work on cell biology.

Fluorescent probes for hydrogen polysulfides (H2Sn, n > 1): from design rationale to applications.

Hydrogen polysulfides (H2Sn, n > 1) being active members of the reactive sulphur species family are gaining much research interest these days due to their involvement in signal transduction, anti-cancer activity and cytoprotection. The importance of H2Sn particularly in human health makes it desirable to explore selective and sensitive techniques for their detection. Out of the several techniques and tools available, fluorescence spectroscopy seems advantageous due to its high sensitivity, non-destructive nature and applicability to living systems. Remarkable progress has been made in this field and different approaches have been adopted for the selective detection of H2Snin vitro as well as in vivo. The present review discusses the importance of H2Sn in living systems and highlights recent advances in the design of fluorescent probes for the detection of H2Sn. It offers a critical overview of the designing strategies adopted for H2Sn detection with emphasis on various ways for achieving selectivity over other reactive sulphur species (RSS) and their applications in biological systems. Further, the fundamental challenges and future prospects in this field are also discussed.

A Highly Selective Fluorescent Probe for Detection of Hydrogen Sulfide in Living Systems: In Vitro and in Vivo Applications.

A fluorescein-based fluorescent probe has been designed and synthesised that selectively detects H2 S in aqueous medium, among various analytes tested. This fluorescein-based fluorescent probe has also been successfully utilised for real-time imaging of exo- and endogenously produced H2 S in cancer cells and normal cells. Moreover, the probe can also detect H2 S in the rat brain hippocampus at variable depths and in living nematodes.

A lysosome targetable fluorescent probe for endogenous imaging of hydrogen peroxide in living cells.

A lysosome targetable naphthalimide based fluorescent probe (LyNC) has been designed and synthesized which detects hydrogen peroxide (H2O2) with high selectivity and sensitivity in brain tissues and in living nematodes among various ROS/RNS tested. Further, the probe LyNC was successfully employed in exogenous and endogenous imaging of H2O2 in living cell lines.

A highly selective fluorescent probe for Fe3+ in living cells: a stress induced cell based model study.

A rhodamine-phenanthroline dyad based fluorescent probe 4 has been designed and synthesized which selectively monitors Fe3+ ions among the various metal ions tested. Furthermore, probe 4 has been explored for monitoring of dynamic changes in the Fe3+ ion level under aggressive Fenton reaction conditions using a cell based model study. More significantly, probe 4 has also been utilized for real time imaging of endogenous Fe3+ ions in living C6 cell lines, the results of which demonstrated that probe 4 acts as an efficient fluorescent tool for Fe3+ ion detection in biological systems.

A TICT based NIR-fluorescent probe for human serum albumin: a pre-clinical diagnosis in blood serum.

A TICT based NIR-fluorescent probe 3 has been designed and synthesized, which selectively detects HSA with fluorescence enhancement in blood plasma with a detection limit of 11 nM among the various proteins, nucleotides and thiols tested.

A bodipy based fluorescent probe for evaluating and identifying cancer, normal and apoptotic C6 cells on the basis of changes in intracellular viscosity.

The applications of a bodipy based probe 1 for the identification of diseased cell population out of normal cells on the basis of changes in intracellular viscosity have been explored. Probe 1 works on the principle of restriction of rotation in viscous medium and the molecular rotor nature of probe 1 is supported by low temperature 1H NMR and variable dihedral angle DFT and TD-DFT studies. More importantly, probe 1 is the first probe which shows its practical application in monitoring micro-viscosity changes in a cell based model system of undifferentiated, differentiated and apoptotic C6 glial cells. Further, probe 1 can effectively monitor the apoptosis pathway by showing an increase in fluorescence intensity from cancerous cells to apoptotic cells via real time live-cell video imaging. Moreover, the viscosity changes in living cells were proved by fluorescence lifetime imaging (FLIM) studies, flow cytometry using Annexin-V and Bcl-xl expression by immunocytofluorescence (ICC) and western blot analysis.

A bodipy based dual functional probe for the detection of hydrogen sulfide and H2S induced apoptosis in cellular systems.

A bodipy based dual functional probe 1 has been designed and synthesized, which selectively detects H2S as well as monitors H2S induced apoptosis in cells.

A thiacalix[4]crown based chemosensor for Zn²âº and H2PO4⁻: sequential logic operations at the molecular level.

A thiacalix[4]crown based di-topic receptor 3 possessing two types of binding sites viz. crown-5 ring and imino moieties has been synthesized which undergoes fluorescence enhancement in the presence of Zn(2+) ions. The selective binding of Zn(2+) to compound 3 does not allow the K(+) ions to bind with the crown-5 ring and thus a negative allosteric behaviour has been observed between Zn(2+)/K(+) ions. In addition, the 3-Zn(2+) complex can be used for the detection of H2PO4(-) ions with a fluorescence "turn-off" response. Furthermore, based on the fluorescence response, a two input and one output sequential logic circuit has been constructed.

A new thiacalix[4]arene-fluorescein based probe for detection of CN(-) and Cu(2+) ions and construction of a sequential logic circuit.

A new thiacalix[4]arene-fluorescein based fluorescent probe was synthesized, which shows a turn-on fluorescence response in the presence of CN(-) ions attributed to the nucleophilic addition of cyanide ions and the resulting cyanide adduct was used for the selective detection of copper ions. Furthermore, based on the fluorescence response a two input, one output, sequential logic circuit was constructed in the presence of CN(-) and Cu(2+) ions.

A highly selective fluorescent probe for hypochlorite and its endogenous imaging in living cells.

An oxime based fluorescent probe has been designed and synthesized, which detects free as well as enzymatically generated hypochlorite with a low detection limit and high sensitivity. In addition, the probe was successfully utilized for the monitoring of endogenously produced hypochlorite in LPS stimulated cell lines, C6 glioma and BV2 microglia.

Synthesis, and the antioxidant, neuroprotective and P-glycoprotein induction activity of 4-arylquinoline-2-carboxylates.

An efficient formic acid catalyzed one-pot synthesis of 4-arylquinoline 2-carboxylates in water via three-component coupling of arylamines, glyoxylates and phenylacetylenes has been described. 4-Arylquinoline 2-carboxylates 1o and 1q displayed significant antioxidant activity as indicated by their Fe-reducing power in the ferric reducing ability of plasma (FRAP) assay. The compounds were found to react directly with hydrogen peroxide, which might be one of the mechanisms of their antioxidant effect. Compounds 1o and 1q effectively quenched H2O2 and amyloid-ß-generated reactive oxygen species (ROS) and also displayed significant protection against H2O2-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Additionally, all compounds exhibited promising P-glycoprotein induction activity in human adenocarcinoma LS-180 cells, indicating their potential to enhance amyloid-ß clearance from Alzheimer's brains. Furthermore, all compounds were relatively non-toxic to SH-SY5Y and LS-180 cells (IC50 > 50 µM). The promising antioxidant, ROS quenching, neuroprotective and Pgp-induction activity of these compounds strongly indicate their potential as anti-Alzheimer's agents.

A charge transfer amplified fluorescent Hg2+ complex for detection of picric acid and construction of logic functions.

A chemosensor 3 based on the N,N-dimethylaminocinnamaldehyde has been synthesized which shows fluorescence turn-on response with Hg(2+) ions, and the in situ prepared 3-Hg(2+) complex has been used for detection of picric acid via electrostatic interaction and construction of a combinatorial logic circuit with NOR and INHIBIT logic functions.