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Formaldehyde (FA), a reactive carbonyl species, is classified as Group 1 carcinogen by International Agency for Research on Cancer (IARC) in 2004. In addition, clinical studies have implicated that elevated levels of FA have been associated with different kinds of diseases, such as neurodegenerative diseases, diabetes, and chronic liver and heart disorders. However, in addition to the direct inhalation of FA in the environment, most organisms can also produce FA endogenously by demethylases and oxidases during the metabolism of amino acids and xenobiotics. Since FA plays an important role in physiological and pathological processes, developing reliable and efficient methods to monitor FA levels in biological samples is crucial. Reaction-based fluorescent/chemiluminescent probes have provided robust methods for FA detection and real-time visualization in living organisms. In this highlight, we will summarize the major developments in the structure design and applications of FA probes in recent years. Three main strategies for designing FA probes have been discussed and grouped by different reaction mechanisms. In addition, some miscellaneous reaction mechanisms have also been discussed. We also highlight novel applications of these probes in biological systems, which offer powerful tools to discover the diverse functions of FA in physiology and pathology processes. [ABSTRACT FROM AUTHOR]

Fluorescent stapled peptides are important chemical tools for detecting intracellular distribution, protein–protein interactions, and localization of target proteins. These peptides are usually labeled with bulky sized fluorophores through reactive functional groups, which may alter the physical properties and biological activities of peptides. Herein, a unique strategy is developed for synthesizing new stapled peptides with built‐in fluorescence. The stapled peptides were prepared through Rh‐catalyzed C(sp2)−H olefination in tryptophan (Trp) residues by using pyridine/pyrimidine as the directing groups under mild conditions. This method displays good regioselectivity and high efficiency. Furthermore, as a proof of concept for its biological applications, stapled peptides without additional fluorophore 9 a and 9 b were constructed for a cell imaging study. These peptides displayed strong binding affinity toward integrin αvβ3 in A549 cells by cell imaging experiments. Notably they demonstrated even better anticancer activity than commercial antagonist cyclic (RGDfK). The method will provide robust tools for the peptide macrocyclization field. [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of important biological processes such as cell differentiation and bone mineralization. Monitoring the activity of ALP in serum can help diagnose a variety of diseases including bone and liver diseases. There has been growing interest in developing new chemical tools for monitoring ALP activity in living systems. Such tools will help further delineate the roles of ALP in biological and pathological processes. Previously reported fluorescent probes has a number of disadvantages that limit their application, such as poor selectivity and short-wavelength excitation. In this work, we report a new two-photon fluorescent probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It offers a number of advantages over previously reported probes, such as fast reaction kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe. The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the probe and the active site of phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging properties such as increased penetration depth and lower tissue autofluorescence. TP-Phos was successfully used to image the endogenous ALP activity of hippocampus, kidney and liver tissues from rat. [ABSTRACT FROM AUTHOR]

Thiophenols are widely utilized for producing pesticides, polymers and pharmaceuticals. Studies have shown that prolonged exposure of human to thiolphenol can result in serious health problems such as significant damage of the central nervous system. Herein, we have developed a novel turn-on two-photon fluorescent probe TP-ASH for selective and sensitive detection of thiophenols. We first constructed a new two-photon dye by connecting a coumarin fluorophore and a naphthalene fluorophore through 2,5- disubstituted-1,3,4-oxadiazole ring to afford d -π-A-π- d -structure. Fluorescent studies showed that this new dye possess large two-photon active absorption across-section (250 GM) and high fluorescence quantum yield (0.85). Encouraged by the results, we proceeded to conjugate this novel dye to a thiophenol recognition moiety, 4-dinitrobenzene-ether (DNB). The resulting probe displayed high selectivity towards thiophenols over their similar analogues, aliphatic thiols. The probe also exhibited high sensitivity, with a detection limit of 6.7 nM and a fluorescence enhancement of 163-fold. In addition, we have successfully applied the probe for two-photon imaging studies of thiphenols in living cells and mice tissues. We envision that the probe will add useful tools in detecting thiophenols in environmental samples and biological systems. [ABSTRACT FROM AUTHOR]

We report a novel turn-on two-photon fluorescent probe ( TPNF ) for detecting formaldehyde based on aza-Cope rearrangement and intramolecular charge transfer (ICT). The probe, containing a 1,8-naphthalimide dye with a homoallylamino group, has was shown with high selectivity, sensitivity and low cytotoxicity and it was capable of detecting formaldehyde in live cells. Moreover, a time-dependent density functional theory (TD-DFT) calculation was performed to demonstrate the turn on mechanism of the probe via ICT effect. Finally, a zebrafish larvae imaging experiment reveals that TPNF displayed a significant signal change when treated with FA in vivo. These results indicated that TPNF is a promising tool in more biomedical researches. [ABSTRACT FROM AUTHOR]

Intracellular pH plays a pivotal role in various biological processes. Herein, we adopted the through bond energy transfer (TBET) strategy to design a unique type of ratiometric two-photon fluorescent probe for imaging of lysosomal pH in live cells and tissues, termed Np-Rh-Lys. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore were directly connected, and dimethylamino moiety served as a lysosomal targeting-group. The experiments demonstrate new probe Np-Rh-Lys was a reliable and specific probe for labeling lysosomes in living cells with two well-resolved emission peaks separated by 80 nm, and which showed high ratiometric imaging resolution and deep-tissue imaging depth over 180 μm, we thus may expect the new platform prompt the development of a wide variety of ratiometric two-photon fluorescent probes application in biological systems. [ABSTRACT FROM AUTHOR]

In contrast to other sirtuins (NAD+-dependent class III lysine deacylases), inhibition of Sirt5 is poorly investigated, yet. Our present work is based on the recently identified Sirt5 inhibitor balsalazide, an approved drug with negligible bioavailability after oral administration. After gaining first insights into its structure-activity relationship in previous work, we were able to now develop heteroaryl-triaryls as a novel chemotype of drug-like, potent and subtype-selective Sirt5 inhibitors. The unfavourable azo group of the lead structure was modified in a systematic and comprehensive manner, leading us to a few open-chained and, most importantly, five-membered heteroaromatic substitutes (isoxazole CG_209 , triazole CG_220 , pyrazole CG_232) with very encouraging in vitro activities (IC 50 on Sirt5 in the low micromolar range, <10 μM). These advanced inhibitors were free of cytotoxicity and showed favourable pharmacokinetic properties, as confirmed by permeability into mitochondria using live cell imaging experiments. Furthermore, results from calculations of the relative free binding affinities of the analogues compared to balsalazide as reference compound agreed well with the trends for inhibitory activities obtained in the in vitro experiments. Therefore, this method can be used to predict the affinity of closely related future potential Sirt5 inhibitors. Encouraged by our findings, we employed chemoproteomic selectivity profiling to confirm Sirt5 as main target of balsalazide and one of its improved analogues. An immobilised balsalazide-analogue specifically pulled down Sirt5 from whole cell lysates and competition experiments identified glutaryl-CoA dehydrogenase (GCDH) and nucleotide diphosphate kinase (NME4) as potential off-targets, once again confirming the selectivity of the novel balsalazide-derived Sirt5 inhibitors. In summary, a combination of targeted chemical synthesis, biological work, and computational studies led to a new generation of tailored Sirt5 inhibitors, which represent valuable chemical tools for the investigation of the physiological role of Sirt5, but could also serve as advanced lead structures for drug candidates for systemic use. [Display omitted] • Systematic variation of balsalazide gave potent, subtype-selective Sirt5 inhibitors. • Replacement of the azo group by five-membered heteroarenes gave drug-like properties. • Chemoproteomic selectivity profiling confirmed Sirt5 as main target of balsalazide. • Two additional target proteins of balsalazide and ist analogues were identified. [ABSTRACT FROM AUTHOR]

In this study, Cu(II)-mediated differential alteration of cysteine (Cys) reactivity is reported by using a Cys-specific fluorescent probe. The probe could react with Cys to give out strong fluorescence. When Cys was preincubated with Cu(II), the fluorescence of the probe was decreased due to the inhibition of Cys's reactivity by Cu(II). Remarkably, experimental results reveal that the probe could detect Cu(II) at subnanomolar concentrations. In contrast, Cu(II) could only partially inhibit the reaction between Cys and Ellman's reagent (DTNB). Furthermore, selectivity experiments show that Cu(II) is a much more potent inhibitor for Cys compared to other metal ions. Cell imaging experiments also confirm the inhibitory effects of Cu(II) on Cys's reactivity in living cells. We envision that the probe could add a useful tool for sensitive and selective detection of Cu(II) for biomedical research. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S) has recently been identified as the third gaseous signaling molecule that is involved in regulating many important cellular processes. We report herein a novel fluorescent probe for detecting H 2 S based on iminocoumarin benzothiazole scaffold. The probe displayed high sensitivity and around 80-fold increment in fluorescence signal after reacting with H 2 S under physiological condition. The fluorescent intensity of the probe was linearly related to H 2 S concentration in the range of 0–100 μM with a detection limit of 0.15 μM (3σ/slope). The probe also showed excellent selectivity towards H 2 S over other biologically relevant species, including ROS, RSS and RNS. Its selectivity for H 2 S is 32 folds higher than other reactive sulfur species. Furthermore, the probe has been applied for imaging H 2 S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H 2 S concentrations in living cells. We envisage that this probe can provide useful tools to further elucidate the biological roles of H 2 S. [ABSTRACT FROM AUTHOR]

• A robust strategy for probe design with dual-quenching groups in one probe. • The strategy has been applied for designing HOCl fluorescent probe successfully. • The strategy can both enhance the probe's sensitivity and selectivity significantly. • The detection limit for HOCl is as low as 3.5 nM. • Two-photon probe has been used to detect HOCl in living cells and kidney tissues. We report herein fluorescent probes equipped with dual-quenching groups exhibiting superior sensitivity than probes with mono-quenching groups. Importantly, with this strategy, the probe with dual-quenching groups react with HOCl through two distinct reaction mechanisms, which reduce the plausible side reactions with other competing analytes and enhance the probe's selectivity. As a proof-of-concept study, we designed and synthesized a probe with dual-quenching groups DQ-HOCl to detect HOCl, which is one of the most important ROS and linked with a number of diseases. In addition, two control probes with mono-quenching groups, MQ-HOCl-1 and MQ-HOCl-2 , were also synthesized for comparison purpose. Fluorescent assays demonstrated that DQ-HOCl indeed shows ultra-high sensitivity and selectivity compared with probes with mono-quenching groups. Furthermore, the probe has been successfully utilized to imaging exogenous/endogenous HOCl in living cells. Moreover, DQ-HOCl was applied to visualize HOCl in kidney tissues from rat due to the increased penetration depth and lower tissue autofluorescence from the nature of two-photon probes. [ABSTRACT FROM AUTHOR]