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This tutorial review provides a general overview for the design of prodrugs and activatable phototherapeutics which enables the development of improved therapies.

We would like to welcome you to the wonderful world of fluorescent chemosensors. This book consists of 16 chapters that take you on a journey from receptor-based to reaction-based sensing systems. The quest includes both molecular-based and nanoparticle-based systems that can be used for medical diagnostics, theranostics and environmental sensing applications. The techniques included embrace two-photon fluorescence, ratiometric and aggregation-induced emission.

The occurrence of endoplasmic reticulum (ER) stress is the main cause of a variety of biological processes that are closely related to numerous diseases. The homeostasis of the ER microenvironment can be disrupted under ER stress. In this research, by linking a pentafluorophenyl to the green fluorescent protein chromophore, we have developed a new ER-targeting fluorescent probe (GE-Y) for measuring changes of intracellular ER viscosity caused by ER stress. Importantly, an increase in ER viscosity was observed using GE-Y in cells undergoing autophagy. As such, our research provides an ideal tool for studying ER stress and autophagy.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO–) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO– and ATP. ONOO– selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO– and ATP products, ATP-LW allows ONOO– levels to be monitored in the green channel (λex = 488 nm, λem = 500–575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575–650 nm). The use of ATP-LW as a combined ONOO– and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO– donor).

Background PD-1 antibodies have been approved for treating several cancers. However, data regarding the safety and efficacy of these agents in HIV-infected patients with cancers is lacking, because these patients are frequently omitted from clinical trials. Objectives The primary aim of our research is to assess the safety, activity, and long-term outcomes of PD-1 inhibitors in the treatment of HIV-infected patients with advanced cancers. Method We retrospectively analyzed data from HIV-infected patients with advanced cancers who were treated with PD-1 inhibitors at Shanghai Public Health Clinical Center, Shanghai, China. Results Fifteen HIV-infected patients (all are men; yellow race; median age, 44)with cancers who were treated with chemotherapy and/or combined the other oncology treatments (along with combined antiretroviral therapy (cART)) prior to Sintilimab (12 out of 15) or Nivolumab (1 out of 11) or Camrelizumab (2 out of 11) injection were identified. Eight patients responded to treatment (disease control rate 53.3%), with 1 got partial response (PR) and 7 were stable. Most treatment-emergent adverse events (TEAEs) were grade 1 or 2including anemia, leukopenia, hyperglycemia, granulocytopenia, and thrombocytopenia. Eight patients (53.3%) experienced treatment-related AEs (TRAEs) with grades 3/4including myelosuppression, infection, and neurological disorders. CD4 count and HIV RNA remained stable throughout the treatment. Conclusions When used in HIV-infected patients with advanced malignancies, PD-1 inhibitors tend to have favorable efficacy, manageable side effects, and no deteriorated impacts on HIV-RNA and CD4+ T count.

Vicinal dithiol-containing proteins (VDPs) are of considerable importance due to their role in regulating cellular functions through the reversible dithiol/disulfide inter-conversion reaction. However, there are currently no effective tools for the simultaneous tracing of endogenous VDPs in live cells. Here, we report an “off-on” fluorescent probe (RhQ) for the selective detection of VDPs and illustrate its utility for the long-term tracing of protein vicinal dithiols with simultaneous fluorescence imaging in various cell lines. Importantly, our study provides the first-time valuable insight into the localization of VDPs in whole cells, dynamically visualizing that presence of cell-surface protein vicinal dithiols in MCF-7 cells. Interestingly, some functional cell-surface VDPs have been reported for MCF-7 cells using proteomic analysis. Furthermore, the gradually released fluorescence from RhQ after its interaction with VDPs provides an overall picture of the cellular protein vicinal dithiol profile for different cell lines. This technology shows great promise as a visualization tool for revealing the role of cellular protein vicinal dithiols, especially VDPs on the cell-surface, and stimulating the design of VDP related drug candidates and vectors.

Optical imaging probes allow us to detect and uncover the physiological and pathological functions of an analyte of interest at the molecular level in a non-invasive, longitudinal manner. By virtue of simplicity, low cost, high sensitivity, adaptation to automated analysis, capacity for spatially resolved imaging and diverse signal output modes, optical imaging probes have been widely applied in biology, physiology, pharmacology and medicine. To build a reliable and practically/clinically relevant probe, the design process often encompasses multidisciplinary themes, including chemistry, biology and medicine. Within the repertoire of probes, dual-locked systems are particularly interesting as a result of their ability to offer enhanced specificity and multiplex detection. In addition, chemiluminescence is a low-background, excitation-free optical modality and, thus, can be integrated into dual-locked systems, permitting crosstalk-free fluorescent and chemiluminescent detection of two distinct biomarkers. For many researchers, these dual-locked systems remain a ‘black box’. Therefore, this Review aims to offer a ‘beginner’s guide’ to such dual-locked systems, providing simple explanations on how they work, what they can do and where they have been applied, in order to help readers develop a deeper understanding of this rich area of research. Dual-locked optical probes change their optical signals when they respond to two biomarkers of interest. This facilitates real-time imaging of multiple interrelated biomarkers in living systems and, thus, provides opportunities to better understand pathological events and enhanced diagnostic specificity.

Hepatic ischemia-reperfusion injury (HIRI) is responsible for postoperative liver dysfunction and liver failure. Precise and rapid navigation of HIRI lesions is critical for early warning and timely development of pretreatment plans. Available methods for assaying liver injury fail to provide the exact location of lesions in real time intraoperatively. HIRI is intimately associated with oxidative stress which impairs lysosomal degradative function, leading to significant changes in lysosomal viscosity. Therefore, lysosomal viscosity is a potential biomarker for the precise targeting of HIRI. Hence, we developed a viscosity-activatable second near-infrared window fluorescent probe (NP-V) for the detection of lysosomal viscosity in hepatocytes and mice during HIRI. A reactive oxygen species-malondialdehyde-cathepsin B signaling pathway during HIRI was established. We further conducted high signal-to-background ratio NIR-II fluorescence imaging of HIRI mice. The contour and boundary of liver lesions were delineated, and as such the precise intraoperative resection of the lesion area was implemented. This research demonstrates the potential of NP-V as a dual-functional probe for the elucidation of HIRI pathogenesis and the direct navigation of HIRI lesions in clinical applications.

Viscosity is a pivotal factor for indicating the dysfunction of the mitochondria. To date, most of the fluorescent probes developed for mitochondrial viscosity have been designed using BODIPY, hemicyanine, or pyridine-based molecular rotors as part of the core structure. Our aim with this research was to extend the range of suitable fluorophores available for the construction of such fluorescent molecular rotors for evaluating the viscosity of mitocondria. Herein, we have developed a green fluorescent protein (GFP)-chromophore-based fluorescent probe (MIT-V) for the detection of mitochondrial viscosity in live cells. MIT-V exhibited a high sensitivity toward viscosity (from 7.9 cP to 438.4 cP). The "off-on"sensing mechanism of MIT-V was ascribed to the restricted rotation of single bonds and excited-state C= C double bonds of MIT-V. Cell studies indicated that MIT-V targets the mitochondria and that it was able to monitor real-time changes in the viscosity of live HeLa cell mitochondria. Therefore, we propose that MIT-V can be used as an effective chemosensor for the real-time imaging of mitochondrial viscosity in live cells. Our results clearly demonstrate the utility of such GFP-chromophore-based derivatives for the development of viscosity-sensitive systems.

In this tutorial review, we will explore recent advances for the design, construction and application of two-photon excited fluorescence (TPEF)-based small-molecule probes. The advantages of TPEF-based probes include deep tissue penetration and minimal photo-damage. We discuss the underlying two-photon (TP) fluorophores including hemicyanine and design strategies such as Förster resonance energy transfer (FRET). Moreover, we emphasize applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvironments, subcellular localization and dual-responsive systems. Examples of photodynamic therapy (PDT) using TP irradiation are also illustrated.

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems. With this perspective we highlight sensing mechanisms including Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited state intramolecular proton transfer (ESIPT), aggregation induced emission (AIE) and multiple modality fluorescence approaches including dual/triple sensing mechanisms (DSM or TSM). Throughout the perspective we highlight the remaining challenges and suggest potential directions for development towards improved small-molecule fluorescent probes suitable for biosensing., Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems.

Drug-induced liver injury (DILI) is an important cause of potentially fatal liver disease. Herein, we report the development of a molecular probe (LW-OTf) for the detection and imaging of two biomarkers involved in DILI. Initially, primary reactive oxygen species (ROS) superoxide (O2˙−) selectively activates a near-infrared fluorescence (NIRF) output by generating fluorophore LW-OH. The C Created by potrace 1.16, written by Peter Selinger 2001-2019 C linker of this hemicyanine fluorophore is subsequently oxidized by reactive nitrogen species (RNS) peroxynitrite (ONOO−), resulting in cleavage to release xanthene derivative LW-XTD, detected using two-photon excitation fluorescence (TPEF). An alternative fluorescence pathway can occur through cleavage of LW-OTf by ONOO− to non-fluorescent LW-XTD-OTf, which can react further with the second analyte O2˙− to produce the same LW-XTD fluorescent species. By combining NIRF and TPEF, LW-OTf is capable of differential and simultaneous detection of ROS and RNS in DILI using two optically orthogonal channels. Probe LW-OTf could be used to detect O2˙− or O2˙− and ONOO− in lysosomes stimulated by 2-methoxyestradiol (2-ME) or 2-ME and SIN-1 respectively. In addition, we were able to monitor the chemoprotective effects of tert-butylhydroxyanisole (BHA) against acetaminophen (APAP) toxicity in living HL-7702 cells. More importantly, TPEF and NIRF imaging confirmed an increase in levels of both O2˙− and ONOO− in mouse livers during APAP-induced DILI (confirmed by hematoxylin and eosin (H&E) staining)., Drug-induced liver injury (DILI) is an important cause of potentially fatal liver disease.

Nitroxyl (HNO) is closely linked with numerous biological processes. Fluorescent probes provide a visual tool for determining HNO. Due to fluorescence quenching by HNO-responsive recognition groups, most of the current fluorescent probes exhibit an "off-on"fluorescence response. As such, the single fluorescence signal of these probes is easily affected by external factors such as the microenvironment, sensor concentration, and photobleaching. Herein, we have developed a ratiometric fluorescent probe (CHT-P) based on our previously developed two-isophorone fluorophore. CHT-P could be used to determine HNO through ratiometric signal readouts with high selectivity and sensitivity, ensuring the accurate quantitative detection of HNO. Additionally, the probe exhibited low cytotoxicity, was cell permeable, and could be used for ratiometric imaging of HNO in cells. Finally, CHT-P-coated portable test strips were used to determine HNO using the solid-state fluorescence signal readout.

Excited-state intramolecular proton transfer (ESIPT)-based fluorophores with two-photon excitation fluorescence (TPEF) are rare. Our aim with this research was to develop ESIPT-based fluorophores exhibiting TPEF. Herein, we used 4-hydroxyisoindoline-1,3-dione as a scaffold to develop a two-photon fluorescent probe BHID-Bpin, for the detection of peroxynitrite (ONOO−). BHID-Bpin exhibits excellent selectivity, sensitivity, and fast response towards ONOO− in PBS buffer solution (10 mM, pH = 7.40). Additionally, BHID-Bpin displays high photo-stability under two-photon irradiation at 750 nm. Furthermore, the probe can image endogenous ONOO− in HeLa cells and exogenous ONOO− in rat hippocampal slices at a depth of 110 μm.

The microenvironment (local environment), including viscosity, temperature, polarity, hypoxia, and acidic-basic status (pH), plays indispensable roles in cellular processes. Significantly, organelles require an appropriate microenvironment to perform their specific physiological functions, and disruption of the microenvironmental homeostasis could lead to malfunctions of organelles, resulting in disorder and disease development. Consequently, monitoring the microenvironment within specific organelles is vital to understand organelle-related physiopathology. Over the past few years, many fluorescent probes have been developed to help reveal variations in the microenvironment within specific cellular regions. Given that a comprehensive understanding of the microenvironment in a particular cellular region is of great significance for further exploration of life events, a thorough summary of this topic is urgently required. However, there has not been a comprehensive and critical review published recently on small-molecule fluorescent chemosensors for the cellular microenvironment. With this review, we summarize the recent progress since 2015 towards small-molecule based fluorescent probes for imaging the microenvironment within specific cellular regions, including the mitochondria, lysosomes, lipid drops, endoplasmic reticulum, golgi, nucleus, cytoplasmic matrix and cell membrane. Further classifications at the suborganelle level, according to detection of microenvironmental factors by probes, including polarity, viscosity, temperature, pH and hypoxia, are presented. Notably, in each category, design principles, chemical synthesis, recognition mechanism, fluorescent signals, and bio-imaging applications are summarized and compared. In addition, the limitations of the current microenvironment-sensitive probes are analyzed and the prospects for future developments are outlined. In a nutshell, this review comprehensively summarizes and highlights recent progress towards small molecule based fluorescent probes for sensing and imaging the microenvironment within specific cellular regions since 2015. We anticipate that this summary will facilitate a deeper understanding of the topic and encourage research directed towards the development of probes for the detection of cellular microenvironments.

Conspectus This Account describes a range of strategies for the development of fluorescent probes for detecting reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive (redox-active) sulfur species (RSS). Many ROS/RNS have been implicated in pathological processes such as Alzheimer’s disease, cancer, diabetes mellitus, cardiovascular disease, and aging, while many RSS play important roles in maintaining redox homeostasis, serving as antioxidants and acting as free radical scavengers. Fluorescence-based systems have emerged as one of the best ways to monitor the concentrations and locations of these often very short lived species. Because of the high levels of sensitivity and in particular their ability to be used for temporal and spatial sampling for in vivo imaging applications. As a direct result, there has been a huge surge in the development of fluorescent probes for sensitive and selective detection of ROS, RNS, and RSS within cellular environments. However, cellular environments are extremely complex, often with more than one species involved in a given biochemical process. As a result, there has been a rise in the development of dual-responsive fluorescent probes (AND-logic probes) that can monitor the presence of more than one species in a biological environment. Our aim with this Account is to introduce the fluorescent probes that we have developed for in vitro and in vivo measurement of ROS, RNS, and RSS. Fluorescence-based sensing mechanisms used in the construction of the probes include photoinduced electron transfer, intramolecular charge transfer, excited-state intramolecular proton transfer (ESIPT), and fluorescence resonance energy transfer. In particular, probes for hydrogen peroxide, hypochlorous acid, superoxide, peroxynitrite, glutathione, cysteine, homocysteine, and hydrogen sulfide are discussed. In addition, we describe the development of AND-logic-based systems capable of detecting two species, such as peroxynitrite and glutathione. One of the most interesting advances contained in this Account is our extension of indicator displacement assays (IDAs) to reaction-based indicator displacement assays (RIAs). In an IDA system, an indicator is allowed to bind reversibly to a receptor. Then a competitive analyte is introduced into the system, resulting in displacement of the indicator from the host, which in turn modulates the optical signal. With an RIA-based system, the indicator is cleaved from a preformed receptor–indicator complex rather than being displaced by the analyte. Nevertheless, without a doubt the most significant result contained in this Account is the use of an ESIPT-based probe for the simultaneous sensing of fibrous proteins/peptides AND environmental ROS/RNS.

The concentrations of ATP and ONOO− have been correlated with the progression a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Here, we report the development of fluorescent probe, ATP-LW, which enables the simultaneous detection of ONOO− and ATP. ONOO− selectively oxidises the boronate pinacol ester of ATP-LW, to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). While, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO− and ATP products, ATP-LW exhibits the unique ability to image ONOO− levels in the green channel (λex = 488 nm, λem = 500-575 nm) and ATP concentrations using the red channel (λex = 514 nm, λem = 575-650 nm). This was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. The treatment of HL-7702 cell line with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of ATP in the red channel and increase in ONOO− green channel. While, the presence of SIN-1 (an exogenous ONOO− donor) results in an increase of ONOO−, and decrease in ATP. Significantly, when HL-7702 cells were treated with acetaminophen as a biological model for drug-induced liver injury, an increase in ONOO− green and decrease in ATP red channel fluorescence was observed. These results illustrate the utility of ATP-LW as a chemical tool to simultaneously monitor ATP and ONOO− concentrations in cellular-based applications.

In this tutorial review, we will explore recent advances in the construction and application of Forster resonance energy transfer (FRET)-based small-molecule fluorescent probes. The advantages of FRET-based fluorescent probes include: a large Stokes shift, ratiometric sensing and dual/multi-analyte responsive systems. We discuss the underlying energy donor–acceptor dye combinations and emphasise their applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvionments and dual/multi-analyte responsive systems.

With this research we set out to develop a number of coumarin-based ‘AND’ logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by attaching the ONOO� reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH. Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when exposed to either HCys and ONOO� (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their effectiveness to function as AND logic gates for HCys and a chosen analyte.

A coumarin-based novel ‘AND’ logic fluorescent probe ROS-AHC has been developed for the simultaneous detection of ONOO− and biological thiols. ROS-AHC was shown to exhibit only a very small fluorescence response upon addition of a single GSH or ONOO− analyte. Exposure to both analytes, however, resulted in a significant fluorescence enhancement.

Herein, we report the evaluation and synthesis of a reaction based fluorescent probe DCM‐Bpin for the detection of Peroxynitrite (ONOO−). DCM‐Bpin exhibits selective fluorescence off‐on response for ONOO− over other reactive oxygen species, including H2O2. Moreover, DCM‐Bpin is biocompatible and has been used to visualize exogenous ONOO− in HeLa cells., Turn on the light: A 50‐fold “turn‐on” fluorescence response at 667 nm was observed for DCM‐Bpin upon the addition of ONOO− (0–27 equiv.) using an excitation wavelength of 560 nm.

ESIPT-based fluorescence probes are emerging as an attractive tool for the detection of biologically relevant analytes owing to their unique photophysical properties. In this work, we have developed an ESIPT-based fluorescence probe (TCBT-OMe) for the detection of HClO/ClO- through the attachment of a bioorthogonal dimethylthiocarbamate linker. TCBT-OMe was shown to rapidly detect HClO/ClO- (10 s) at biologically relevant concentrations (LoD = 0.16 nM) and have an excellent selectivity towards others ROS/RNS and amino acids. Therefore, TCBT-OMe was tested in live cells and was successfully shown to be able to detect endogenous and exogenous HClO/ClO- in HeLa cells. Additionally, TCBT-OMe acts as a dual input logic gate for Hg2+ and H2O2. Interestingly, Hg2+ alone gradually causes a fluorescence response but requires30 min to produce a fluorescence response. Test strips containing TCBT-OMe were prepared and were demonstrated as an effective way to detect HClO/ClO- in water. Furthermore, TCBT-OMe was shown to detect exogenously added HClO/ClO- in three different water samples with little interference thus demonstrating the effectiveness as a method for the detection of HClO/ClO- in drinking water samples.

Heat stroke is a lethal condition which can cause dysfunction in the central nervous system, multi-organ damage and even death. However, there is still limited knowledge of the detailed mechanism about the roles of lysosomes in heat stroke due to lack of effective tools. Herein, we introduce our previously developed hemicyanine with a large D-π-A structure as the key fluorophore to develop a new fluorescent probe (CPY) for ratiometric mapping of lysosomal pH changes in live cells under a heat shock stimulus.

A simple ESIPT-based fluorescence probe (HMBT-LW) was developed for the detection of superoxide (O2˙−). HMBT-LW was synthesised over two steps and was shown to rapidly detect low concentrations of O2˙− (limit of detection = 7.4 μM), fully reacting within two minutes. Furthermore, HMBT-LW demonstrated excellent selectivity and sensitivity towards O2˙−.

Correction for ‘ESIPT-based ratiometric fluorescence probe for the intracellular imaging of peroxynitrite’ by Luling Wu et al., Chem. Commun., 2018, 54, 9953–9956.

A novel dual-function fluorescent probe (ATP-LW) was developed for the detection of ONOO- and ATP.

With this research we set out to develop a coumarin-based novel fluorescent probe NTR-AHC for the detection of biological thiols and nitroreductase (NTR). Probe NTR-AHC was constructed by attaching the NTR trigger moiety (p-nitrobenzyl) and biothiol acceptor (maleic acid monoamide), to the core fluorophore AHC. In the presence of both glutathione (GSH) and NTR, probe NTR-AHC produced a >8-fold increase in fluorescence intensity at 463 nm.

Background Past investigations showed inconsistent results for diagnostic and prognostic predictive values of Krebs von den Lungen-6 (KL-6) for interstitial lung disease (ILD). Methods Web of Science and PubMed were systematically searched on for articles exploring the association of KL-6 and ILDs published between September 1993 and March 2019. For comparisons between-groups, the standard mean difference and 95% confidence intervals (CIs) were computed as the effect sizes. For diagnostic studies, a summary of sensitivity, specificity, positive likelihood ratios, negative likelihood ratios, and diagnostic odds ratio, which indicated the accuracy of KL-6 in the differentiation of ILDs and no ILDs, were calculated from the true positive, true negative, false positive, and false negative of each study. In addition, the summary receive-operating characteristics curve was constructed to summarize the TP and FP rates. For follow-up study, we computed hazard ratios (HRs) and 95% CIs for mortality. ILD patients showed elevated concentrations of KL-6, compared to healthy controls and patients without ILD. Results The meta-analysis showed a sensitivity (0.85 [95% CI: 0.77-0.91]) and specificity (0.97 [95% CI: 0.90-0.99]) of KL-6 for ILDs. In addition, it showed elevated baseline circulating levels of KL-6 in subsequent active ILD, compared to subsequent inactive ILD. Moreover, there was a significant association between baseline levels of circulating KL-6 and mortality of ILD (HR 2.95, 95% CI 2.45-3.55, I = 65.9%, P = .032). Conclusion In conclusion, the study suggested that circulating KL-6 showed diagnostic and prognostic predictive values for ILDs.

A simple ESIPT-based fluorescence probe (HMBT-LW) was developed for the detection of superoxide (O2⸱-). HMBT-LW was synthesised over two steps and was shown to rapidly detect low concentrations of O2⸱- (limit of detection = 7.4 μM), fully reacting within two minutes. Furthermore, HMBT-LW demonstrated excellent selectivity and sensitivity towards O2⸱-.

In this review we will explore recent advances in the design and application of excited-state intramolecular proton-transfer (ESIPT) based fluorescent probes. Fluorescence based sensors and imaging agents (probes) are important in biology, physiology, pharmacology, and environmental science for the selective detection of biologically and/or environmentally important species. The development of ESIPT-based fluorescence probes is particularly attractive due to their unique properties, which include a large Stokes shift, environmental sensitivity and potential for ratiometric sensing.

A series of 3-hydroxyflavone (3-HF) ESIPT (excited-state intramolecular proton transfer) boronate-based fluorescent probes have been developed for the detection of peroxynitrite (ONOO

An ESIPT-based 'AND' logic fluorescence probe (GSH-ABAH) was developed for the simultaneous detection of ONOO- and biological thiols. GSH-ABAH was shown to have good cell permeability and with the addition of just SIN-1 (ONOO- donor) or GSH, no fluorescence response was observed in live cells. However, in the presence of both analytes GSH-ABAH could be used to image exogenous ONOO- 'AND' GSH added to RAW264.7 cells.

In this work, we set out to develop an endoplasmic reticulum (ER) directed ESIPT-based ONOO− ratiometric fluorescent probe (ABAH-LW). ABAH-LW was synthesized in four steps and found to have a high sensitivity and selectivity towards the detection of ONOO−. ABAH-LW was able to detect low concentrations of ONOO− (limit of detection = 21.4 nM) within seconds producing a ratiometric change in fluorescence intensity. ABAH-LW further demonstrated the ability to ratiometrically image endogenous and exogenous ONOO− in HeLa cells. Moreover, co-localization experiments were carried out using commercially available ER-Tracker Red, Lyso-Tracker Red and Mito-Tracker-Red, which were co-stained with ABAH-LW in HeLa cells. For ER-Tracker Red, Pearson's correlation co-efficient of 0.93 was determined and 3D surface plot analysis illustrated a large overlap between ABAH-LW and ER-Tracker Red using both red and blue channels. In addition some co-localisation with Mito-Tracker Red and ABAH-LW was observed (0.73).

Based on the NCL reaction, we report a novel “off–on” fluorescent probe BQ for the sensing of Cys/Hcy by a FRET strategy. Owing to the quenching effect of azo-based naphthalimide, BQ showed nonfluorescence. Upon the interaction of Cys/Hcy, a dramatic increase of the fluorescence signal was observed, with a high selectivity toward Cys/Hcy over GSH. Additionally, the discrimination of Cys from Hcy also could be achieved through the different rates of the transthioesterification reaction between thiols and BQ, especially when the concentration of Cys and Hcy was below 100 μM. The Cys/Hcy could be specifically labeled by the BODIPY fluorophore of BQ after the NCL reaction. Living cell imaging experiments demonstrated that BQ can selectively label Cys/Hcy and distinguish Cys/Hcy from other bioreagents including GSH in living systems.

A ligand-controlled regioselectivity switch of ring-opening coupling reaction of diarylmethylenecyclopropa[b]naphthalenes with Grignard reagents providing differently substituted β-vinylic naphthalenes in moderate to excellent yields was reported: when Pd(OAc)2 was used, the aromatic group from the Grignard reagent regioselectively coupled to the naphthyl ring after the ring-opening of three-membered cycle, which is different from the Pd(PPh3)2Cl2-catalyzed reaction. Based on a careful NMR study, we concluded that it may be explained by the ligand effect.

A cationic gold(I)-catalyzed decarboxylative etherification of propargyl carbonates to selectively produce propargyl ethers is reported. In the reaction the gold(I) catalyst shows a distinct σ-Lewis acidity rather than the commonly observed π-Lewis acidity, and thus catalyzes the decarboxylation of a variety of propargyl carbonates to give the corresponding propargyl ethers with high selectivity. This reaction represents a rare example of the tunable reactivity of cationic gold(I) complexes between σ-Lewis acidity and π-Lewis acidity.

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

A gold-catalyzed cyclization of 3-(2'-azidoaryl)-1-arylpropargyl carbonates to generate substituted quinolines via a sequence of 3,3-rearrangement, 6-endo-trig cyclization and denitrogenation has been developed. Similar products could be obtained from 3-aryl-1-(2'-azidoaryl)propargyl carbonates under different gold catalytic conditions via a sequential 6-endo-dig cyclization, denitrogenation, and 1,2-H shift process.

The construction of 3-oxabicyclo[3.2.0]heptanes is developed using the electrophilic addition of carbocations generated from functionalized allylic alcohols to cyclopropylidene substituted ethanols (I).

An unexpected hydroxyphosphinylation of 3-cyclopropylideneprop-2-en-1-ones with phosphines has been observed. This method provides a unique regio- and stereoselective synthesis of highly functionalized 1-dialkylphinyl-3-oxo-(1Z)-alkenyl cyclopropanols with important potentials. The reaction displays an unusual mechanistic feature--a highly selective cleavage of C-P σ bonds in phosphines.

As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.