Bio-orthogonally Activatable Fluorescent Probe for Specific Imaging of Myeloid Cell Leukemia 1 Protein.

The antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) has been increasingly identified as a promising potential therapeutic target attributed to its critical regulation effect in diverse cellar physiopathological events. Current fluorescence imaging strategies tend to be susceptible to the cellular microenvironment, and straightforward mapping of Mcl-1's level variation remains challenging. In this paper, an activatable "off-on" fluorescence strategy for Mcl-1 specific labeling was presented based on bio-orthogonal chemistry by introducing tetrazine-functionalized borondipyrromethene (TB) as a fluorescent reporter and trans-cyclooctyne-derived indole-2-carboxylic acid (TI) as an Mcl-1 targeting moiety. With the click pair of TB and TI, the Mcl-1 expression level in vitro and in vivo was successfully mapped straightforward. Also, the level changes of Mcl-1 upon drug challenge were demonstrated. This work provides a robust fluorescence strategy for Mcl-1 in situ imaging, and the results would further facilitate the comprehensive revelation of the Mcl-1 biological effect.

Endoplasmic Reticulum-Targeted Carbon Monoxide Photoreleaser for Drug-Induced Hepatotoxicity Remediation.

The alleviation of drug-induced liver injury has been a long-term public health concern. Growing evidence suggests that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of drug-induced hepatotoxicity. Therefore, the inhibition of ER stress has gradually become one of the important pathways to alleviate drug-induced liver injury. In this work, we developed an ER-targeted photoreleaser, ERC, for controllable carbon monoxide (CO) release with a near-infrared light trigger. By employing peroxynitrite (ONOO-) as an imaging biomarker of hepatotoxicity, the remediating effect of CO was mapped upon drug acetaminophen (APAP) challenge. The direct and visual evidence of suppressing oxidative and nitrosative stress by CO was obtained both in living cells and in mice. Additionally, the ER stress inhibiting the effect of CO was verified during drug-induced hepatotoxicity. This work demonstrated that CO may be employed as a potent potential antidote for APAP-related oxidative and nitrative stress remediation.

In Situ Fluorescence Imaging Reveals Contribution of Cerebral Hydroxyl Radicals in Hyperhomocysteinemia-Induced Alzheimer-like Dementia.

Elevated plasma level of homocysteine, also termed as hyperhomocysteinemia, is acknowledged as a significant and independent risk factor of Alzheimer's disease. However, the mechanistic insight has not been thoroughly elucidated yet. In this work, 3,5-dihydroxybenzyloxy was explored as the unique reaction trigger and integrated into the naphthalimide fluorophore via a carbamate linker to afford a new probe for •OH imaging. •OH treatment induced aromatic hydroxylation and subsequent elimination reaction to release the caged fluorophore, accompanied with a highly specific and sensitive turn-on fluorescence response. Cell imaging results revealed that excess homocysteine triggered overwhelming •OH production, which was mediated by N-methyl-d-aspartate receptor and NADPH oxidase, and the resultant •OH stress further initiated neuronal ferroptosis, also confirmed by western blot analyses. Additionally, hyperhomocysteinemic mouse models were established, and Alzheimer-like dementia of the mice was observed from behavioral tests. Most importantly, with this probe, cerebral •OH fluctuation was in situ visualized in live mice, which positively correlated with the severity of Alzheimer-like dementia induced by hyperhomocysteinemia. These results reveal that cerebral •OH stress may be the critical nexus linking hyperhomocysteinemia and Alzheimer's disease. This work provides a robust fluorescence probe for in situ visualizing the cerebral •OH fluctuations and illuminating critical insights into •OH contributions in brain disorders.

Photocontrollable Fluorescence Imaging of Mitochondrial Peroxynitrite during Ferroptosis with High Fidelity.

Ferroptosis, a new regulatory cell death modality, underlies the pathogenesis of a broad range of disorders. Although much efforts have been made to uncover the molecular mechanisms, some mechanistic details of ferroptosis still remain poorly understood. Particularly, the functional relevance of mitochondrial reactive oxygen species (ROS) in ferroptosis is still highly controversial, which is partially due to the fact that it still remains puzzled how the mitochondrial ROS level varies during ferroptosis. The conventional mitochondria-targeted probes may react with cytosolic ROS and show fluorescence variation before entering mitochondria, thus probably giving a false result on the mitochondrial ROS level and leading to the misjudgment on its biofunction. To circumvent this issue, we rationally designed a photocontrollable and mitochondria-targeted fluorescent probe to in situ visualize the mitochondrial peroxynitrite (ONOO-), which is the ROS member and mediator of ferroptosis. The photoactivated probe was endowed with a highly specific and sensitive fluorescence response to ONOO-. Notably, the response activity could be artificially regulated with light irradiation, which ensured that all the probe molecules passed through the cytosol in the locked status and were then photoactivated after reaching mitochondria. This photocontrolled fluorescence imaging strategy eliminated the interference of ONOO- outside the mitochondria, thus potentially afforded improved fidelity for mitochondrial ONOO- bioimaging in live cells and animal models. With this probe, for the first time, we revealed the mitochondrial ONOO- flux and its probable biological source during erastin-induced ferroptosis. These results suggest a tight correlation between mitochondrial ONOO-/ROS and ferroptotic progression, which will further facilitate the comprehensive exploration and manipulation of ferroptosis.

Dual-Channel Imaging of Amyloid-ß Plaques and Peroxynitrite To Illuminate Their Correlations in Alzheimer's Disease Using a Unimolecular Two-Photon Fluorescent Probe.

Alzheimer's disease (AD) involves multiple pathological factors that mutually cooperate and closely contact to form interaction networks for jointly promoting the AD progression. Therefore, the comonitoring of different factors is particularly valuable for elucidating their level dynamics and complex interactions. However, such significant investigations remain a major challenge due to the lack of unimolecular fluorescent probes capable of simultaneous and discriminative visualization of multiple targets. To address this concern, as proof of principle, we rationally designed a unimolecular fluorescent probe to discriminate and simultaneously profile amyloid-ß (Aß) plaques and peroxynitrite (ONOO-), which are both the pronounced AD pathological factors. Herein, a novel ONOO- reaction trigger was installed onto an Aß plaque binding fluorophore to generate a dual functional fluorescent probe, displaying completely separate spectral responses to Aß plaques and ONOO- with high selectivity and sensitivity. With this probe, for the first time, we comonitored the distribution and variation of Aß plaques and ONOO- through two independent fluorescence channels, demonstrating their close apposition and tight correlation during AD course in live cell and mouse models through two-photon imaging mode. Notably, Aß aggregates induce the neuronal ONOO- generation, which conversely facilitates Aß aggregation. The two critical events, ONOO- stress and Aß aggregation, mutually amplify each other through positive feedback mechanisms and jointly promote the AD onset and progression. Furthermore, by coimaging of the level dynamics of Aß plaques and ONOO-, we found that the cerebral ONOO- is a potential biomarker, which emerges earlier than Aß plaques in transgenic mouse models. Overall, the dual-channel responsive performance renders this probe as a powerful imaging tool to decipher Aß plaque-ONOO- interactions, which will facilitate AD-associated molecular pathogenesis elucidation and multitarget drug discovery.

Reactivity Modulation of Benzopyran-Coumarin Platform by Introducing Electron-Withdrawing Groups: Specific Detection of Biothiols and Peroxynitrite.

A variety of fluorophores have been designed and created to fabricate organic fluorescent probes. Among these fluorophores, benzopyran-coumarin (BC) based fluorescent platform has attracted increasing attention as it shows multiple appropriate fluorescent and imaging capacities. Nevertheless, the analytical potential of BC is still urgently needed to be further excavated as its detection performance is hindered by the inherent drawbacks of current BC skeleton, that is, limited number of reactive sites. As such, in this work, by simply introducing electron-withdrawing (EW) substituent groups, we reconstructed BC skeleton to afford two fluorescent probes, BCB (-Br substitued) and BCN (-NO2 substitued), both of which featured two highly reactive sites. These two probes were capable of detecting peroxynitrite (ONOO-) and biothiols (hydrogen sulfide, glutathione, cysteine, and homocysteine) through naked eye and UV-vis absorption analysis in buffer solution. In addition, BCB was able to specifically sense biothiols with fluorescent analysis while BCN, with - NO2 instead of -Br, displayed more prominent fluorescent specificity toward ONOO-. This work provided a new strategy for the reactivity regulation of fluorophore through EW group introduction, as well as an alternative approach and method for the construction of fluorescent probes for other important biological species.

A Facile, Versatile, and Highly Efficient Strategy for Peroxynitrite Bioimaging Enabled by Formamide Deformylation.

Peroxynitrite (ONOO-) is attracting increasing attention due to its involvement in multiple facets of pathophysiological processes. However, ONOO- bioimaging is still challenging due to (1) the lack of highly specific reaction triggers, (2) the tedious and low-yielding synthesis of current sophisticated probes, and (3) the lack of availability of a versatile chemical strategy. To address these challenges, on the basis of amine formylation/deformylation chemistry, we have developed a novel strategy for ONOO- bioimaging. As proof of principle, we designed, synthesized, and evaluated four novel fluorescent probes equipped with the formamide functionality. Although they feature distinctly different fluorophore classes, all probes can be synthesized in one step in high yields and exhibit particularly specific, highly sensitive, and rapid responses to ONOO-. The bioimaging capability is well demonstrated by successfully visualizing ONOO- fluctuation in live cells and major organs of mice suffering from paraquat poisoning. The proposed strategy has proved to be a facile, versatile, and highly efficient methodology for ONOO- visualization, which will greatly facilitate ONOO- biochemistry and pathophysiology.

Evaluating Drug-Induced Liver Injury and Its Remission via Discrimination and Imaging of HClO and H2S with a Two-Photon Fluorescent Probe.

Drug-induced liver injury (DILI) has aroused wide concern. Finding new markers or indicators as well as detoxification molecules for DILI is of great significance and good application prospect, which can help develop effective preclinical screening methodology and corresponding treatment protocols. Herein, in this article, DILI caused by antidepressant drugs of duloxetine and fluoxetine and its remission were evaluated by a two-photon fluorescent probe, RPC-1, through discriminating and imaging HClO and H2S simultaneously. By being applied both in vitro and in vivo, RPC-1 revealed slight up-regulation of HClO and negligible liver damage after administration of either of the two drugs. In contrast, an apparent up-regulation of HClO and obvious liver damage was observed after combined administration of the drugs. Meanwhile, the pretreatment of N-acetyl cysteine (NAC) resulted in the increasing of endogenous H2S level, which contributed to the remission of DILI. The histological analysis and serological test both gave good consistency with the imaging results. These findings demonstrate that HClO may be an appropriate indicator of DILI, and H2S plays an important role in the antidotal effect of NAC. We envision that RPC-1 can be used as a powerful tool to predict clinical DILI and study the effect of antidote, as well as explore the molecular mechanisms involved.

Mitochondrial Peroxynitrite Mediation of Anthracycline-Induced Cardiotoxicity as Visualized by a Two-Photon Near-Infrared Fluorescent Probe.

Anthracyclines rank among the most efficacious anticancer medications. However, their clinical utility and oncologic efficacy are severely compromised by the cardiotoxicity risk facing the early-diagnosis difficulty and their unclear molecular mechanism. Herein, a two-photon-excitable and near-infrared-emissive fluorescent probe, TPNIR-FP, was fabricated and endowed with extraordinary specificity and sensitivity and a rapid response toward peroxynitrite (ONOO-), as well as mitochondria-targeting ability. With the aid of TPNIR-FP, we demonstrate that mitochondrial ONOO- is upregulated in the early stage and contributes to the onset and progression of anthracycline cardiotoxicity in cardiomyocyte and mouse models; therefore, it represents an early biomarker to predict subclinical cardiotoxicity induced by drug challenge. Furthermore, TPNIR-FP is proved to be a robust imaging tool to provide critical insights into drug-induced cardiotoxicity and other ONOO--related pathophysiological processes.

Gadolinium-Labeled Aminoglycoside and Its Potential Application as a Bacteria-Targeting Magnetic Resonance Imaging Contrast Agent.

Magnetic resonance imaging (MRI) is a powerful diagnostic technique that can penetrate deep into tissue providing excellent spatial resolution without the need for ionizing radiation or harmful radionuclides. However, diagnosing bacterial infections in vivo with clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capabilities, and bacterial penetration and specificity. Here, we report the development of the first gadolinium (Gd)-based bacteria-specific targeting MRI contrast agent, probe 1, by conjugating neomycin, an aminoglycoside antibiotic, with Dotarem (Gd-DOTA, an FDA approved T1-weighted MRI contrast agent). The T1 relaxivity of probe 1 was found to be comparable to that of Gd-DOTA; additionally, probe 1-treated bacteria generated a significantly brighter T1-weighted MR signal than Gd-DOTA-treated bacteria. More importantly, in vitro cellular studies and preliminary in vivo MRI demonstrated probe 1 exhibits the ability to efficiently target bacteria over macrophage-like cells, indicating its great potential for high-resolution imaging of bacterial infections in vivo.

A Two-Photon H2 O2 -Activated CO Photoreleaser.

Carbon monoxide (CO) is proposed as an active pharmaceutical agent with promising pharmaceutical prospects, as it has been involved in multifaceted modulation of diverse physiological and pathological processes. However, questions remain for therapeutic application of inhaled CO attributed to the inherent great affinity between CO and hemoglobin. Therefore, a robust platform with the function of CO transport and controllable release, depending on the local status of an organism, is of prominent significance for effectively avoiding the side effects of CO inhalation and optimizing the biological regulation function of CO. Utilizing the oxidative stress biomarker H2 O2 as a trigger and combining with photo-control, a two-photon H2 O2 -activated CO photoreleaser, FB, featuring highly sensitive and specific H2 O2 sensing and photocontrollable CO release, was developed and the vasodilation effect of CO against angiotensin II was demonstrated.

Simultaneous Fluorescence and Chemiluminescence Turned on by Aggregation-Induced Emission for Real-Time Monitoring of Endogenous Superoxide Anion in Live Cells.

Biological sensors with simultaneous turn-on signals of fluorescence (FL) and chemiluminescence (CL) triggered by one single species are supposed to integrate spatiotemporally resolved FL imaging with dynamic CL sensing into one luminescent assay. Efficiently increased accuracy can be expected based on complementary information simultaneously obtained from two independent modes, which is crucial in disease detection and diagnosis. However, very few examples can be found to date because of the key challenges in the rational design of sensing structures. Herein, aggregation-induced emission (AIE) was employed to develop a novel organic platform TPE-CLA with simultaneous turn-on FL/CL signals specifically modulated by O2•- in cells, which can be attributed to the activation of AIE resulted from the decreasing solubility after recognition. Using imidazopyrazinone (CLA) as the reactive motif and tetraphenylethene (TPE) as FL/CL enhancing skeleton, TPE-CLA is sensitive enough to image native O2•- in Raw264.7 cells and lipopolysaccharide stimulated O2•- in mice. Endogenous O2•- in HL-7702 cells induced by acetaminophen (APAP) was uninterruptedly monitored for 7200 s with CL and the results were further confirmed by FL imaging. Accordingly, TPE-CLA turns out to be a reliable candidate for real-time and continuous monitoring of endogenous O2•- in live cells. The strategy utilizing AIE to accomplish the FL/CL dual detection is expected to extend the application of AIE as reaction-activated biosensors.

Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and Thiophenol Based on Diversified Pyrimidine Moieties and Their Biological Applications.

We present a feasible paradigm of developing original fluorescent probes for target biomolecules via combinatorial chemistry. In this developmental program, pyrimidine moieties were investigated and optimized as unique recognition units for thiols for the first time through a parallel synthesis in combination with a rapid screening process. This time-efficient and cost-saving process effectively facilitated the developmental progress and provided detailed structure-reactivity relationships. As a result, Res-Biot and Flu-Pht were identified as optimal fluorescent probes for biothiol and thiophenol, respectively. Their favorable characteristics and superior applicability have been well demonstrated in both chemical and biological contexts. In particular, Res-Biot enables the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis, indicating its suitability in elucidation of a specific pathophysiological process in both living cells and living animals. Meanwhile, Flu-Pht is competent to visualize thiophenols without the interference from endogenous biothiols in living cells.

Fluorescent Probe Based on Azobenzene-Cyclopalladium for the Selective Imaging of Endogenous Carbon Monoxide under Hypoxia Conditions.

Carbon monoxide (CO), a crucial gas message molecule, plays an important role in the regulation of physiological and pathological process. Hypoxia-induced CO is involved in modulating various cellular activities, including signal transduction, proliferation, and apoptosis. However, tracking CO fluctuation in the hypoxic cells is still a challenge due to lack of straightforward, visualized, and noninvasive tools. In this work, based on metal palladium-catalyzed reaction, we present the rational design, synthesis, and biological utility of an azobenzene-cyclopalladium-based fluorescent probe, ACP-2, for CO monitoring. ACP-2 exhibits capacity of detecting CO in aqueous buffer solution and live cells with high sensitivity and specificity. Utilizing ACP-2, we displayed a direct and visual evidence of endogenous CO up-regulation in live cells induced by hypoxia. Moreover, CO up-regulation during oxygen-glucose deprivation/reperfusion (OGD/R) was also imaged and certified by ACP-2.

Rational Design of an α-Ketoamide-Based Near-Infrared Fluorescent Probe Specific for Hydrogen Peroxide in Living Systems.

Hydrogen peroxide, an important biomolecule, receives earnest attention because of its physiological and pathological functions. In this Article, we present the rational design, characterization, and biological application of a mitochondria-targetable NIR fluorescent sensor, Mito-NIRHP, for hydrogen peroxide visualization. Mito-NIRHP utilizes a unique reaction switch, α-ketoamide moiety, to turn on a highly specific, sensitive, and rapid fluorescence response toward hydrogen peroxide coupled with the intramolecular charge transfer strategy. Mito-NIRHP is competent to track endogenously produced hydrogen peroxide in both living cells and living animals. In addition, utilizing Mito-NIRHP, overgeneration of hydrogen peroxide during ischemia-reperfusion injury was directly visualized at both cell and organ levels.

Identification of 3,5,6-substituted indolin-2-one's inhibitors of Aurora B by development of a luminescent kinase assay.

Aurora B kinase plays an important role in the cell normal mitosis and overexpresses in a variety of tumors. Inhibition of Aurora B kinase resulted in an apoptosis of cancer cells, which prevented tumor growth in xenograft models. In this Letter, we developed a luminescent kinase assay to perform high-throughput screening for identification of small molecule Aurora B inhibitors. Two 3,5,6-substituted indolin-2-one derivatives were identified within an in-house compound library. Their new derivatives were then designed and synthesized that resulting two new inhibitors of Aurora B kinase with improved potency. Docking simulation further demonstrated the proposed binding modes between indolin-2-one inhibitor and Aurora B.

Deuterium Editing of Small Molecules: A Case Study on Antitumor Activity of 1,4-Benzodiazepine-2,5-dione Derivatives.

Substituting hydrogen with deuterium in drug molecules is an appealing bioisosteric strategy for the generation of novel chemical entities in drug development. Optimizing lead compounds through deuteration has proven to be challenging and unpredictable, particularly for compounds with multiple metabolic sites. This study presents the pioneering achievement of substituting up to 19 hydrogen atoms with deuterium on 1,4-benzodiazepine-2,5-dione derivatives, shedding light on the structure-metabolism relationship and the impact of multiple deuterations on drug-like properties. Notably, the deuterated compound 3f exhibited remarkable antitumor activity in vivo and demonstrated favorable drug-like properties as a drug candidate.

Quinazoline-2,4(1H, 3H)-diones inhibit the growth of multiple human tumor cell lines.

Quinazoline-2,4(1H, 3H)-diones exhibit a wealth of biological activities including antitumor proliferation. We established an improved method for the synthesis of quinazoline-2,4(1H, 3H)-dione derivatives with three points of molecular diversity. Data indicate that compounds 60 (average logGI50=−6.1), 65 (average logGI50=−6.13), 69 (average logGI50 = −6.44), 72 (average logGI50 = −6.39), and 86 (average logGI50 = −6.45) significantly inhibited the in vitro growth of 60 human tumor cell lines tested. Structure­activity relationship analyses indicate that chlorophenethylureido is the necessary substituent at the D3 diversity point (7-position of quinazoline-2,4(1H, 3H)-dione), in particular, o-chlorophenethylurea (69) achieved optimal activity. o- or m-Chlorophenethyl substitutions (69 and 72) at the D2 diversity point (3-position of quinazo line-2,4(1H, 3H)-dione) gave the most potent compounds. Methoxyl and 4-methylpiperazin-1-yl substitution at the D1 diversity point (6-position of quinazoline-2,4(1H, 3H)-dione skeleton) may yield better activity than other groups. The quinazoline-2,4(1H, 3H)-dione scaffold can be effectively replaced by 2H-benzo[b][1,4]thiazin-3(4H)-one.

Modified preparation of Si@C@TiO2 porous microspheres as anodes for high-performance lithium-ion batteries.

Microscale porous silicon materials have shown great application potential as anodes for next-generation lithium-ion batteries (LIBs); however, they face significant challenges, including mechanical structure instability, low intrinsic conductivity, and uncontrollable processing. In this study, a modified etching strategy combined with a facile sol-gel method is demonstrated to prepare microscale porous Si microspheres encapsulated by an inner amorphous carbon shell (≈10 nm) and an outer rigid anatase titanium oxide (TiO2) shell (≈20 nm) (PSi@C@TiO2), with the intact porous framework and core-shell-shell spherical structure. The interconnected pores can sufficiently accommodate the expansion of the Si core during lithiation. Moreover, the double shells can not only enhance the kinetic behavior of the PSi@C@TiO2 microspheres, but can act as a compact fence to force the Si core to expand toward the internal pores during lithiation, ensuring the integrity of the porous spherical structure. As a result, the PSi@C@TiO2 anodes show greatly superior high specific capacity, excellent rate capability, stable solid-electrolyte interphase (SEI) films and steady mechanical structure. It delivers a high reversible capacity of 1004 mA h g-1 after 250 cycles at 0.5 A g-1. This study provides a modified method to prepare microscale porous Si anodes with a stable mechanical structure and long cycle life for LIBs.