Fluorescent Chemosensors as Future Tools for Cancer Biology

Reviews Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

Fluorescent Chemosensors as Future Tools for Cancer Biology Kamalpreet Singh, Adrian M. Rotaru, and Andrew A. Beharry* Department of Chemistry and Department of Chemical and Physical Sciences, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada ABSTRACT: It is well established that aberrant cellular biochemical activity is strongly linked to the formation and progression of various cancers. Assays that could aid in cancer diagnostics, assessing anticancer drug resistance, and in the discovery of new anticancer drugs are highly warranted. In recent years, a large number of small molecule-based fluorescent chemosensors have been developed for monitoring the activity of enzymes and small biomolecular constituents. These probes have shown several advantages over traditional methods, such as the ability to directly and selectively measure activity of their targets within complex cellular environments. This review will summarize recently developed fluorescent chemosensors that have potential applications in the field of cancer biology.

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and anticancer resistance often fails since the act of providing resistance depends on active protein and not amount.8 For cancer therapeutics, the discovery of new inhibitors/drugs using a single step assay would significantly reduce time and labor, thereby permitting the use of high throughput screening platforms. Furthermore, if the assay directly measured activity, false positives and false negatives, often observed with multistep, indirect assays, would be minimal.9,10 Fluorescent chemosensors (or fluorescent probes) have emerged as an attractive alternative to conventional biochemical methods for delineating enzyme activity.11 In principle, a fluorescent probe could be used to monitor specific enzyme activity in complex mixtures with minimal disruption to the studied system, thereby providing a more accurate depiction of their native roles. Depending on the design, the fluorescent probe can report directly on activity in a single step, thereby minimizing time and labor and eliminating the need for numerous controls for making clinical connections. Ideally, the probe would also report on real-time changes in activity in response to external stimuli (e.g., drugs or new inhibitors). This feature will greatly increase the confidence of assigning connections between enzyme activity and anticancer drug resistance and also in the discovery of new inhibitors for potential cancer therapeutics. Our aim in this review is to highlight some efforts in developing fluorescent chemosensors that are promising for 1) making new clinical connections between aberrant activity and cancer, 2) cancer diagnostics and in assessing anticancer drug resistance, and 3) the discovering of new inhibitors through high throughput screening. In particular our review will focus

iological activity is highly dependent on enzyme/protein expression levels, the availability of substrates, and the presence of other associated enzymes/proteins, all of which can vary depending on the cell type and state.1 Complexity further arises within certain diseases such as cancer, whereby point mutations in enzymes and/or associated proteins can lead to aberrant activity, leading to abnormal levels of their products thereby affecting cellular homeostasis.1,2 Abnormal DNA repair, cell death inhibition, and epigenetic control are some examples of defective pathways that are strongly linked to cancer formation and progression.3−5 Within these pathways certain enzymes often said to be “overexpressed” or “underexpressed” are compared to their normal cell counterpart. Several diagnostic methods have exploited these abnormalities for diagnostics, specifically to answer whether a patient has a certain cancer type and to assess staging (e.g., antibody-based diagnostics), and in providing links between anticancer drug resistance for personalized medicine.6,7 Although traditional biochemical methods such as Western blots, PCR, gene knockouts, and siRNA knockdowns have made many of the connections between aberrant enzymes and cancer, these methods are an indirect measure of protein activity, laborious, multistep (i.e. discontinuous), and lowthroughput. As a result, connecting an enzymes role with cancer has been time-consuming and highly prone to error, as numerous controls and multiple methods are required to validate the result. A challenge in cancer biology has been to monitor enzymatic activity directly with minimal perturbing conditions. The use of fusion proteins to GFP (or its variants) has provided valuable information regarding expression levels in cancer and normal cells. However, a drawback with this technique is the requirement for genetic manipulation and the inability of these fluorescent proteins to report on real-time dynamic changes in enzymatic activity. For cancer diagnostics, associating protein levels with cancer types is generally acceptable; however, correlations between protein amounts © XXXX American Chemical Society

Special Issue: Sensors Received: January 4, 2018 Accepted: March 26, 2018 Published: March 26, 2018 A

DOI: 10.1021/acschembio.8b00014 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology on small-molecule (or low molecular weight) fluorescent chemosensors for monitoring cancer related targets. General Probe Considerations. A significant advantage of using small-molecule based fluorescent chemosensors is their ability to be synthetically tuned to achieve properties for a given application. As you will see in the examples below, most fluorescent chemosensors developed to date are at the early stages, as specific chemical design and “proof-of-principle” type experiments are required in order to prove their suitability for cancer biology. Therefore, it is important to first highlight a few practical considerations that goes into designing fluorescent chemosensors: (i) The probe is to be water-soluble and stable under physiological buffer conditions. (ii) Fluorophores used in the chemosensor should preferably be excited and undergo emission at long wavelengths to avoid interference from background fluorescence. In the case for in vivo applications, long wavelengths (e.g., NIR) would also permit deeper tissue penetration. (iii) Preferably the probe functions within live cells or in animal models, and so cell permeability is highly desirable. Depending on the cellular localization of the target, the probe must also localize in that area for efficient reporting. (iv) Probes must be constructed so that it is highly selective for only its target. (v) For a high detection limit and large dynamic range, probes must initially have low fluorescence and exhibit high fluorescence in the presence of its target (i.e., undergo a large fluorescence fold change). Several photophysical mechanisms have been used to transduce a fluorescence signal in response to the active target. Some of these include FRET, photoinduced electron transfer (PeT), and intramolecular charge transfer (ICT). (vi) For in vivo use, ratiometric chemosensors are preferred as one emission channel can be used to correct for any artifacts such as intensity increases due to simple localization and not activation. The following sections will describe fluorescent chemosensors for directly monitoring the activity of enzymes. We also discuss the development of probes for monitoring small biomolecular constituents produced in abundance in cancer cells as a consequence of aberrant enzyme activity. The descriptions will highlight challenges and efforts in chemically constructing probes that meet the above requirements.

permeability.16,17 Their best probe, QMeNN (λex = 438 nm and λem = 527 nm), exhibited a 136-fold fluorescence light-up with hNQO1 and was capable of selectively imaging hNQO1 activity in numerous cancer cell lines (Prasai, Silvers, and McCarley 2015) (Figure 1).

Figure 1. Fluorescently silent QMeNN chemosensor was activated via reduction of quinone to hydroquinone by hNQO1 to induce spontaneous cyclization of Q3PA and subsequent liberation of the active fluorophore, NN. Figure adapted from Prasai et al. 2015.16

Given the success of the activation strategy, hNQO1 fluorescent chemosensors that can be excited and imaged with longer wavelength of light were recently developed to permit in vivo imaging. The Yoon group used a two-photon active naphthalene derivative (λex = 740/400 nm and λem = 520 nm) which was modified with the Q3PA hQNO1 substrate/ PeT quencher.18 Although two-photon activation was possible, only a 9-fold increase in fluorescence emission was observed. The probe was later improved by the Jiang group who achieved a 25-fold increase in fluorescence; however, two-photon activation was only demonstrated in cultured cells and not in animal models.19 Recently, the McCarley group synthesized a cy7 based NIR activatable fluorescent chemosensor called Q3STCy (λex = 605 nm and λem = 765 nm).20 Upon hNQO1 reduction of the quinone quencher group, the hydroquinone and subsequently the linker were eliminated, resulting in a 240fold increase in fluorescence intensity. Heterogeneous hNQO1 activity was successfully imaged in 3D colorectal tumor models, and metastases in preclinical mouse models of human ovarian serous adenocarcinoma were successfully identified using fluorescence generated by hNQO1 (Figure 2). Reductases Associated with Hypoxia. Tumors with hypoxia often exhibit resistance to radiotherapy and chemotherapy.21 The stress induced by hypoxia is accompanied by an increase in expression of various reductases, including nicotinamide adenine dinucleotide (NADH) dependent, Nitroreductase (NTR)22 and Azoreductase (AR).23 Development of fluorescent chemosensors for NTR and AR would therefore provide a direct measure of hypoxia which can be used to predict the effect of anticancer drug therapies. Nitroreductase (NTR). In the presence of the electron donor, NADH, NTR was found to efficiently reduce various nitroarene compounds to their amino counterparts.24 This conversion of an electron withdrawing group to an electron donating group upon NTR activity has made nitroarenes very attractive to be used as a substrate and PeT quencher in the construction of various fluorescent chemosensors. For example, the Ma group incorporated a nitrofuran moiety on the 7-hydroxyl on the xanthene-based dye, Resorufin.24 The dye was found to initially

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FLUORESCENT CHEMOSENSORS FOR MONITORING ENZYMATIC ACTIVITY Human NAD(P)Quinone Oxidoreductase Isozyme I (hNQO1). Human NAD(P)H:Quinone oxidoreductase I (hNQO1) is a cytosolic flavoprotein responsible for the twoelectron reduction of quinones to hydroquinone.12 It is upregulated 50-fold in cancer cells as compared to their healthy counterparts and is therefore attractive as a cancer biomarker.12−14 Work in developing a fluorescent chemosensor for hNQO1 was initiated by the McCarley group, who used quinone propionic acid (Q3PA) as a substrate for hNQO1which also acted as a PeT quencher for the fluorophore, Nmorpholino-capped rhodamine.15 Reduction of the quinone to hydroquinone by hNQO1 triggered spontaneous intramolecular cyclization causing elimination of the Q3PA quencher, resulting in a 96-fold increase in fluorescence emission. Exchanging the polar rhodamine dye to a series of neutral, hydrophobic naphthalamide derivatives resulted in better cell B


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nitrobenzene substrate/quencher.25 In the presence of NTR, a rapid 110-fold increase in fluorescence was observed. The probe was successfully used to image NTR in tumor xenograft mouse models and report on hypoxia in cultured cells. A similar approach was utilized by the Kim group, who conjugated a nitrobenzyl scaffold to a meso-bodipy dye.26 Their probe showed a large 220-fold light-up in fluorescence upon action of NTR and was used to report on varying degrees of hypoxia (5% to 1% O2) in A549 cells. NTR-responsive hypoxia chemosensors capable of twophoton excitation have also been synthesized. The Tan group synthesized a naphthalene-based turn-on chemosensor (λex = 780 nm and λem = 494 nm) that featured a self-eliminating nitrobenzyl quencher group.27 Their probe was found to exhibit a 70-fold enhancement in fluorescence which was used to differentiate hypoxic levels in Hela cells using one- and twophoton excitation (Figure 4). Later, the Lin and Liu group replaced naphthalene with a coumarin derivative and a fluorene dye, respectively, resulting in red-shifted two-photon NTR fluorescent chemosensors.28,29 Azoreductase (AR). AR is responsible for the reduction of azo linkages to their amine counter parts.30 The Nagano group has exploited AR activity, by synthesizing an AR-responsive hypoxia probe by linking the NIR dye, Cy5, to black hole quencher 3 (BHQ3) via an azo linkage.23 The close proximity to BHQ-3 quenched the fluorescence of Cy5 via FRET. Reduction of the azo bond by AR separated Cy5 from BHQ-3 resulting in a 100-fold increase in fluorescence. The chemosensor was successfully used in imaging ischemic liver in mice models. The Nagano group also developed a sensitive AR chemosensor by directly conjugating azobenzene to a xanthene dye. Fluorescence quenching was achieved due to rapid rotation

Figure 2. Fluorescent imaging of peritoneal metastasis in SHIN3 xenograft mice models 1 h postadministration with a NIR Q3STCy chemosensor for hNQO1. The presence of SHIN3 cells was confirmed using RFP. Fluorescent emission was not observed in a xenograft deficient mouse model (control). Figure adapted from Shen et al. 2017.20

be quenched until NTR-mediated reduction to the aminofuran triggered a rearrangement and elimination, releasing the native fluorophore causing a red-shift in the absorption spectrum and a 100-fold increase in fluorescence (Figure 3). The chemosensor was found to have no fluorescence under normoxic conditions (20% O2) and could successfully detect various extents of hypoxia (5% to 1% O2) in Hela and A549 cells.24 The success of the activation strategy was built upon by the Li group, who synthesized an ultrasensitive NIR NTRresponsive hypoxia chemosensor (λex = 769 nm and λem = 788 nm) using the cyanine fluorophore, cy7, conjugated to a

Figure 3. (Top) The mechanism of activation of a Resorufin-based nitroreductase chemosensor. The free fluorophore is released upon nitro reduction followed by 1,6-elimination of the nitrofuran quencher group. (Bottom) Absorption and fluorescence spectra for the quenched probe (black line) and the activated probe (red line). Figure adapted from Z. Li et al. 2013.24 C


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Figure 4. (A) Fluorescent imaging of nitroreductase activity using one-photon microscopy (OPM) and two-photon microscopy (TPM) in Hela cells under various extents of hypoxia. (B) Relative pixel intensity under various extents of hypoxia in one- and two-photon fluorescence imaging of nitroreductase. Figure adapted from J. Zhang et al. 2015.27

Figure 5. (Left) MAR is activated via liberation of the free fluorophore via azoreductase-mediated reductive cleavage of the azo bond. (Right) Fluorescent imaging of local hypoxia in rat models with branch retinal artery occulusion after 2 h of incubation with MAR. The fluorescence intensity (or azoreductase activity) was lower in regions of normoxia versus hypoxia. Reprinted with permission from ref 31. Copyright 2013 Wiley-VCH.

Figure 6. (A) HMRef-βgal is activated upon cleavage of the β-galactopyranoside by β-gal to release the ring-opened active rhodol based fluorophore. (B) Comparison of the fluoresence enhancement of the first generation chemosensor, HMDER-βgal, with HMRef-βgal. The second generation derivative exhibited a much greater increase in fluoresence upon addition of β-gal. Adapted with permission from ref 38. Copyright 2015 Nature Publishing Group.

around the azo-bond upon excitation.31 One probe called MAR, consisted of dimethylamino-azobenzene conjugated to the 7-position of a 2-methyl-rhodamine derivative. MAR was initially quenched but lit up 630-fold upon AR-mediated reductive cleavage of the azo bond. AR activity was successfully imaged in A549 cells under hypoxic conditions of up to 5% of O2 and was then used to differentiate retinal hypoxia from normoxia regions in a rat model of retinal artery occlusion31 (Figure 5). This activation strategy was later employed by the Romieu group32 who synthesized an AR activatable chemosensor based upon an unsymmetrical aniline derivative of Texas red, a red-shifted derivative of rhodamine. The AR induced cleavage of the azo bond induced a 94-fold increase in fluorescence of the chemosensor. Recently, the above-mentioned activation methodology has been extended by Tung and associates, who have conjugated an azobenzene to a piperazine modified Bodipy dye in order to

synthesize a lysosome targeted AR chemosensor.33 Upon AR cleavage, an aminobenzene is produced, which undergoes spontaneous rearrangement and elimination to release a piperazine conjugated Bodipy dye. Protonation of the piperazine moiety permitted localization and imaging of lysosomes in HepG2 cells under hypoxic conditions. β-Galactosidase (β-gal). β-Galactosidase (β-gal) is a hydrolase enzyme responsible for cleaving the glycosidic bond of the disaccharide lactose into glucose and galactose monomers.34 It is often overexpressed in prostate35 and ovarian cancer36 and can therefore be employed as a diagnostic tool for cancer detection. The Urano group conjugated a β-galactopyranoside, a substrate for β-gal, to the phenolic hydroxyl of a hydroxymethyl variant of a rhodol based dye.37 In this form, the dye underwent spirocyclization at physiological pH, which diminished its absorbance and fluorescence quantum yield. In the presence of D


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Figure 7. (Left) Separation of β-galactopyranoside from a DCM fluorophore by β-gal resulted in emission in the NIR. (Right) DCM-βgal probe was used to image tumors in nude mouse models containing βgal (A) and without βgal (B). Figure adapted from Gu et al. 2015.39

β-gal, β-galactopyranoside was hydrolyzed from the rhodol fluorophore resulting in spontaneous ring opening and a 76fold increase in fluorescence. Although successful in measuring β-gal activity in cultured cells, detection in vivo was limited due to high background fluorescence of the probe itself. Later, density functional theory calculations indicated that the modified dye can be more so driven toward the spirocyclized form by addition of electron withdrawing substituents to the amine of the rhodol.38 To this regard, a second generation β-gal chemosensor called HMRef-βgal, containing a methyl carbontrifluoride to the aniline moiety of the rhodol, was developed. A 1420-fold increase in fluorescence was now observed in the presence of β-gal which was used successfully in identifying peritoneal metastasis in cancer mouse models38 (Figure 6). The success of the above activation strategy led to the development of β-gal chemosensors utilizing NIR fluorophores. Zhu and colleagues developed a β-gal responsive NIR ratiometric chemosensor called DCM-βgal by conjugating a dicyanomethylene-4H-pyran (DCM) fluorophore to the βgalactopyranoside β-gal substrate.39 When conjugated, the chemosensor was found to emit in the visible spectrum (λem = 500 nm). Upon β-gal hydrolysis, the chemosensor exhibited a red shift in emission (λem = 685 nm) accompanied by a 34-fold increase in fluorescence. The effectiveness of the sensor was demonstrated in cultured cells and in vivo by imaging β-gal activity in colorectal tumor bearing mouse models39 (Figure 7). A similar approach has also been recently utilized by the Kim group to conduct in vivo imaging of hepatocellular carcinoma.40 Thioredoxin Reductase (TrxR). Thioredoxin reductase (TrxR) is an intracellular reductase that is involved in a variety of cellular events such as gene transcription, apoptosis, and cell proliferation. It is responsible for the reduction of various proteins including thioredoxin, a key player in redox homeostasis.41 TrxR is overexpressed in multiple cancers including thyroid, prostate, and colorectal carcinomas and is therefore a potential biomarker whose activity can be exploited for tumor imaging.42 Fang and colleagues developed a TrxR activatable fluorescent chemosensor, TRFS-green, containing a 1,2dithiolane moiety as the TrxR substrate conjugated to the amine handle of a naphthalamide fluorophore (λex = 438 nm and λem = 538 nm) via a carbamate linkage.43 The disulfide in 1,2-dithiolane was found to be selectively recognized and reduced by TrxR while exhibiting relative inertness toward endogenous thiols and reductases. The reductive cleavage of the disulfide by TrxR resulted in the liberation of free thiolates which underwent spontaneous intramolecular cyclization and elimination to release the free fluorophore, yielding a 35-fold increase in emission (Figure 8). The group recently expanded

Figure 8. TRFS-green is activated upon TrxR mediated reduction of the disulfide bond leading to intramolecular cyclization and selfelimination of 1,2-dithiolane, liberating the active green-emitting fluorophore. Figure adapted from Zhang et al. 2014.43

this work by constructing a red-shifted TrxR chemosensor by exchanging the naphthalamide dye for Nile blue (λex = 615 nm and λem = 661 nm).44 Their chemosensor exhibited a 90-fold increase in fluorescence, and TrxR activity could be readily detected in cultured cells. Glutathione-S-Transferase (GST). Glutathione-S-Transferase (GST) belongs to a family of dimeric proteins involved in the detoxification of endogenous and exogenous compounds.45 To achieve this, GST catalyzes the nucleophilic attack of glutathione (GSH) at electrophilic sites of potential toxins, reducing their reactivity and hence toxicity.45 GST is often overexpressed in cancers and plays an active role in anticancer drug resistance.46 Classical GST responsive fluorescent chemosensors suffer from selectivity, as they readily react with glutathione (GSH) alone. To resolve this limitation, the Nagano group conjugated a 3,4-diinitrobenzanilide moiety to an aminofluoroscein dye.47 The dye was found to be quenched by the 3,4-dinitrobenzanilide group through PeT. In the presence of GST, the nitro group at the 4-position was replaced by GSH which disrupted efficient PeT quenching, resulting in a 528-fold increase in fluorescence. Activation of their probe required GST-catalyzed GSH addition (no reaction with free GSH) and was used to selectively image GST activity in cultured cells. A similar approach was later employed by the Morgenstern group to develop a series of GST activatable fluorescent chemosensors (Zhang et al. 2011). One of these probes, DNs-CV, utilized a cresyl violet fluorophore conjugated to a 2,4-dinitrobenzenesofonyl quencher group via an electrophilic sulfonamide linkage. Upon the selective action of GST, the sulfonamide linkage was cleaved, separating the fluorophore from the quencher moiety to yield a 580-fold increase in fluoresence emission. The chemosensor was subsequently used to image microsomal membrane bound GST activity in living cells. Recently, Feng and associates have further extended this E


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photon, ratiometric hCES2 fluorescent chemosensor called NCEN.53 NCEN was comprised of a naphthalamide-based fluorophore conjugated to a chloroacetyl group resulting in blue fluorescence. In the presence of hCES2, the chloroacetyl group was selectively cleaved, liberating a green emitting fluorophore, NAH. NCEN was used to image hCES2 activity in HepG2 cells and mouse liver slices from depths of 10 to 50 μm using two-photon excitation (λex = 800 nm) (Figure 10). Recently, the Urano group developed a fluorescent-substratelibrary-based enzyme discovery approach that identified pyruval anilide groups specifically hydrolyzed by hCES2. Conjugation of the pyruvyl group to the amine on a 7-amino-4methylcoumarin (AMC) produced a nonfluorescent compound but was found to be selectively hydrolyzed by hCES2 resulting in an increase in AMC fluorescence.54 Fluorometric assays were successfully performed in various mouse tissue lysates. Peptidases. Strong associations between the overexpression of peptidases (or proteases) and cancer progression have been well established due to their ability to degrade extracellular matrices to allow for cancer invasion and metastasis.55 Although a number of peptide-based fluorescent chemosensors exist for a variety of proteases, we will limit our discussion to two targets, GGT and APN, whereby small molecule-based probes have been developed. γ-Glutamyltranspeptidase (GGT). γ-Glutamyltranspeptidase (GGT) is a cell-surface bound enzyme involved in cellular glutathione (GSH) homeostasis and cysteine salvaging.56 GGT is overexpressed in ovarian and cervical cancer and is known to aid in tumor progression, invasion and in conferring anticancer drug resistance.56,57 The first GGT fluorescent chemosensor, gGlu-HMRG, was developed by Urano and Kobayashi by conjugating glutamic acid (Glu) to an amine on the xanthene moiety of rhodamine which produced a colorless, nonfluorescent, and noncell permeable spirocyclized probe (Figure 11).57 Upon enzymatic action by GGT, the amide bond conjugating the Glu trigger group to the dye was cleaved, liberating a colored, fluorescent and cell permeable open form of the dye (λex = 495 nm and λem = 520 nm). gGlu-HMRG was used to image GGT activity in 11 different human ovarian cancer cell lines, including mice models with human peritoneal ovarian cancer xenografts. Later, the Urano group developed a more red-shifted analogue of gGlu-HMRG, called gGlu-HMJSiR, by conjugating the glutamic acid trigger to a red-shifted silicated analogue of rhodamine (λex = 637 nm and λem = 662 nm).58 The Wu group

activation approach by synthesizing a two-photon fluorescent chemosensor for GST called P-GST (λex = 420/820 nm and λem = 550 nm).48 Their probe contained a 2,4-dinitrobenzenesulfonate group as the GST substrate/PeT quencher to a naphthalamide derivative. Upon GST action, the 2,4dinitrobenzenesulfonate group was transferred to GSH, liberating the free fluorophore and yielding a 40-fold increase in fluorescence (Figure 9). Two-photon imaging of GST activity was successfully observed in MCF-7 cells.

Figure 9. A two-photon excited P-GST probe is activated upon GST mediated transfer of GSH to the 2,4-dinitrobenzenesulfonate PeT quencher group, thereby liberating the active fluorophore. Figure adapted from J. Zhang et al. 2017.48

Human Carboxylesterase 2 (hCES2). Human carboxylesterase 2 (hCES2) is a serine-dependent hydrolyze involved in the metabolism of lipids. Since hCES2 is highly active in many cancers, it has been used to activate prodrugs of anticancer agents such as Capecitabine and Irinotecan.49−51 As a result, prior knowledge of hCES2 activity in cancer cells can be used to predict the efficacy of pre-existing anticancer agents. To this regard, the Yang group52 developed a fluorescent chemosensor for hCES2 using a TCF fluorophore called TCFB. The probe was made by conjugation of a benzoyl quencher group to the phenolic hydroxide of TCF, producing a nonfluorescent chemosensor through an ICT quenching mechanism. Upon the selective action of hCES2, the benzoyl trigger group was cleaved, liberating the free fluorophore with a fluorescence increase at 612 nm. The chemosensor was successfully used to image endogenous hCES2 in A549 cells. The group later extended this work by constructing a two-

Figure 10. (Left) The blue emitting hCES2 chemosensor, NCEN, is activated via cleavage of the chloroacetyl trigger group to yield the green emitting fluorophore, NAH. (Right) Endogenous CES2 was fluorescently imaged via two-photon microscopy in mouse liver slices at various depths. Figure adapted from Jin et al. 2015.53 F


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Figure 11. Conjugation of Glu to a xanthene amine on rhodamine results in a noncell permeable, spirocyclized colorless and nonfluorescent molecule. Cell surface GGT results in cleavage of the γ-glutamyl bond converting the molecule to a cell permeable, open, colored, and fluorescent form. “R” refers to the glutamic acid trigger group. Reprinted with permission from ref 57. Copyright 2011 AAAS.

developed a two-photon excited chemosensor, DCM-GA (λex = 490/800 nm and λem = 635 nm), by conjugating the glutamic acid trigger group to a dicyanomethylene-4H-pyran (DCM) fluorophore.59 The Ma group expanded upon this work by developing a NIR GGT chemosensor, which utilized the native substrate of GGT, GSH.60 Their probe had GSH conjugated to a NIR hemicyanine dye, HXPI (λex = 680 nm and λem = 708 nm), via an acrylyl linker. Acylation of the HXPI hydroxide with the acrylyl linker was found to render the dye weakly fluorescent. Upon enzymatic cleavage of GSH by GGT, the probe underwent a spontaneous rearrangement leading to the elimination of the acrylyl linker, liberating the HXPI hydroxide to yield the fluorescent dye. Fluorescent chemosensing of GGT activity was demonstrated in vivo using zebrafish animal models. Aminopeptidase N (APN). Aminopeptidase N (APN) is a ubiquitous integral membrane protein that is overexpressed in tumor vasculature of multiple cancers such as ovarian, liver, and nonsmall cell lung cancer.61−63 Its C-terminal extracellular domain allows it to function as an exopeptidase, cleaving amino acids from the N-terminal end of peptide chains. Specifically, APN prefers neutral or basic amino acids with a special preference for alanine (Ala). The Li group has exploited this substrate preference by developing a set of ratiometric chemosensors for detection of activity.64 Their best probe, Ala-PABA-7HC, contained Ala conjugated to a 7-hydroxy coumarin (7HC) fluorophore via a self-eliminating p-aminobenzyl alcohol (PABA) linker. Upon interaction with APN, the Ala trigger group is cleaved to liberate the free amino group of PABA that spontaneously self-eliminated to release the free fluorophore. This enzyme induced rearrangement resulted in a shift in fluorescence emission from 390 to 450 nm (λex = 330 nm) accompanied by an 11-fold increase in intensity at 450 nm.64 Later, their probe was improved by replacing 7HC with a red-shifted 1,8-naphthalimide fluorophore (λex = 400 nm and λem = 550 nm) which successfully imaged APN activity in a human ovarian cancer cell line.65 More recently, the Ma group conjugated the Ala trigger group to cresyl violet (CV) to develop a long wavelength ratiometric chemosensor, CVN (λex = 525 nm and λem = 575 nm, 626 nm) (Figure 12).66 Conversion of the amine on CV to an amide by Ala conjugation resulted in a blue shift in its emission to 575 nm. APNmediated hydrolysis liberated the native CV fluorophore producing emission at 626 nm. The ratiometric response was used to reveal higher APN levels in liver cancer cells (HepG2) than in normal liver cells (LO2). More recently, the same group developed a NIR APN chemosensor, HCAN, by conjugating Ala to a hemicyanine fluorophore. HCAN was successfully used to image APN activity in cellulo and in vivo.67

Figure 12. Conjugating the carboxyl group of Ala to Cresyl violet (CV) resulted in a blue-shift in CV’s emission from 626 to 575 nm. Upon APN hydrolysis of the amide bond, the fluorescence of CV at 626 nm is retrieved leading to a ratiometric response. Figure adapted from He et al. 2017.66

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FLUORESCENT CHEMOSENSORS FOR MONITORING SMALL BIOMOLECULAR CONSTITUENTS Glutathione (GSH). Glutathione (GSH) is the most abundant thiol containing molecule found in mammalian cells.68,69 It is involved in many vital cell processes including protein synthesis, modulation of enzyme activity, and in the antioxidation of the cell.68,70 Abnormal levels of glutathione are found in various types of cancers, conferring resistance to radiation or chemotherapy.69 For this reason, there have been several strategies in developing fluorescent chemosensors for GSH, all of which have exploited the intrinsic reactivity of its thiolate. For example, the Kim group71 used a heptamethanebased NIR fluorophore modified with a nitro-azo group that acted as a reactive substrate for GSH and a PeT quencher for the heptamethane dye. In the presence of GSH, the nitro-azo group is substituted by GSH causing a large 460-fold fluorescence increase along with a red-shift in the emission spectrum within the NIR region.71,72 Their probe was found to image GSH within the mitochondria of HeLa cells. Realizing interference from other thiol-containing compounds (e.g., Cysteine (Cys) and homocysteine (Hcy)) is a problem, several groups have constructed probes that can discriminate a GSH reaction by having it produce a distinct emission color from other thiols. For example, the probe developed by the Guo group73 used a chlorinated coumarinhemicyanine dye that contained multiple reactive sites. The first reaction site is thiolate substitution at the chlorinated position on coumarin, by which GSH, Hcy, and Cys will attack to produce a thiol-coumarin. However, since the distance between the thiolate and terminal amine all differ with these molecules, kinetically favorable rearrangement to an amino-coumarin hemicyanine could take place with Cys and Hcy but not GSH. Furthermore, by incorporating two more reactive sites at increasing distances away from the first site, two different cyclization products were produced via Michael additions with Cys and GSH (none with Hcy). Importantly, since each G


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Figure 13. Left, structure of a chlorinated coumarin-hemicyanine probe depicting three reaction sites. Right, since Cys, Hcy, and GSH all differ in the distance between their amine and thiol, they will give rise to different products each producing a distinct fluorescence color allowing them to be distinguished from one another. Figure adapted from J. Liu et al. 2014.73

Figure 14. Top, a ratiometric FRET-based probe for sensing GSH reversibly. Bottom left, real-time ratiometric imaging of GSH in A549 cells under a glucose deprivation perfusion system, whereby low glucose decreases GSH and high glucose increases GSH. Condition 1 (top row): 0−3 min (high glucose, 25 mM), 3−12 min (glucose-free, 0 mM), 12−30 min (high glucose, 25 mM). Conditions 2 (bottom row): 0−3 min (high glucose, 25 mM), 3−30 min (glucose-free, 0 mM). Bottom right, time-course of GSH concentration under the glucose deprivation system. Red is condition 1; blue is condition 2. Reprinted with permission from ref 75. Copyright 2017 Nature Publishing Group.

emission. At low GSH concentrations, FRET occurred, and high emission from SiR was observed. Their ratiometric probe successfully measured real-time GSH dynamics within seconds in HeLa, ovarian cancer (SHIN3 and SKOV3), and lung cancer cells (A549 and H226) (Figure 14). Nitric Oxide. Nitric oxide (NO) plays important roles in the human body, such as acting as a messenger in the nervous system, a smooth muscle relaxant in the cardiovascular system, and in prevention of thrombosis.76−78 To date, the role of NO as a cancer-promoting agent or as an anticancer agent remains unclear.79 To this regard, the development of a NO fluorescent chemosensor would help in making better clinical connections in establishing its role in various cancers.80 Nearly two decades ago, Nagano and colleagues designed a fluorescent probe for NO using fluorescein that would weakly fluoresce due to PeT from a diaminophenyl groups such as o-phenylenediamino (OPD). In the presence of NO, the amines would cyclize into a less electron donating triazole ring, resulting in loss of its

substitution, rearrangement, and cyclization product effects overall π-conjugation (or the absorption spectra) differently, a different excitation and fluorescence color is produced for each thiol (blue for Cys, green for GSH, no color for Hcy) (Figure 13). Indeed, their probe successfully measured both GSH and Cys simultaneously in COS-7 cells. Later, a similar strategy to achieve discrimination was used by the Liang group74 who employed a cyano-aminobenzothiazole dye. In the above strategies, the mechanism of activation involves an irreversible nucleophilic substitution reaction with GSH. Thus, measuring real-time dynamic changes of glutathione in cells is not possible with those probes. To overcome those limitations, recently Urano and colleagues75 developed a FRETbased probe consisting of a silicon-substituted Rhodamine (SiR) dye fused to TetramethylRhodamine (TMR). The SiR (acceptor dye) underwent an intermolecular equilibrium attack with GSH, and as a result, at high GSH concentrations, the emission of SiR decreased causing an increase in the TMR H


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Figure 15. Left, structure of a Bodipy-based dye containing an o-phenylenediamino quencher group and its reaction with NO to produce a fluorescent species. Right, structure of a quinolone-based dye containing a dimethylaminophenyl group and its reaction with NO to produce a new conjugated fluorescent dye. Figure on the left adapted from Zhang et al. 2014.83 Figure on the right adapted from Dai et al. 2017.85

quenching ability.81,82 The principle behind these diaminofluoresceins formed the basis for many subsequent fluorescent probes for NO. For example, Zhang and colleagues incorporated a diaminophenyl group on a Bodipy-based dye83 which exhibited a low fluorescence quantum yield but increased up to 550-fold in the presence of NO (Figure 15). The probes were tested in human vascular endothelial cells and indeed could measure NO concentrations. The excellent photostability and long intracellular retention made their probes suitable for monitoring NO over long periods of time. Another example of a recent OPD-based probe is the one developed by Guo and colleagues84 which contained a positive charge on the scaffold to drive uptake into the mitochondria. The probe was based off a pyronin dye, and in the presence of Cys and GSH, measurements of NO could be observed in the green and red emission channels, respectively. A large 833-fold increase in quantum yield was observed in the presence of NO, and when tested in B16 cells, a strong fluorescence response was produced. More recently, several groups have used an alternative NO reactive group than OPD. Song’s group85 developed a probe consisting of a dimethylaminophenyl group that reacts intramolecularly with the amine from quinolone in the presence of NO enabling the chromophore to fluoresce (Figure 15). No interferences from other reactive species like GSH, reactive oxygen species, dehydroascorbic acid (DHA), and ascorbic acid (AA), were observed.85 The probe was also capable of measuring NO concentrations as low as 15 nM which was comparable to a probe developed around the same time by Liu and colleagues.86 The Zhihong group87 developed a SiR-based probe modified with a 4-methoxy-N-methylaniline moiety to quench the fluorescence of SiR through PeT. In the presence of NO, a N-nitrosation of the methylamino group occurs which suppresses the PeT process, causing SiR to fluoresce. Their probe underwent two-photon excitation in the NIR and emission in the red, permitting NO imaging in cultured cells and in tumor tissues of a xenograft mouse model (Figure 16). Hydrogen Sulfide. In recent years, high levels of hydrogen sulfide (H2S) have been found to enhance tumor growth of cancers of the colon and ovaries.88 As a result, many approaches have been taken to construct fluorescent chemosensors to measure this important molecule in biological systems. For example, Cui and colleagues.89 developed a probe based off of 1,8-naphthalimide modified with a dinitrophenyl group as a PeT quencher. In the presence of H2S, the quencher is eliminated via thiolysis resulting in a 42-fold increase in fluorescence intensity. A 4-(2-aminoethyl)morpholine moiety was attached to the scaffold to promote lysosome targeting in

Figure 16. Two-photon fluorescence microscopy of a NO NIR probe. Left, xenograft mouse model tissue slices postinjection for (a) healthy tissue, (b) 2-week-old tumors, (c) 4-week-old tumors, and (d) after injection with an NO scavenger. Right, relative fluorescence intensities for each image on the left is compared. Figure adapted from ref 87.

MCF-7 cells, whereby H2S levels were successfully measured. Yoon and colleagues90 used a similar strategy by also using a naphthalimide fluorophore but containing an electron withdrawing 7-nitro-1,2,3-benzoxadiazole (NBD) quencher group. In the presence of H2S, NBD is selectively removed through thiolysis thereby producing a 68-fold increase in naphthalimide fluorescence. Their probe was further designed to specifically target the mitochondria through incorporation of a triphenylphosphonium cation moiety and was successful in measuring H2S levels in HeLa cells (Figure 17). Guo and colleagues91 recently developed a BODIPY-based probe containing two phenylselenide groups. Substitution of one of the phenylselenide groups with H2S resulted in a decrease in BODIPY fluorescence, and a further decrease was observed with substitution at the second phenylselenide site, resulting in an overall 49-fold decrease in red-emission. The probe was highly selective for H2S and could be imaged in BHK cells. Reactive Oxygen Species. Reactive oxygen species (ROS) comprises of a group of reactive ions derived from molecular oxygen. Some of these include hydroxyl radicals, superoxide anion, and hydrogen peroxide (H2O2). Due to an enhanced I


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Figure 17. Left, the structure and reaction of a mitochondria-targeted naphthalimide based probe selectively activated by H2S. Right, the fluorescence spectrum of the probe before and after addition of H2S. Figure adapted from Pak et al. 2016.90

Figure 18. Top, structure and reaction of a fluorescent chemosensor for hydroxyl radicals and hypochlorous acid occurring through two separate pathways producing a different florescence signal (cyan emission for hydroxyl radical, green emission for hypochlorous acid). Bottom, fluorescence imaging in macrophage cells shows simultaneous detection of both ROS when localized in the mitochondria. Figure adapted from Zhang et al. 2016.97

carbazole-based dye via PeT. In the presence of H2O2, the boronate of the p-pinacolborylbenzyl is oxidized, followed by a 1,6-rearrangement elimination releasing the cationic quinolone to a neutral species causing a 33-fold increase in fluorescence intensity. The probe exhibited good selectivity for H2O2 and could report intracellular H2O2 levels within the mitochondria of HeLa cells. The Yang group96 developed fluorescein-based probes for the detection of superoxide. They are initially nonfluorescent due to lack of conjugation from lactone formation induced by the electron-withdrawing nature of attached trifluoromethanesulfonate groups on the xanthene portion. The trifluoromethyl groups also serve to activate the sulfonate ester toward nucleophilic attack by superoxide, which removed the

metabolism and dysfunctional mitochondria, many cancer cells have elevated levels of ROS compared to their normal cell counterparts. As a result, high levels of ROS promote tumor formation and progression as well as contributing to tumor resistance to chemotherapy.92 To this regard, several groups aimed to develop fluorescent chemosensors for ROS for cancer diagnostics and predicting the efficacy of anticancer drugs. Since the major source of ROS production occurs in the mitochondria,93,94 many fluorescent probes have been designed to specifically target the mitochondria in order to become sufficiently activated to report levels of ROS. The Shao group95 used a p-pinacolborylbenzyl moiety as a reaction site for the ROS, H2O2, which was linked at a quaternarized quinolone cationic moiety to target mitochondria and to also quench a J


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ACS Chemical Biology ORCID

trifluoromethanesulfonate groups, opening up the lactone ring and restoring conjugation. As a result, a 654-fold increase in fluorescence was observed. One of the probes, HKSOX-1m, contained a triphenylphosphonium group used to drive uptake into the nitochondria. Indeed their probes could detect superoxide in various cell lines with high selectivity and was further used to measure endogenous superoxide in vivo using zebrafish animal models. Recently, the Zhang group97 developed a single fluorescent chemosensor for measuring two ROS independently, hydroxyl radicals and hypochlorous acid (HClO) (Figure 18). Derived from fluorescein, one side of the xanthene ring was modified with a triethylene glycol chain, while the phenyl ring contained a hydrazine motif resulting in formation of a five-membered heterocyclic ring. Initially, the probe is nonfluorescent due to the newly formed five-membered ring breaking π conjugation of xanthene. However, in the presence of hydroxyl radicals, the five-membered ring is broken along with one of the xanthene rings leaving two aromatic rings in conjugation to produce cyan fluorescence. In the presence of HClO, only the five-membered ring is broken leaving a conjugated xanthene producing green fluorescence. Their probe successfully measured both ROS simultaneously within the mitochondria of cancer cells using the well separated emission channels. In addition, their probe could profile hydroxyl radicals and HClO within metabolic organs in zebrafish and could measure hydroxyl radical production at the fresh wounds of zebrafish (Figure 18).

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Andrew A. Beharry: 0000-0003-1505-2375 Notes

The authors declare no competing financial interest.

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OUTLOOK

The past several years has yielded numerous fluorescent chemosensors that can successfully monitor enzymatic and small biomolecular activity. By using a combination of organic synthesis and photophysical chemistry, researchers have devised creative chemical designs to achieve desirable properties for sensing targets in vitro, cultured cells and in vivo. Although these probes have shown the potential to be used for cancer diagnosis, linking aberrant activity to anticancer drug resistance and to aid in the discovery of new inhibitors using high throughput screening methods, many of them have not moved beyond proof-of-principle experiments. In order to advance, chemists need to begin communicating and collaborate with cancer biologists. This will ensure that developed probes can start to answer questions pertaining to their biological targets. Much like GFP, fluorescent chemosensors ideally should be used alongside conventional biochemical tools but are more powerful in making accurate, faster assessments with biological aberrant activity and cancer. In addition, the power of fluorescent chemosensors may assist with addressing complex questions that would otherwise be difficult to explore due to limitations of current techniques. Although we have witnessed several generations of probes for a given target, stronger relationships with the end user will better guide chemists toward a specific set of chemical refinement. For example, while using a chemist’s new tool, cancer biologists may request larger fluorescence fold-changes, longer emission wavelengths, faster kinetics, etc. Once basic science applications are in place, it seems likely that fluorescent chemosensors for answering clinical-based questions will start to arise.

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ACKNOWLEDGMENTS

We thank the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery) for support.

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KEYWORDS Fluorophore: a molecule that absorbs light of a given wavelength and emits light at a different (longer) wavelength Enzyme: a protein that catalyzes a biochemical reaction Fluorescent chemosensor: a molecule that undergoes changes in fluorescence in response to certain stimuli Real-time monitoring: live changes in signal detection without the need of workups Cancer biomarker: a measured molecule or cellular process that is distinctly different in cancer cells versus their normal cell counterpart Cancer diagnostics: procedures that confirm the presence of cancer and its type, location, and stage Hypoxia: a condition in which certain regions of tissues are deprived of oxygen leading less than normal levels of oxygen typically observed Anticancer drug resistance: a means in which the cancer treatment is rendered ineffective due to underlying mechanisms that ultimately diminish the effect of the drug REFERENCES

(1) Martin, G. S. (2003) Cell signaling and cancer. Cancer Cell 4, 167−174. (2) Panieri, E., and Santoro, M. M. (2016) ROS homeostasis and metabolism: a dangerous liason in cancer cells. Cell Death Dis. 7, e2253. (3) Helleday, T., Petermann, E., Lundin, C., Hodgson, B., and Sharma, R. A. (2008) DNA repair pathways as targets for cancer therapy. Nat. Rev. Cancer 8, 193. (4) Sharma, S., Kelly, T. K., and Jones, P. A. (2010) Epigenetics in cancer. Carcinogenesis 31, 27−36. (5) Labi, V., and Erlacher, M. (2015) How cell death shapes cancer. Cell Death Dis. 6, e1675. (6) Volm, M., and Efferth, T. (2015) Prediction of Cancer Drug Resistance and Implications for Personalized Medicine. Front. Oncol. 5, 282. (7) Zhang, X., Soori, G., Dobleman, T. J., and Xiao, G. G. (2014) The application of monoclonal antibodies in cancer diagnosis. Expert Rev. Mol. Diagn. 14, 97−106. (8) Housman, G., Byler, S., Heerboth, S., Lapinska, K., Longacre, M., Snyder, N., and Sarkar, S. (2014) Drug resistance in cancer: An overview. Cancers 6, 1769−1792. (9) Acker, M. G., and Auld, D. S. (2014) Considerations for the design and reporting of enzyme assays in high-throughput screening applications. Perspect. Sci. 1, 56−73. (10) An, W. F., and Tolliday, N. (2010) Cell-Based Assays for HighThroughput Screening. Mol. Biotechnol. 45, 180−186. (11) Wu, D., Sedgwick, A. C., Gunnlaugsson, T., Akkaya, E. U., Yoon, J., and James, T. D. (2017) Fluorescent chemosensors: the past, present and future. Chem. Soc. Rev. 46, 7105−7123. (12) Cresteil, T., and Jaiswal, A. K. (1991) High levels of expression of the NAD(P)H: Quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin. Biochem. Pharmacol. 42, 1021−1027.

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Reviews

ACS Chemical Biology (13) Cui, X., Li, L., Yan, G., Meng, K., Lin, Z., Nan, Y., Jin, G., and Li, C. (2015) High expression of NQO1 is associated with poor prognosis in serous ovarian carcinoma. BMC Cancer 15, 244. (14) Awadallah, N. S., Dehn, D., Shah, R. J., Russell Nash, S., Chen, Y. K., Ross, D., Bentz, J. S., and Shroyer, K. R. (2008) NQO1 Expression in Pancreatic Cancer and Its Potential Use as a Biomarker. Appl. Immunohistochem. Mol. Morphol. 16, 24−31. (15) Silvers, W. C., Payne, A. S., and McCarley, R. L. (2011) Shedding light by cancer redox-human NAD(P)H:quinone oxidoreductase 1 activation of a cloaked fluorescent dye. Chem. Commun. 47, 11264−11266. (16) Prasai, B., Silvers, W. C., and McCarley, R. L. (2015) Oxidoreductase-Facilitated Visualization and Detection of Human Cancer Cells. Anal. Chem. 87, 6411−6418. (17) Silvers, W. C., Prasai, B., Burk, D. H., Brown, M. L., and McCarley, R. L. (2013) Profluorogenic Reductase Substrate for Rapid, Selective, and Sensitive Visualization and Detection of Human Cancer Cells that Overexpress NQO1. J. Am. Chem. Soc. 135, 309−314. (18) Kwon, N., Cho, M. K., Park, S. J., Kim, D., Nam, S.-J., Cui, L., Kim, H. M., and Yoon, J. (2017) An efficient two-photon fluorescent probe for human NAD(P)H:quinone oxidoreductase (hNQO1) detection and imaging in tumor cells. Chem. Commun. 53, 525−528. (19) Pan, D., Luo, F., Liu, X., Liu, W., Chen, W., Liu, F., Kuang, Y.Q., and Jiang, J.-H. (2017) A novel two-photon fluorescent probe with a long Stokes shift and a high signal-to-background ratio for human NAD(P)H:quinone oxidoreductase 1 (hNQO1) detection and imaging in living cells and tissues. Analyst 142, 2624−2630. (20) Shen, Z., Prasai, B., Nakamura, Y., Kobayashi, H., Jackson, M. S., and McCarley, R. L. (2017) A Near-Infrared, Wavelength-Shiftable, Turn-on Fluorescent Probe for the Detection and Imaging of Cancer Tumor Cells. ACS Chem. Biol. 12, 1121−1132. (21) Strese, S., Fryknäs, M., Larsson, R., and Gullbo, J. (2013) Effects of hypoxia on human cancer cell line chemosensitivity. BMC Cancer 13, 331. (22) Brown, J. M., and Wilson, W. R. (2004) Exploiting tumour hypoxia in cancer treatment. Nat. Rev. Cancer 4, 437−447. (23) Kiyose, K., Hanaoka, K., Oushiki, D., Nakamura, T., Kajimura, M., Suematsu, M., Nishimatsu, H., Yamane, T., Terai, T., Hirata, Y., and Nagano, T. (2010) Hypoxia-Sensitive Fluorescent Probes for in Vivo Real-Time Fluorescence Imaging of Acute Ischemia. J. Am. Chem. Soc. 132, 15846−15848. (24) Li, Z., Li, X., Gao, X., Zhang, Y., Shi, W., and Ma, H. (2013) Nitroreductase Detection and Hypoxic Tumor Cell Imaging by a Designed Sensitive and Selective Fluorescent Probe, 7-[(5-Nitrofuran2-yl)methoxy]-3H-phenoxazin-3-one. Anal. Chem. 85, 3926−3932. (25) Li, Y., Sun, Y., Li, J., Su, Q., Yuan, W., Dai, Y., Han, C., Wang, Q., Feng, W., and Li, F. (2015) Ultrasensitive Near-Infrared Fluorescence-Enhanced Probe for in Vivo Nitroreductase Imaging. J. Am. Chem. Soc. 137, 6407−6416. (26) Kim, T.-I., Kim, H., Choi, Y., and Kim, Y. (2017) meso-ester BODIPYs for the imaging of hypoxia in tumor cells. Sens. Actuators, B 249, 229−234. (27) Zhang, J., Liu, H.-W., Hu, X.-X., Li, J., Liang, L.-H., Zhang, X.-B., and Tan, W. (2015) Efficient Two-Photon Fluorescent Probe for Nitroreductase Detection and Hypoxia Imaging in Tumor Cells and Tissues. Anal. Chem. 87, 11832−11839. (28) Xu, A., Tang, Y., Ma, Y., Xu, G., Gao, S., Zhao, Y., and Lin, W. (2017) A fast-responsive two-photon fluorescent turn-on probe for nitroreductase and its bioimaging application in living tissues. Sens. Actuators, B 252, 927−933. (29) Zhai, B., Hu, W., Sun, J., Chi, S., Lei, Y., Zhang, F., Zhong, C., and Liu, Z. (2017) A two-photon fluorescent probe for nitroreductase imaging in living cells, tissues and zebrafish under hypoxia conditions. Analyst 142, 1545−1553. (30) Chevalier, A., Renard, P.-Y., and Romieu, A. (2017) Azo-Based Fluorogenic Probes for Biosensing and Bioimaging: Recent Advances and Upcoming Challenges. Chem. - Asian J. 12, 2008−2028. (31) Piao, W., Tsuda, S., Tanaka, Y., Maeda, S., Liu, F., Takahashi, S., Kushida, Y., Komatsu, T., Ueno, T., Terai, T., Nakazawa, T.,

Uchiyama, M., Morokuma, K., Nagano, T., and Hanaoka, K. (2013) Development of Azo-Based Fluorescent Probes to Detect Different Levels of Hypoxia. Angew. Chem., Int. Ed. 52, 13028−13032. (32) Chevalier, A., Piao, W., Hanaoka, K., Nagano, T., and Renard, P.-Y. (2015) Azobenzene-caged sulforhodamine dyes: a novel class of “turn-on” reactive probes for hypoxic tumor cell imaging. Methods Appl. Fluoresc. 3, 044004. (33) Luo, S., Liu, Y., Wang, F., Fei, Q., Shi, B., An, J., Zhao, C., and Tung, C.-H. (2016) A fluorescent turn-on probe for visualizing lysosomes in hypoxic tumor cells. Analyst 141, 2879−2882. (34) Juers, D. H., Matthews, B. W., and Huber, R. E. (2012) LacZ βgalactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 21, 1792−1807. (35) Wagner, J., Damaschke, N., Yang, B., Truong, M., Guenther, C., McCormick, J., Huang, W., and Jarrard, D. (2015) Overexpression of the Novel Senescence Marker β-Galactosidase (GLB1) in Prostate Cancer Predicts Reduced PSA Recurrence. PLoS One 10, e0124366. (36) Chatterjee, S. K., Bhattacharya, M., and Barlow, J. J. (1979) Glycosyltransferase and Glycosidase Activities in Ovarian Cancer Patients. Cancer Res. 39, 1943. (37) Kamiya, M., Asanuma, D., Kuranaga, E., Takeishi, A., Sakabe, M., Miura, M., Nagano, T., and Urano, Y. (2011) β-Galactosidase Fluorescence Probe with Improved Cellular Accumulation Based on a Spirocyclized Rhodol Scaffold. J. Am. Chem. Soc. 133, 12960−12963. (38) Asanuma, D., Sakabe, M., Kamiya, M., Yamamoto, K., Hiratake, J., Ogawa, M., Kosaka, N., Choyke, P. L., Nagano, T., Kobayashi, H., and Urano, Y. (2015) Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo. Nat. Commun. 6, 6463. (39) Gu, K., Xu, Y., Li, H., Guo, Z., Zhu, S., Zhu, S., Shi, P., James, T. D., Tian, H., and Zhu, W.-H. (2016) Real-Time Tracking and In Vivo Visualization of β-Galactosidase Activity in Colorectal Tumor with a Ratiometric Near-Infrared Fluorescent Probe. J. Am. Chem. Soc. 138, 5334−5340. (40) Kim, E.-J., Kumar, R., Sharma, A., Yoon, B., Kim, H. M., Lee, H., Hong, K. S., and Kim, J. S. (2017) In vivo imaging of β-galactosidase stimulated activity in hepatocellular carcinoma using ligand-targeted fluorescent probe. Biomaterials 122, 83−90. (41) Zhang, J., Li, X., Han, X., Liu, R., and Fang, J. (2017) Targeting the Thioredoxin System for Cancer Therapy. Trends Pharmacol. Sci. 38, 794−808. (42) Lincoln, D. T., Ali Emadi, E. M., Tonissen, K. F., and Clarke, F. M. (2003) The thioredoxin-thioredoxin reductase system: overexpression in human cancer. Anticancer Res. 23, 2425−2433. (43) Zhang, L., Duan, D., Liu, Y., Ge, C., Cui, X., Sun, J., and Fang, J. (2014) Highly Selective Off−On Fluorescent Probe for Imaging Thioredoxin Reductase in Living Cells. J. Am. Chem. Soc. 136, 226− 233. (44) Ma, H., Zhang, J., Zhang, Z., Liu, Y., and Fang, J. (2016) A fast response and red emission probe for mammalian thioredoxin reductase. Chem. Commun. 52, 12060−12063. (45) Eaton, D. L., and Bammler, T. K. (1999) Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol. Sci. 49, 156−164. (46) Tew, K. D., Monks, A., Barone, L., Rosser, D., Akerman, G., Montali, J. A., Wheatley, J. B., and Schmidt, D. E. (1996) Glutathioneassociated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program. Mol. Pharmacol. 50, 149. (47) Fujikawa, Y., Urano, Y., Komatsu, T., Hanaoka, K., Kojima, H., Terai, T., Inoue, H., and Nagano, T. (2008) Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells. J. Am. Chem. Soc. 130, 14533−14543. (48) Zhang, J., Jin, Z., Hu, X.-X., Meng, H.-M., Li, J., Zhang, X.-B., Liu, H.-W., Deng, T., Yao, S., and Feng, L. (2017) Efficient TwoPhoton Fluorescent Probe for Glutathione S-Transferase Detection and Imaging in Drug-Induced Liver Injury Sample. Anal. Chem. 89, 8097−8103. L


Reviews

ACS Chemical Biology (49) Xu, G., Zhang, W., Ma, M. K., and Mcleod, H. L. (2002) Human Carboxylesterase 2 Is Commonly Expressed in Tumor Tissue and Is Correlated with Activation of Irinotecan 1. Clin Cancer Res. 8, 2605− 2611. (50) Sanghani, S. P., Quinney, S. K., Fredenburg, T. B., Sun, Z., Davis, W. I., Murry, D. J., Cummings, O. W., Seitz, D. E., and Bosron, W. F. (2003) Carboxylesterases Expressed in Human Colon Tumor Tissue and Their Role in CPT-11 Hydrolysis. Clin. Cancer Res. 9, 4983. (51) Yano, H., Kayukawa, S., Iida, S., Nakagawa, C., Oguri, T., Sanda, T., Ding, J., Mori, F., Ito, A., Ri, M., Inagaki, A., Kusumoto, S., Ishida, T., Komatsu, H., Inagaki, H., Suzuki, A., and Ueda, R. (2008) Overexpression of carboxylesterase-2 results in enhanced efficacy of topoisomerase I inhibitor, irinotecan (CPT-11), for multiple myeloma. Cancer Sci. 99, 2309−2314. (52) Feng, L., Liu, Z.-M., Xu, L., Lv, X., Ning, J., Hou, J., Ge, G.-B., Cui, J.-N., and Yang, L. (2014) A highly selective long-wavelength fluorescent probe for the detection of human carboxylesterase 2 and its biomedical applications. Chem. Commun. 50, 14519−14522. (53) Jin, Q., Feng, L., Wang, D.-D., Dai, Z.-R., Wang, P., Zou, L.-W., Liu, Z.-H., Wang, J.-Y., Yu, Y., Ge, G.-B., Cui, J.-N., and Yang, L. (2015) A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues. ACS Appl. Mater. Interfaces 7, 28474−28481. (54) Yoshioka, K., Komatsu, T., Nakada, A., Onagi, J., Kuriki, Y., Kawaguchi, M., Terai, T., Ueno, T., Hanaoka, K., Nagano, T., and Urano, Y. (2015) Identification of Tissue-Restricted Bioreaction Suitable for in Vivo Targeting by Fluorescent Substrate Library-Based Enzyme Discovery. J. Am. Chem. Soc. 137, 12187−12190. (55) López-Otín, C., and Matrisian, L. M. (2007) Emerging roles of proteases in tumour suppression. Nat. Rev. Cancer 7, 800−808. (56) Pompella, A., De Tata, V., Paolicchi, A., and Zunino, F. (2006) Expression of γ-glutamyltransferase in cancer cells and its significance in drug resistance. Biochem. Pharmacol. 71, 231−238. (57) Urano, Y., Sakabe, M., Kosaka, N., Ogawa, M., Mitsunaga, M., Asanuma, D., Kamiya, M., Young, M. R., Nagano, T., Choyke, P. L., and Kobayashi, H. (2011) Rapid Cancer Detection by Topically Spraying a γ-Glutamyltranspeptidase−Activated Fluorescent Probe. Sci. Transl. Med. 3, 110−119. (58) Iwatate, R. J., Kamiya, M., Umezawa, K., Kashima, H., Nakadate, M., Kojima, R., and Urano, Y. (2018) Silicon Rhodamine-Based NearInfrared Fluorescent Probe for γ-Glutamyltransferase. Bioconjugate Chem. 29, 241−244. (59) Zhang, P., Jiang, X., Nie, X., Huang, Y., Zeng, F., Xia, X., and Wu, S. (2016) A two-photon fluorescent sensor revealing druginduced liver injury via tracking γ-glutamyltranspeptidase (GGT) level in vivo. Biomaterials 80, 46−56. (60) Li, L., Shi, W., Wu, X., Gong, Q., Li, X., and Ma, H. (2016) Monitoring γ-glutamyl transpeptidase activity and evaluating its inhibitors by a water-soluble near-infrared fluorescent probe. Biosens. Bioelectron. 81, 395−400. (61) Tokuhara, T., Hattori, N., Ishida, H., Hirai, T., Higashiyama, M., Kodama, K., and Miyake, M. (2006) Clinical Significance of Aminopeptidase N in Non−Small Cell Lung Cancer. Clin. Cancer Res. 12, 3971. (62) Cui, S.-X., Qu, X.-J., Gao, Z.-H., Zhang, Y.-S., Zhang, X.-F., Zhao, C.-R., Xu, W.-F., Li, Q.-B., and Han, J.-X. (2010) Targeting aminopeptidase N (APN/CD13) with cyclic-imide peptidomimetics derivative CIP-13F inhibits the growth of human ovarian carcinoma cells. Cancer Lett. 292, 153−162. (63) Wickström, M., Larsson, R., Nygren, P., and Gullbo, J. (2011) Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Sci. 102, 501−508. (64) Chen, L., Sun, W., Li, W., Li, J., Du, L., Xu, W., Fang, H., and Li, M. (2012) The first ratiometric fluorescent probe for aminopeptidase N. Anal. Methods 4, 2661−2663. (65) Chen, L., Sun, W., Li, J., Liu, Z., Ma, Z., Zhang, W., Du, L., Xu, W., Fang, H., and Li, M. (2013) The first ratiometric fluorescent probes for aminopeptidase N cell imaging. Org. Biomol. Chem. 11, 378−382.

(66) He, X., Xu, Y., Shi, W., and Ma, H. (2017) Ultrasensitive Detection of Aminopeptidase N Activity in Urine and Cells with a Ratiometric Fluorescence Probe. Anal. Chem. 89, 3217−3221. (67) He, X., Hu, Y., Shi, W., Li, X., and Ma, H. (2017) Design, synthesis and application of a near-infrared fluorescent probe for in vivo imaging of aminopeptidase N. Chem. Commun. 53, 9438−9441. (68) Meister, A., and Anderson, M. E. (1983) Glutathione. Annu. Rev. Biochem. 52, 711−760. (69) Townsend, D. M., Tew, K. D., and Tapiero, H. (2003) The Importance of Glutathione in Human Disease. Biomed. Pharmacother. 57, 145−155. (70) Kaplowitz, N. (1981) The importance and regulation of hepatic glutathione. Yale J. Biol. Med. 54, 497−502. (71) Lim, S.-Y., Hong, K.-H., Kim, D. Il, Kwon, H., and Kim, H.-J. (2014) Tunable Heptamethine−Azo Dye Conjugate as an NIR Fluorescent Probe for the Selective Detection of Mitochondrial Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 136, 7018−7025. (72) Kobayashi, H., Ogawa, M., Alford, R., Choyke, P. L., and Urano, Y. (2010) New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging. Chem. Rev. 110, 2620−2640. (73) Liu, J., Sun, Y.-Q., Huo, Y., Zhang, H., Wang, L., Zhang, P., Song, D., Shi, Y., and Guo, W. (2014) Simultaneous Fluorescence Sensing of Cys and GSH from Different Emission Channels. J. Am. Chem. Soc. 136, 574−577. (74) Miao, Q., Li, Q., Yuan, Q., Li, L., Hai, Z., Liu, S., and Liang, G. (2015) Discriminative Fluorescence Sensing of Biothiols in Vitro and in Living Cells. Anal. Chem. 87, 3460−3466. (75) Umezawa, K., Yoshida, M., Kamiya, M., Yamasoba, T., and Urano, Y. (2017) Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics. Nat. Chem. 9, 279−286. (76) Snyder, S. H. (1992) Nitric oxide: first in a new class of neurotransmitters? Science (Washington, DC, U. S.) 257, 494. (77) Yuan, S., Patel, R. P., and Kevil, C. G. (2015) Working with nitric oxide and hydrogen sulfide in biological systems. Am. J. Physiol. Lung Cell. Mol. Physiol. 308, L403. (78) Pacher, P., Beckman, J. S., and Liaudet, L. (2007) Nitric Oxide and Peroxynitrite in Health and Disease. Physiol. Rev. 87, 315. (79) Vahora, H., Khan, M. A., Alalami, U., and Hussain, A. (2016) The Potential Role of Nitric Oxide in Halting Cancer Progression Through Chemoprevention. J. Cancer Prev. 21, 1−12. (80) Korde Choudhari, S., Chaudhary, M., Bagde, S., Gadbail, A. R., and Joshi, V. (2013) Nitric oxide and cancer: a review. World J. Surg. Oncol. 11, 118. (81) Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y., and Nagano, T. (1998) Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins. Anal. Chem. 70, 2446−2453. (82) Chan, J., Dodani, S. C., and Chang, C. J. (2012) Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 4, 973−984. (83) Zhang, H.-X., Chen, J.-B., Guo, X.-F., Wang, H., and Zhang, H.S. (2014) Highly Sensitive Low-Background Fluorescent Probes for Imaging of Nitric Oxide in Cells and Tissues. Anal. Chem. 86, 3115− 3123. (84) Sun, Y.-Q., Liu, J., Zhang, H., Huo, Y., Lv, X., Shi, Y., and Guo, W. (2014) A Mitochondria-Targetable Fluorescent Probe for DualChannel NO Imaging Assisted by Intracellular Cysteine and Glutathione. J. Am. Chem. Soc. 136, 12520−12523. (85) Dai, C.-G., Wang, J.-L., Fu, Y.-L., Zhou, H.-P., and Song, Q.-H. (2017) Selective and Real-Time Detection of Nitric Oxide by a TwoPhoton Fluorescent Probe in Live Cells and Tissue Slices. Anal. Chem. 89, 10511−10519. (86) Mao, Z., Jiang, H., Li, Z., Zhong, C., Zhang, W., and Liu, Z. (2017) An N-nitrosation reactivity-based two-photon fluorescent probe for the specific in situ detection of nitric oxide. Chem. Sci. 8, 4533−4538. M


Reviews

ACS Chemical Biology (87) Mao, Z., Jiang, H., Song, X., Hu, W., and Liu, Z. (2017) Development of a Silicon-Rhodamine Based Near-Infrared Emissive Two-Photon Fluorescent Probe for Nitric Oxide. Anal. Chem. 89, 9620−9624. (88) Hellmich, M. R., and Szabo, C. (2015) Hydrogen sulfide and cancer. Handb. Exp. Pharmacol. 230, 233−241. (89) Liu, T., Xu, Z., Spring, D. R., and Cui, J. (2013) A LysosomeTargetable Fluorescent Probe for Imaging Hydrogen Sulfide in Living Cells. Org. Lett. 15, 2310−2313. (90) Pak, Y. L., Li, J., Ko, K. C., Kim, G., Lee, J. Y., and Yoon, J. (2016) Mitochondria-Targeted Reaction-Based Fluorescent Probe for Hydrogen Sulfide. Anal. Chem. 88, 5476−5481. (91) Gong, D., Zhu, X., Tian, Y., Han, S.-C., Deng, M., Iqbal, A., Liu, W., Qin, W., and Guo, H. (2017) A Phenylselenium-Substituted BODIPY Fluorescent Turn-off Probe for Fluorescence Imaging of Hydrogen Sulfide in Living Cells. Anal. Chem. 89, 1801−1807. (92) Galadari, S., Rahman, A., Pallichankandy, S., and Thayyullathil, F. (2017) Reactive oxygen species and cancer paradox: To promote or to suppress? Free Radical Biol. Med. 104, 144−164. (93) Ott, M., Gogvadze, V., Orrenius, S., and Zhivotovsky, B. (2007) Mitochondria, oxidative stress and cell death. Apoptosis 12, 913−922. (94) Gomes, A., Fernandes, E., and Lima, J. L. F. C. (2005) Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 65, 45−80. (95) Xu, J., Zhang, Y., Yu, H., Gao, X., and Shao, S. (2016) Mitochondria-Targeted Fluorescent Probe for Imaging Hydrogen Peroxide in Living Cells. Anal. Chem. 88, 1455−1461. (96) Hu, J. J., Wong, N.-K., Ye, S., Chen, X., Lu, M.-Y., Zhao, A. Q., Guo, Y., Ma, A. C.-H., Leung, A. Y.-H., Shen, J., and Yang, D. (2015) Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and. J. Am. Chem. Soc. 137, 6837−6843. (97) Zhang, R., Zhao, J., Han, G., Liu, Z., Liu, C., Zhang, C., Liu, B., Jiang, C., Liu, R., Zhao, T., Han, M.-Y., and Zhang, Z. (2016) RealTime Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe. J. Am. Chem. Soc. 138, 3769−3778.

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