Fluorescent Probes for Selective Recognition of Hypobromous Acid: Achievements and Future Perspectives

molecules

Review Fluorescent Probes for Selective Recognition of Hypobromous Acid: Achievements and Future Perspectives

Yuyu Fang 1,2 and Wim Dehaen 2,*

1 State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China; [email protected] 2 Department of Chemistry, KU Leuven, Celestijnenlaan 200f-bus 02404, 3001 Leuven, Belgium * Correspondence: [email protected]

Abstract: Reactive oxygen species (ROS) have been implicated in numerous pathological processes and their homeostasis facilitates the dynamic balance of intracellular redox states. Among ROS, hypobromous acid (HOBr) has a high similarity to hypochlorous acid (HOCl) in both chemical and physical properties, whereas it has received relatively little attention. Meanwhile, selective recognition of endogenous HOBr suffers great challenges due to the fact that the concentration of this molecule is much lower than that of HOCl. Fluorescence-based detection systems have emerged as very important tools to monitor biomolecules in living cells and organisms owing to distinct advantages, particularly the temporal and spatial sampling for in vivo imaging applications. To date, the development of HOBr-specific fluorescent probes is still proceeding quite slowly, and the research related to this area has not been systematically summarized. In this review, we are the first to review the progress made so far in fluorescent probes for selective recognition and detection of HOBr. The molecular structures, sensing mechanisms, and their successful applications of these probes as bioimaging agents are discussed here in detail. Importantly, we hope this review will call for more attention to this rising field, and that this could stimulate new future achievements. Keywords: fluorescent probes; selective recognition; ROS; hypobromous acid Citation: Fang, Y.; Dehaen, W. Fluorescent Probes for Selective Recognition of Hypobromous Acid: Achievements and Future 1. Introduction Perspectives. Molecules 2021, 26, 363. https://doi.org/10.3390/ Nature contains a complex collection of elements, molecules, and ions that play irre- molecules26020363 placeable roles in a wide range of chemical and biological processes. Selective recognition of these specific guests is a significant research area in supramolecular chemistry [1,2]. Received: 8 December 2020 Among the various guests, reactive oxygen species (ROS), which have higher reactivity Accepted: 7 January 2021 than molecular oxygen in the ground state, as their name suggests, are groups of reactive Published: 12 January 2021 neutral and anionic small molecules [3]. In particular, intracellular ROS are produced within many cell types upon incomplete reduction of oxygen through multiple electron Publisher’s Note: MDPI stays neu- transfer reactions, depending on the cell and tissue types [4]. tral with regard to jurisdictional clai- One of the major sources of intracellular ROS is NADPH oxidase process (Figure1a ), ms in published maps and institutio- and the commonly seen ROS include hydrogen peroxide (H2O2), hypohalous acids (HOCl/ nal affiliations. − − 1 ClO ), superoxide anion (O2• ), hydroxyl radical (•OH), singlet oxygen ( O2), peroxyni- − trite (ONOO ) and ozone (O3)[5]. It has been widely demonstrated that endogenous ROS are involved in numerous biological signaling pathways [6–8]. However, excess production of ROS can lead to oxidative damage to a wide range of biomolecules such as carbohydrates, Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. proteins, nucleic acids, and lipids, which has been implicated in physiological and patho- This article is an open access article physiological processes such as cancer, aging, cardiovascular disease, diabetes mellitus, distributed under the terms and con- gastrointestinal diseases, Alzheimer’s disease (AD), and so on [9–11]. ROS concentrations ditions of the Creative Commons At- within an expected threshold facilitate the dynamic balance of intracellular redox state. tribution (CC BY) license (https:// Undoubtedly, selective recognition and detection of those species have attracted a great creativecommons.org/licenses/by/ deal of attention [12–14]. Not surprisingly, biologists and chemists have made joint efforts 4.0/). to conceive feasible ROS-related therapeutic approaches in the past decades. Nevertheless,

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chemists have made joint efforts to conceive feasible ROS-related therapeutic approaches in the past decades. Nevertheless, the monitoring and quantitation of ROS remains a challenging taskthe monitoringnot only due and to quantitationtheir high re ofactivity ROS remains and short a challenging lifetime, but task also not to only the due to their low concentrationshigh reactivity in vivo. and short lifetime, but also to the low concentrations in vivo.

Figure 1. (a) TheFigure intracellular 1. (a) The generationintracellular of generation reactive oxygen of reactive species oxygen (ROS) species and (b )(ROS) the formation and (b) the of endogenousformation of hypobromous acid (HOBr)endogenous catalyzed hypobromous by eosinophil acid peroxidase (HOBr) catalyzed (EPO) or myeloperoxidaseby eosinophil peroxidase (MPO). (EPO) or myeloperoxidase (MPO). Hypobromous acid (HOBr) is an important ROS with a high similarity to HOCl Hypobromousin both acid chemical (HOBr) and is physicalan important properties. ROS with In a organelles, high similarity HOBr to is HOCl generated in from the − both chemicalperoxidation and physical of bromideproperties. anions In (Brorganelles,) with H HOBr2O2 (Figure is generated1b), whose from reaction the pe- is catalyzed by roxidation ofthe bromide heme peroxidasesanions (Br−) suchwith as H eosinophil2O2 (Figure peroxidase 1b), whose (EPO) reaction or myeloperoxidase is catalyzed by (MPO) [15]. the heme peroxidasesImportantly, such HOBr as eosinophil is a potent peroxidase oxidizer with(EPO) effective or myeloperoxidase antibacterial activity,(MPO) and is also [15]. Importantly,regarded HOBr as anis a integral potent factoroxidizer in thewith neutrophil effective hostantibacterial defense system.activity, Forand instance, is HOBr also regardedparticipates as an integral in the factor formation in the ofneutrophil sulfilimine host cross-links defense insystem. collagen For IV, instance, whose scaffolds are HOBr participatesessential in the to theformation formation of sulfil and functionimine cross-links of basement in collagen membranes IV, whose (BMs) scaf-in vivo [16]. In a folds are essentialsimilar to fashion the formation to other and ROS, function excessive of generationbasement membranes of HOBr will (BMs) injure in organisms,vivo which [16]. In a similaralways fashion results to in other inflammatory ROS, excessive tissue damagegeneration and of a varietyHOBr will of diseases injure organ- [17–19]. Increasing isms, which alwaysevidence results reveals in thatinflammatory the EPO levels tissue in damage serum ofand asthmatic a variety patients of diseases is three [17– times higher 19]. Increasingthan evidence that in healthyreveals individualsthat the EP [20O ].levels Considering in serum that of theasthmatic bromide patients concentration is in blood three times higherand plasma than that is far in lower healthy than individuals that of chloride [20]. Considering (ca. 1000-fold) that [the21], bromide the concentration of concentrationendogenous in blood and HOBr plasma is relatively is far lower lower than than that HOCl, of chloride which renders(ca. 1000-fold) the selective [21], recognition the concentrationof endogenous of endogenous HOBr HOBr more is challenging relatively lower than other than ROS.HOCl, which renders the selective recognitionAmong of endogenous various analytical HOBr more techniques, challenging fluorescent than other probes ROS. have emerged as indispens- Among ablevarious tools analytical to monitor techniques, biomolecules fluorescent in living cellsprobes and have organisms emerged owing as toindis- the high sensitiv- pensable toolsity, to non-invasive monitor biom imaging,olecules real-time in living detection, cells and low organisms cost, and owing superb to spatio-temporal the high resolu- sensitivity, non-invasivetion [22–25]. Particularly,imaging, real-t the probesime detection, with near-infrared low cost, (NIR) and emissionsuperb (650–900spa- nm) are tio-temporal highlyresolution favored [22–25]. for in Particularly, bio-imaging the due probes to their with distinct near-infrared ability of tissue (NIR) penetration emis- [26–28]. sion (650–900Meanwhile, nm) are highly ratiometric favored fluorescent for in bio-imaging probes are due highly to their desirable distinct because ability these of molecules tissue penetrationcould [26–28]. overcome Meanwhile, the ambiguities ratiomet ofric single fluorescent fluorescence probes intensity are highly and desira- offer quantitative measurements via self-calibration of two emission bands [29]. In addition, in contrast to ble because these molecules could overcome the ambiguities of single fluorescence in- aggregation-caused quenching (ACQ) that is quite common in most conjugated fluorescent tensity and offer quantitative measurements via self-calibration of two emission bands molecular systems, the aggregation-induced emission (AIE) first discovered by Tang’s [29]. In addition, in contrast to aggregation-caused quenching (ACQ) that is quite com- group in 2001 has become a major player in chemosensors [30–34]. mon in most conjugated fluorescent molecular systems, the aggregation-induced emis- To date, various classic fluorescence dyes, acting as signal reporters such as coumar- ins [35], 1,8-naphthalimides [36,37], rhodamines [38], difluoroboron dipyrromethenes (better known as BODIPY) [39–42], cyanine dyes [43], pyrene [44–46], AIE-active lumino-

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sion (AIE) first discovered by Tang’s group in 2001 has become a major player in chemosensors [30–34]. To date,gens various [47–49 classic] and fluorescence so on [50 dyes,–52], acting have as been signal widely reporters developed such as couma- to construct fluorescent rins [35], 1,8-naphthalimides [36,37], rhodamines [38], difluoroboron dipyrromethenes chemosensors for broad and exciting applications. Recently, we have summarized small- (better known as BODIPY) [39–42], cyanine dyes [43], pyrene [44–46], AIE-active lu- minogensmolecule-based [47–49] and so on fluorescent[50–52], have probesbeen widely for f-blockdeveloped metal to construct ions [53 fluorescent] and pillararene-based recep- chemosensorstors forfor bindingbroad and of differentexciting app metallications. ions [54Recently,] as well we as have fluorescent summarized chemosensors and smart small-molecule-basedmaterials constructed fluorescent probes from for macrocyclic f-block metal arenes ions [53] that and incorporate pillararene-based BODIPY [55]. Specifically, receptorsthe for binding reaction-based of differen fluorescentt metal ions probes[54] as well (also as knownfluorescent as chemodosimeters),chemosensors and whose recognition smart materialsevents constructed involve irreversible from macrocyclic chemical arenes reactions that incorporate as induced BODIPY by a [55]. target Spe- analyte, have received cifically, thegreat reaction-based attention duringfluorescent the pr lastobes decade (also known as this as ch promisingemodosimeters), and whose attractive strategy always recognition events involve irreversible chemical reactions as induced by a target analyte, have receivedoffers great high attention selectivity during and the sensitivity last decade [56 as–59 this]. promising and attractive strategy alwaysWith offers regard high se tolectivity ROS, and selective sensitivity recognition [56–59]. of these reactive species using fluorescent Withdetectors regard to ROS, has beenselective well recognition documented of these in reactive several species excellent using reviews, fluorescent which mainly focus on − − 1 detectors• hasOH been [60 ],well HOCl/ClO documented [in61 several], H2O excellent2 [62], ONOO reviews, which[63] and mainlyO 2focus[64]. on Hitherto, the research •OH [60],related HOCl/ClO to fluorescent- [61], H2O2 probes[62], ONOO for- selective [63] and recognition1O2 [64]. Hitherto, of HOBr the research has not been systematically related tosummarized. fluorescent probes Despite for selective the growing recognition interest, of HOBr the developmenthas not been systemati- of HOBr-specific fluorescent cally summarized. Despite the growing interest, the development of HOBr-specific flu- probes is still evolving very slowly. Obviously, all the reported fluorescent probes for ROS orescent probes is still evolving very slowly. Obviously, all the reported fluorescent probes forfunction ROS function in a in reaction-based a reaction-based manner manner [65–67], [65–67 ],and and HOBr HOBr is not is an not excep- an exception. There are tion. Therethree are three strategies strategies to constructto construct a a suitable suitable fluorescentfluorescent probe probe for for HOBr HOBr (Figure (Figure 2): (a) oxidation 2): (a) oxidationreactions reactions caused caused by HOBr, by HOBr, where where HOBr HOBr acts acts as a as strong a strong oxidant; oxidant; (b) (b) coupling cyclization of coupling ancyclization amino groupof an amino and S-methyl group and moiety S-methyl catalyzed moiety catalyzed by HOBr; by (c) HOBr; HOBr-induced (c) substitution HOBr-inducedreactions. substitution In light reactions. of the recent In light achievements of the recent achievements and deficiency and deficiency in HOBr-specific fluorescence in HOBr-specificprobes, fluorescence this is a timely probes, review this is of athe timely advances review madeof the advances so far in made constructions so far and applications in constructions and applications of fluorescent probes for HOBr using diverse fluoro- of fluorescent probes for HOBr using diverse fluorophores. phores.

FigureFigure 2. ThreeThree reported reported strategies strategies to construct to construct a suitable a suitable fluorescent ﬂuorescent probe for HOBr. probe for HOBr.

In this mini review, we will provide all the literature of ﬂuorescent probes for selective recognition and detection of HOBr, which represents the ﬁrst comprehensive summary

related to this attractive research area. The main text will be organized based on the three HOBr-sensing strategies as described above. The molecular structures, sensing mechanisms, and their successful applications as bioimaging agents will be discussed in detail. The purpose of this review is not only to provide a general overview of the design and development of ﬂuorescent probes for HOBr, but more importantly, it calls for more attention to this rising ﬁeld. Molecules 2021, 26, 363 4 of 18

2. Probes Based on the Oxidation Reactions Caused by HOBr Not until 2012 did Han’s group report the construction of cyanine-based fluorescent probes 1 and 2 for the selective recognition of HOBr (Figure3), which is the first example of a fluorescent probe for HOBr [68]. In this system, the cyanine platform served as a signaling fluorophore, while the 4-hydroxylamino-2,2,6,6-tetramethylpiperidine-N-oxyl (TemOH) moiety was incorporated both as the reaction site to HOBr and an effective fluorescent modulator. Despite an almost complete lack of changes in the absorption spectrum for the two probes upon treatment with HOBr under simulated physiological conditions (0.2 M PBS, pH = 7.4, 10 µM probes), a remarkable ratiometric fluorescence response for 1 and fluorescence quenching behavior for 2 were observed. The probe 1 exhibited two absorption maxima at 445 nm and 610 nm, together with the corresponding fluorescence maxima at 550 nm and 632 nm. The addition of HOBr did not cause a change of the fluorescence intensity at 550 nm but led to a decrease of the fluorescence intensity at 630 nm (quantum yield varying from 0.10 to 0.02), from which a 13-fold-decrease of the ratiometric fluorescence response F632 nm/F550 nm could be deduced while increasing the amount of HOBr from 0 to 110 µM. This unique recognition mechanism could be ascribed to HOBr-mediated oxidation of the TemOH moiety to the corresponding oxyammonium cation, which resulted in a donor-excited PET (d-PET) quenching effect. In addition, further addition of ascorbic acid to the in situ system gave rise to the recovery of its fluorescence emission due to the ascorbic acid-induced reduction of the oxyammonium cation, resulting from the inhibition of the d-PET process. The fluorescence intensity could also be turned off and on repeatedly with the alternate addition of HOBr and ascorbic acid in at least three cycles. With regard to cyanine probe 2, it exhibited NIR absorption and emission located at 702 nm and 755 nm (Φ = 0.11) in the same media, respectively. Similarly to 1, compound 2 responded to the HOBr/ascorbic acid redox cycle in a sequential manner, but the latter dye suffered from serious bleaching after three cycles. Importantly, the two probes showed Molecules 2021, 26, x FOR PEER REVIEW 5 of 19 low toxicity to the RAW264.7 cell line and localized in the cytoplasm, and so they could be successfully used to monitor intracellular HOBr.

FigureFigure 3.3.Chemical Chemical structuresstructures ofof cyanine-basedcyanine-based ﬂuorescentfluorescent probesprobes1 1and and2 2 forfor selectiveselective recognitionrecognition ofof HOBr.HOBr.

Subsequently, the same group developed BODIPY-based fluorescent probe 3 for HOBr in H2O-CH3CN (4:1, v/v, 20 mM PBS, pH = 7.4) solution [69]. Structurally, probe 3 contained the 4-methoxyphenylselenide unit as a modulator (Figure 4a), which could not only extend the π-conjugation system of the fluorophore but also facilitated the fluorescence of the probe to tune to the NIR region owing to the strong electron-donating affinity of the Se atom. As expected, compound 3 showed very weak fluorescence with the maximum emission wavelength located NIR region (λem = 711 nm, Φ = 0.00083) due to the heavy atom effect and efficient PET process from the diarylselenides to the BODIPY backbone. In the presence of HOBr, the fluorescence emission was significantly blue-shifted to 635 nm (Φ = 0.206) with a 118-fold ratiometric (F635 nm/F711 nm) enhancement, which was also accompanied by an obvious color change from green to blue, indicating that probe 3 was an excellent colorimetric and ratiometric fluorescent sensor for HOBr. This recognition process could be attributed to the oxidation of selenide to selenoxide by HOBr, causing a shortening of the donor-acceptor π-conjugated system as the selenoxide has much higher electron-withdrawing ability. Actually, selenium-incorporating fluorescent probes have been widely demonstrated to be potent chemosensors for several ROS, including peroxynitrite and hypochlorous acid [70–74]. However, in this system, probe 3 was found to be highly selective towards recognition of HOBr and not liable to interference by other ROS (Figure 4b). Additionally, only the addition of H2S (with high selectivity over other reactive sulfur species such as Cys, Hcys and GSH) induced the reduction of selenoxide to the corresponding selenide, leading to recovery of its original fluorescence. The redox recognition event mediated by HOBr and H2S, whose detection limit is determined to be 50 nM and 0.1 μM respectively, could be repeated at least five cycles. The probe possessed good cell membrane permeability and was applied to continuously detect intracellular HOBr/H2S redox cycle replacement in RAW264.7 cells.

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Subsequently, the same group developed BODIPY-based fluorescent probe 3 for HOBr in H2O-CH3CN (4:1, v/v, 20 mM PBS, pH = 7.4) solution [69]. Structurally, probe 3 contained the 4-methoxyphenylselenide unit as a modulator (Figure4a), which could not only extend the π-conjugation system of the fluorophore but also facilitated the fluorescence of the probe to tune to the NIR region owing to the strong electron-donating affinity of the Se atom. As expected, compound 3 showed very weak fluorescence with the maximum emission wavelength located NIR region (λem = 711 nm, Φ = 0.00083) due to the heavy atom effect and efficient PET process from the diarylselenides to the BODIPY backbone. In the presence of HOBr, the fluorescence emission was significantly blue-shifted to 635 nm (Φ = 0.206) with a 118-fold ratiometric (F635 nm/F711 nm) enhancement, which was also accompanied by an obvious color change from green to blue, indicating that probe 3 was an excellent colorimetric and ratiometric fluorescent sensor for HOBr. This recognition process could be attributed to the oxidation of selenide to selenoxide by HOBr, causing a shortening of the donor-acceptor π-conjugated system as the selenoxide has much higher electron-withdrawing ability. Actually, selenium-incorporating fluorescent probes have been widely demonstrated to be potent chemosensors for several ROS, including peroxynitrite and hypochlorous acid [70–74]. However, in this system, probe 3 was found to be highly selective towards recognition of HOBr and not liable to interference by other ROS (Figure4b). Additionally, only the addition of H 2S (with high selectivity over other reactive sulfur species such as Cys, Hcys and GSH) induced the reduction of selenoxide to the corresponding selenide, leading to recovery of its original fluorescence. The redox recognition event mediated by HOBr and H2S, whose detection limit is determined to be 50 nM and 0.1 µM respectively, could be repeated at least five cycles. The probe possessed Molecules 2021, 26, x FOR PEER REVIEW 6 of 19 good cell membrane permeability and was applied to continuously detect intracellular HOBr/H2S redox cycle replacement in RAW264.7 cells.

Figure 4. (a) Ratiometric ﬂuorescentfluorescent probe 3 on Se-BODIPY for selective recognition of HOBr followed by H2S in a redox cycle; (b) fluorescence intensity ratio (F635 nm/F711 nm) of 3 upon reacting with various ROS for 60 min. Reproduced with cycle; (b) ﬂuorescence intensity ratio (F635 nm/F711 nm) of 3 upon reacting with various ROS for 60 min. Reproduced with permission from Reference [[69].69]. CopyrightCopyright 2013 RoyalRoyal SocietySociety ofof Chemistry.Chemistry.

InIn 2020, 2020, Zeng Zeng and and coworkers coworkers reported reported a coumarin-based a coumarin-based fluorescent ﬂuorescent probe probe 4 for4 forse- lectiveselective detection detection of of HOBrHOBr (Figure(Figure5 a),5a), whose whose sensing sensing mechanism mechanism was basedwas onbased HOBr- on HOBr-mediatedmediated oxidation oxidation of amidoxime of amidoxime to the to corresponding the corresponding cyano cyano group group [75]. [75]. Probe Probe4 had 4 hadgood good water water solubility solubility and exhibitedand exhibited a 5 nm a red5 nm shift red (from shift 395(from to 400395 nm)to 400 in nm) its absorption in its ab- sorptionspectrum spectrum in PBS buffer in PBS solution buffer (pHsolution = 7.4) (pH upon = 7.4) addition upon ofaddition HOBr. Onof HOBr. the other On hand,the other the hand,ﬂuorescence the fluorescence intensity of intensity4 at 460 nmof 4 was at 460 greatly nm enhancedwas greatly with enhanced the quantum with yieldthe quantum varying yieldfrom varying 0.0445 to from 0.79 0.0445 in the presenceto 0.79 in ofthe HOBr, presen whichce of HOBr, could bewhich ascribed could to be the ascribed ICT effect to the of ICTthe resultanteffect of the product. resultant The product. sensing abilityThe sensing was demonstrated ability was demonstrated to be optimal to in be the optimal pH range in theof 7.0 pH to range 8.0.This of 7.0 probe to 8.0. had This a quite probe fast had response a quite fast time response (less than time 30 s)(less and than the detection30 s) and thelimit detection of HOBr limit was of calculated HOBr was as lowcalculated as 30.6 as nM. low Probe as 30.64 displayed nM. Probe a good4 displayed selectivity a good for selectivity for HOBr over other potential interferences, including ROS, RNS, various amino acids, and metal ions. Moreover, the content of HOBr in blood was monitored by the probe after successful establishment of an arthritic model mice (Figure 5b). The fluorescence intensity of 4 in the arthritic mice’s blood gradually increased within 6 days and reached a maximum value on the sixth day (Figure 5c), which was 4-fold higher than the value of untreated blood samples, indicating that probe 4 has potential for early diagnosis and evaluation of inflammation in clinical practice.

Figure 5. (a) Coumarin-based fluorescent probe 4 for HOBr; (b) swelling changes of right foot of mice within 10 days after injection of Freund’s adjuvant; (c) fluorescence response of probe 4 (50 μM) added in the blood of arthritis model mice within 10 days (λex = 395 nm, slits: 5/5 nm). Reproduced with permission from Reference [75]. Copyright 2020 Elsevier.

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Figure 4. (a) Ratiometric fluorescent probe 3 on Se-BODIPY for selective recognition of HOBr followed by H2S in a redox cycle; (b) fluorescence intensity ratio (F635 nm/F711 nm) of 3 upon reacting with various ROS for 60 min. Reproduced with permission from Reference [69]. Copyright 2013 Royal Society of Chemistry.

In 2020, Zeng and coworkers reported a coumarin-based fluorescent probe 4 for selective detection of HOBr (Figure 5a), whose sensing mechanism was based on HOBr-mediated oxidation of amidoxime to the corresponding cyano group [75]. Probe 4 had good water solubility and exhibited a 5 nm red shift (from 395 to 400 nm) in its absorption spectrum in PBS buffer solution (pH = 7.4) upon addition of HOBr. On the other hand, the fluorescence intensity of 4 at 460 nm was greatly enhanced with the quantum yield varying from 0.0445 to 0.79 in the presence of HOBr, which could be ascribed to the Molecules 2021, 26, 363 6 of 18 ICT effect of the resultant product. The sensing ability was demonstrated to be optimal in the pH range of 7.0 to 8.0. This probe had a quite fast response time (less than 30 s) and the detection limit of HOBr was calculated as low as 30.6 nM. Probe 4 displayed a good HOBrselectivity over for other HOBr potential over interferences,other potential including interferences, ROS, RNS, including various ROS, amino RNS, acids, various and metalamino ions. acids, Moreover, and metal the ions. content Moreover, of HOBr the content in blood of was HOBr monitored in blood bywas the monitored probe after by successfulthe probe after establishment successful of establishment an arthritic model of an mice arthritic (Figure model5b). mice The ﬂuorescence (Figure 5b). intensityThe fluo- ofrescence4 in the intensity arthritic mice’sof 4 in bloodthe arthritic gradually mice’s increased blood gradually within 6 days increased and reached within a 6 maximum days and valuereached on a the maximum sixth day value (Figure on 5thec), sixth which day was (Fig 4-foldure 5c), higher which than was the 4-fold value higher of untreated than the bloodvalue samples,of untreated indicating blood samp that probeles, indicating4 has potential that probe for early 4 has diagnosis potential and for evaluationearly diagno- of inﬂammationsis and evaluation in clinical of inflammation practice. in clinical practice.

FigureFigure 5.5. ((aa)) Coumarin-basedCoumarin-based ﬂuorescentfluorescent probeprobe4 4for for HOBr;HOBr; ((bb)) swellingswelling changeschanges ofof rightright footfoot ofof micemice withinwithin 1010 daysdays afterafter injectioninjection ofof Freund’sFreund’s adjuvant;adjuvant; ((cc)) ﬂuorescencefluorescence responseresponse ofof probeprobe 44 (50(50 µμM)M) addedadded inin thethe bloodblood ofof arthritisarthritis modelmodel micemice within 10 days (λex = 395 nm, slits: 5/5 nm). Reproduced with permission from Reference [75]. Copyright 2020 Elsevier. within 10 days (λex = 395 nm, slits: 5/5 nm). Reproduced with permission from Reference [75]. Copyright 2020 Elsevier.

3. Coupling and Cyclization of Amino and S-Methyl Groups Catalyzed by HOBr

Inspired by the key role that HOBr plays in the formation of sulfilimine (-S=N-) cross-links in collagen IV [16], Tang and coworkers pioneered the construction of a simple and ultrasensitive fluorescent probe 5 for selective recognition of HOBr (Figure6a) [ 76]. This unique mechanism was based on a specific coupling cyclization between the amino group and S-methyl group catalyzed by HOBr (Figure6b). The probe 5 was easily synthe- sized using commercially available o-(methylthio)-phenylboronic acid and o-bromoaniline through a Suzuki cross-coupling reaction in 85% yield. In PBS solution (pH = 7.4), the maximum excitation and emission wavelength of 5 located at 375 nm and 435 nm respectively, whereas those of the reaction product after treatment of 5 with HOBr were corresponding to 480 and 525 nm (Φ = 0.31). The large red shift of the maximum emission wavelength (ca. 90 nm) can be ascribed to the extended rigid skeleton of the reaction product. The probe featured an ultrasensitive response (detection limit of 17 pM), fast sensing time (approximate 3 min), low cytotoxicity (IC50 = 711.20 µM) and high selectivity for HOBr over other highly active oxidizing species (particularly the HOCl analogue, Figure6c) and active reducing species (Figure6d). Compared with probes 1–3 that detected HOBr only through activation after exposure to EPO, hydrogen peroxide (H2O2), and bromide anions (Br−) in live-cells, probes 5 imaged HOBr without bromine anion stimulation and − − exhibited different intensities of fluorescence emission with Br , Br /H2O2, or HOBr in HepG2 cells and zebrafish. Consequently, this probe ought to be a promising candidate for quantifying changes in endogenous HOBr, and is beneficial for a better understanding of − − the interconversion of Br , Br /H2O2, and HOBr in living organisms. The work shown not only presents the first example of a fluorescent probe for the specific detection of HOBr in vivo, but also it paves the way for acquiring other novel fluorescent sensors for HOBr with tailored properties (see the following examples). Soon afterwards, it was found that only H2Te reductant could trigger the recovery of the fluorescence to the original level Molecules 2021, 26, 363 7 of 18

of 5 as the sulfilimine bond can be easily cleaved by H2Te [77]. Thus, compound 5 was demonstrated to be an excellent sequential fluorescence sensor for HOBr followed by H2Te with high sensitivity and selectivity. The detection limit of H2Te was as low as 8.0 µM, and at least four cycles with a reasonable fluorescence decrement were acquired upon alternate addition of HOBr and H2Te. Furthermore, the cyclized product of 5 formed by HOBr could also be capable of detecting H2Te in HepG2 cells. Importantly, this HOBr/H2Te-mediated conjugated system worked very well in modulating the formation and cleavage of the

Molecules 2021, 26, x FOR PEER REVIEWsulﬁlimine bond in both dipeptide and C-terminal noncollagenous (NC1) hexamers,8 of 19 which offers a better understanding of the physiological function of collagen IV.

FigureFigure 6. (a) Fluorescent6. (a) Fluorescent probe probe5 for 5 selective for selective recognition recognition of HOBr of HOBr followed followed H Te;H2Te; (b) ( theb) the mechanism mechanism of of oxidative oxidative cyclization cyclization of the amino group and S-methyl group catalyzed by HOBr; (c) fluorescence2 intensity of 5 after adding various of theactive amino oxidizing group andspecies S-methyl or (d) active group reducing catalyzed species. by Reproduced HOBr; (c) fluorescence with permission intensity from Reference of 5 after [76]. adding Copyright various 2018 active oxidizingAmerican species Chemical or (d) active Society. reducing species. Reproduced with permission from Reference [76]. Copyright 2018 American Chemical Society. Dicyanomethylene-benzopyran (DCMB) derivatives are well-known NIR fluoro- phoresDicyanomethylene-benzopyran with good stability and large (DCMB)Stokes sh derivativesifts owing to are the well-known distinct push-pull NIR fluorophores effect withand good extended stability π-conjugation, and large which Stokes are shifts ideal owing platforms to theto construct distinct push-pullpotent NIR effectfluores- and ex- tendedcent sensorsπ-conjugation, for various which analytes are ideal [78–81]. platforms Conjugation to construct between potent DCMB NIR and fluorescent the skeleton sensors forof various compound analytes 5 ought [78 to–81 afford]. Conjugation a unique fluorescent between probe DCMB for and HOBr. the As skeleton expected, of compoundcom- 5 oughtpound to6 affordhas demonstrated a unique fluorescentto be an excellent probe fluorescent for HOBr. probe As expected, for markedly compound selective 6 has recognition of HOBr over the ROS, reactive nitrogen species (RNS), reactive sulfur spe- demonstrated to be an excellent fluorescent probe for markedly selective recognition of cies (RSS), common biological amino acids and metal ions (Figure 7), although the probe HOBr over the ROS, reactive nitrogen species (RNS), reactive sulfur species (RSS), com- worked in a fluorescence quenching manner [82]. The absorption centered at 478 nm of mon biological amino acids and metal ions (Figure7), although the probe worked in a free probe 6 in PBS-CH3CN (3:2, v/v, pH 7.4) solution sharply decreased, accompanied by fluorescenceappearance quenching of two new mannerpeaks at 392 [82 ].nm The and absorption 448 nm in the centered presence at of 478 HOBr. nm ofMeanwhile, free probe 6 in PBS-CHprobe 36CN showed (3:2, vstrong/v, pH fluorescence 7.4) solution emission sharply at 655 decreased, nm due to accompanied the efficient ICT by appearanceprocess, of twowhich new was peaks mostly at 392 quenched nm and and 448 nmred-shifted in the presence to 700 nm of together HOBr. Meanwhile,with a remarkable probe fluo-6 showed strongrescence fluorescence color change emission from red at 655to colorles nm dues after to the treatment efficient with ICT HOBr. process, The which correspond- was mostly quencheding sensing and mechanism red-shifted could to 700 also nm be together attributed with to HOBr-triggered a remarkable fluorescence cyclization, and color the change fromreaction red to product colorless lacked after satisfactory treatment ICT. with The HOBr. reaction The kinetics corresponding of 6 with HOBr sensing took mechanismabout could8 min also to level be attributed off. Probe 6 to maintained HOBr-triggered its sensing cyclization, ability in a andwidethe pH reactionrange (pH product = 4.0–8.0) lacked satisfactorywith a detection ICT. The limit reaction of 0.66 kineticsμM. Moreover, of 6 with this HOBr probe tookhad a about low cytotoxicity 8 min to level and off.was Probe successfully applied for monitoring HOBr in MCF-7 cells. 6 maintained its sensing ability in a wide pH range (pH = 4.0–8.0) with a detection limit

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Molecules 2021, 26, x FOR PEER REVIEWof 0.66 µM. Moreover, this probe had a low cytotoxicity and was successfully9 of applied19 for monitoring HOBr in MCF-7 cells. Molecules 2021, 26, x FOR PEER REVIEW 9 of 19

FigureFigure 7. 7. Dicyanomethylene-benzopyranDicyanomethylene-benzopyran (DCMB)-based (DCMB)-based near-infrared near-infrared (NIR) fluorescent (NIR) ﬂuorescent probe 6 probe 6 for

forHOBr HOBr in in a fluorescencea fluorescence quenchingquenching manner. manner. Figure 7. Dicyanomethylene-benzopyran (DCMB)-based near-infrared (NIR) fluorescent probe 6 for HOBr in a fluorescence quenching manner. TakingTaking advantage advantage of the of thesame same coupling coupling cyclization cyclization of the amino of the group amino and group S-methyl and S-methyl group, sulfilimine-based fluorescent probe 7 containing 4-CF3-7-aminoquinoline as the group, sulfilimine-based fluorescent probe 7 containing 4-CF -7-aminoquinoline as the fluorophoreTaking for advantagesensing HOBr of the was same described coupling in cyclization detail by Zhu of the and amino coworkers3 group (Figureand S-methyl 8) fluorophore for sensing HOBr was described in detail by Zhu and coworkers (Figure8 )[83]. [83].group, Compound sulfilimine-based 7 displayed fluorescentthree intense probe absorption 7 containing bands peaking4-CF3-7-aminoquinoline at 275 nm, 335 nm, as the andCompoundfluorophore 380 nm in7 PBS-EtOH displayedfor sensing (6:4, threeHOBr v/v intense,was pH described= 7.4) absorption solution. in detail The bands by addition Zhu peaking and of coworkersHOBr at 275 induced nm, (Figure 335 a nm, 8) and decrease380[83]. nm Compound inof PBS-EtOHthe absorption 7 displayed (6:4, atv /275v, three pHnm =and intense 7.4) 380 solution. absorptionnm, as well The bands additionas the peaking emergence of HOBr at 275 of induced twonm, 335new anm, decrease absorptionof theand absorption380 peaks nm in appearing PBS-EtOH at 275 nm at (6:4, 300 and vnm 380/v, andpH nm, =410 7.4) as nm. well solution. The as strong the The emergence emissionaddition ofpeak of HOBr two of 7new inducedat 505 absorption a nmpeaks decreasewas appearing gradually of the absorptiondecreasing at 300 nm atand and 275 a 410 nmnew and nm. emission 380 The nm, strongsignal as well started emission as the to emergence appear peak of at 7of545at two 505nm new nm was alonggraduallyabsorption with a decreasing sustained peaks appearing increase and a newat of 300 HOBr. emission nm andThe 410 signalratios nm. of startedThe fluorescence strong to appearemission intensity at peak 545 (F 545of nm/F 7 505at along) 505 with nm was gradually decreasing and a new emission signal started to appear at 545 nm offereda sustained a good increase linear correlation of HOBr. with The the ratios amount of of fluorescence HOBr in the intensityrange of 0–20 (F545 μM,/F from505) offered a whichgoodalong linearthe withdetection correlation a sustained limit was with increase estimated the amountof HOBr.to be of92 The HOBrnM. ratios In inaddition, theof fluorescence range the low of 0–20 cytotoxicity intensityµM, from (F and545/F which 505) the offered a good linear correlation with the amount of HOBr in the range of 0–20 μM, from gooddetection biocompatibility limit was estimated of 7 alsoto allowed be 92 nM. the Inprobe addition, to recognize the low and cytotoxicity image exoge- and good bio- which the detection limit was estimated to be 92 nM. In addition, the low cytotoxicity and nous/endogenouscompatibility of HOBr7 also in allowed living RAW the probe 264.7 cells to recognize and zebrafish. and image exogenous/endogenous good biocompatibility of 7 also allowed the probe to recognize and image exoge- HOBrnous/endogenous in living RAW HOBr 264.7 in cellsliving and RAW zebrafish. 264.7 cells and zebrafish.

Figure 8. Ratiometric fluorescence probe 7 for HOBr (top) and its fluorescence imaging of HOBr in RAW 264.7 cells (down). Cells were incubated with (A) control; (B) probe 7; (C) probe 7 then treated with HOBr (50 μM); (D) probe 7 then FiguretreatedFigure 8. withRatiometric 8. NaBr Ratiometric (100 ﬂuorescence μM); fluorescence (E) NaBr probe (100 probe7 μforM) 7 HOBrandfor HOBrNAC (top) (100(top) and μ andM, its a ﬂuorescenceits scavenger fluorescence of imagingHOBr) imaging then of HOBroftreated HOBr in with RAWin RAW probe 264.7 264.7 7; cells(F )cells (down). Fluorescence(down). Cells intensity were ratiosincubated (Fgreen with/Fblue ()A from) control; (A–E ().B Blue) probe channel: 7; (C) probe455 nm–505 7 then nm,treated green with channel: HOBr (50545 μnm–645M); (D) nm,probe λex 7 = then Cells were incubated with (A) control; (B) probe 7;(C) probe 7 then treated with HOBr (50 µM); (D) probe 7 then treated 434 treatednm. Reproduced with NaBr with (100 permission μM); (E) NaBr from (100 Reference μM) and [83]. NAC Copyright (100 μ M,2020 a scavengerElsevier. of HOBr) then treated with probe 7; (F) µ µ µ with NaBrFluorescence (100 M); inte (Ensity) NaBr ratios (100 (FgreenM)/F andblue) from NAC (A (100–E). BlueM, achannel: scavenger 455 ofnm–505 HOBr) nm, then green treated channel: with 545 probe nm–6457;(F nm,) Fluorescence λex = intensity434 ratiosnm. Reproduced (Fgreen/F bluewith) frompermission (A–E from). Blue Reference channel: [83]. 455 Copyright nm–505 2020 nm, Elsevier. green channel: 545 nm–645 nm, λex = 434 nm. Reproduced with permission from Reference [83]. Copyright 2020 Elsevier.

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Molecules 2021, 26, 363 9 of 18

Each of the distinct subcellular organelles plays an indispensable role in cellular processes,Each ofwhich the distinctrequires subcellularan appropriate organelles microenvironment plays an indispensable and specific rolebiological in cellular spe- processes,cies to maintain which their requires cellular an appropriate functions [84, microenvironment85]. Mitochondria, and essential specific organelles biological in species most eukaryoticto maintain organisms their cellular and the functions major contributor [84,85]. Mitochondria, to cellular ROS essential levels, organellesare responsible in most for energyeukaryotic supply organisms and aerobic and the metabolism major contributor [86]. Consequently, to cellular ROS monitoring levels, are mitochondrial responsible microenvironmentsfor energy supply and including aerobic metabolismROS, pH, polarity, [86]. Consequently, viscosity, and monitoring temperature mitochondrial may give moremicroenvironments information on including the status ROS, of this pH, organelle polarity, viscosity,[87–90]. and temperature may give more informationOn the onbasis the of status the same of this coupling/cyclization organelle [87–90]. recognition mechanism, rhodamine derivativeOn the 8 basis(Figure of the9) was same rationally coupling/cyclization constructed by recognition Tang and mechanism,coworkers, which rhodamine is the firstderivative example8 (Figure of a mitochondria-targeting9) was rationally constructed fluorescent by Tangprobe and designed coworkers, for monitoring which is thena- tivefirst HOBr example in vivo of a [91]. mitochondria-targeting Rhodamine 110 can fluorescentserve as both probe the fluorophore designed for and monitoring the mitochondria-targetingnative HOBr in vivo unit,[91 ].avoiding Rhodamine chemical 110 canmodification serve as bothof the the parent fluorophore dye with and addi- the tionalmitochondria-targeting mitochondria-targeting unit, avoiding groups. chemicalIn HEPES modification buffer solution of the (10 parent mM, dye pH with 7.4, addi-con- tainingtional mitochondria-targeting 0.3% DMSO as a cosolvent), groups. Inthe HEPES probe buffer8 displayed solution the (10 excitation mM, pH 7.4,and containing emission peaking0.3% DMSO at 495 as and a cosolvent), 530 nm, therespectively probe 8 ,displayed whereas the reaction excitation product and emission of 8 after peaking treat- mentat 495 with and HOBr 530 nm, showed respectively, the excitation whereas and the emission reaction maxima product in of the8 after NIR treatmentregion, located with atHOBr 624 and showed 663 nm, the excitationrespectively. and Benefitting emission maxima from the in high the NIRfluorescence region, located quantum at 624yield and of the663 nm,reaction respectively. product Benefitting(Φ = 0.68) fromin the the NIR high region, fluorescence which quantumcan greatly yield enlarge of the reactionthe sig- productnal-to-noise (Φ = ratio 0.68) and in theimproves NIR region, the detection which can sensitivity, greatly enlarge probe the8 had signal-to-noise an extremely ratio low detectionand improves limit the for detection HOBr (20 sensitivity, pM) and probefast re8sponsehad an time extremely (ca. 3 lowmin). detection Meanwhile, limit this for moleculeHOBr (20 possessed pM) and fast low response cytotoxicity time (ca.(IC50 3 = min). 650 μ Meanwhile,M) and persistence this molecule of the possessed fluorescence low sensingcytotoxicity ability (IC for50 =HOBr 650 µ inM) a andwide persistence pH range (pH of the = 2.0–12). fluorescence This probe sensing did ability not exhibit for HOBr any obviousin a wide interference pH range (pH by =various 2.0–12). bioanalytes This probe including did not exhibit competing any obvious ROS, RNS, interference and com- by variousmonly-seen bioanalytes metal ions including and amino competing acids. It ROS, was RNS,also successfully and commonly-seen utilized to metal image ions native and HOBramino in acids. mitochondria It was also of successfullyHepG2 cells utilizedand zebrafish. to image Taking native the HOBr imaging in mitochondria of zebrafish ofas anHepG2 example, cells andin comparison zebrafish. Taking to Figure the imaging9a,c, a significant of zebrafish fluorescence as an example, enhancement in comparison was observedto Figure 9witha,c, a the significant feeding fluorescenceof Br- followed enhancement by probe 8 was(Figure observed 9b), demonstrating with the feeding that of 8 Br− followed by probe 8 (Figure9b), demonstrating that 8 was capable of monitoring the was capable of monitoring the exogenous HOBr in vivo as HOBr− can be only generated fromexogenous Br- in HOBrthis case. in vivo as HOBr can be only generated from Br in this case.

Figure 9.9. Mitochondria-targeting NIR fluorescentfluorescent probe 8 forfor HOBrHOBr (left)(left) andand itsits fluorescencefluorescence imagingimaging ofof nativenative HOBrHOBr inin zebrafish (right). (a) Zebrafish fed with 8 for 30 min (50.0 μM); (b) zebrafish fed with Br- (100 μM) for 30 min followed by zebrafish (right). (a) Zebrafish fed with 8 for 30 min (50.0 µM); (b) zebrafish fed with Br− (100 µM) for 30 min followed 8 (50.0 μM) for another 30 min; (c) zebrafish incubated with NAC (a scavenger of HOBr,20.0 μM) for 30 min followed by 8 by 8 (50.0 µM) for another 30 min; (c) zebrafish incubated with NAC (a scavenger of HOBr,20.0 µM) for 30 min followed (50.0 μM) for another 30 min. (d–f) Correspond to bright-field images of (a–c). (λex = 633 nm, λem = 650–750 nm). Repro- µ λ λ byduced8 (50.0 with M)permission for another from 30 Reference min. (d– f[91]) Correspond. Copyright to 2017 bright-field American images Chemical of ( aSociety.–c). ( ex = 633 nm, em = 650–750 nm). Reproduced with permission from Reference [91]. Copyright 2017 American Chemical Society. Apart from rhodamine dyes, these fluorophores decorated with one positively Apart from rhodamine dyes, these fluorophores decorated with one positively charged charged group, e.g., the classic pyridinium cation and triphenylphosphonium (TPP), can group, e.g., the classic pyridinium cation and triphenylphosphonium (TPP), can also also have excellent mitochondrial targeting ability owing to the inherent charge attrac- have excellent mitochondrial targeting ability owing to the inherent charge attraction

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Molecules 2021, 26, x FOR PEER REVIEW 11 of 19 from the negative potential of inner mitochondrial membrane [92]. According to this strategy, Tian and Huang introduced a TPP group onto the backbone of 5, leading to obtain tion fromanother the negative mitochondria-targeting potential of inner mitochondrial ratiometric membrane ﬂuorescent [92]. According probe 9tofor this selective recognition strategy,and Tian biosensingand Huang introduced of HOBr witha TPP highgroup selectivity onto the backbone and sensitivity of 5, leading (Figure to 10)[93]. In PBS obtain anothersolution mitochondria-targeti (pH = 7.4, containingng ratiometric 0.5% DMSO), fluorescent probe probe9 behaved 9 for selective almost in the same fashion recognitionto 5and, indicative biosensing of HOBr the nearly-identical with high selectivity ﬂuorescence and sensitivity emission (Figure 10) centered [93]. at 437 nm. The In PBS solutioninitial emission(pH = 7.4, containing of 9 gradually 0.5% DMSO), decreased probe with 9 behaved the addition almost in of the HOBr, same accompanied by the fashion to 5, indicative of the nearly-identical fluorescence emission centered at 437 nm. enhancement of a new emission peak at 528 nm. Compound 9 had a faster response time The initial emission of 9 gradually decreased with the addition of HOBr, accompanied by the enhancement(30 s) and of a a new higher emission detection peak atlimit 528 nm. (1.8 Compound± 0.2 nM) 9 had for a HOBrfaster response as compared to those of 5. time (30 Thes) and low a higher cytotoxicity, detection goodlimit (1.8 biocompatibility ± 0.2 nM) for HOBr and asappreciable compared to those tolerance of 5. to a wide pH range The low( pHcytotoxicity, = 4.0–9.0 good) allowed biocompatibility probe 9 to and function appreciable very tolerance well in real-timeto a wide imagingpH and biosensing range (pHof = HOBr 4.0–9.0) in allowed the mitochondria probe 9 to function of RAW264.7 very well cells.in real-time More imaging importantly, and bio- the work shown here sensing of HOBr in the mitochondria of RAW264.7 cells. More importantly, the work •− clearly demonstrated that endogenous HOBr could also be generated from O2 -induced shown hereoxidative clearly stressdemonstrated rather that than endoge onlynous the Br HOBr− stimulation could also be in generated mitochondria. from O2•−-induced oxidative stress rather than only the Br− stimulation in mitochondria.

Figure 10.FigureA ratiometric 10. A ratiometric ﬂuorescent fluorescent probe probe9 for 9 for bioimaging bioimaging and and biosensingbiosensing of of HOBr HOBr in mitochon- in mitochondria. dria.

The lysosomeThe lysosomeis the main isdigestive the main compar digestivetment in compartment the eukaryotic cell, in the where eukaryotic nu- cell, where numerous merousmacromolecules macromolecules are degraded are for degradedcellular recycling for cellular [94]. Lysosome-targeting recycling [94]. Lysosome-targeting fluorescentfluorescent probes have probes also been have widely alsobeen constructed widely for constructed various biological for various species biological [95– species [95–97]. 97]. In particular,In particular, lysosome lysosome contains containshydrolytic hydrolyticenzymes with enzymes a high proton with concentra- a high proton concentration tion (pH( pH< 6.0), < 6.0 meaning), meaning that thatit performs it performs its function its function only under only underacidic conditions. acidic conditions. Structurally, Structurally,lysosome-localized lysosome-localized fluorescent fluorescent pr probesobes always always incorporate incorporate lipophilic lipophilic amines amines (e.g., morpho- (e.g., morpholine and tertiary amine) [98]. These moieties can be easily protonated, which facilitateline the positively and tertiary charged amine) probes [98 to]. be These entrapped moieties and diffuse can be in easily the lysosomes. protonated, which facilitate the Almostpositively at the same charged time, probestwo 1,8-na to bephthalimide-based entrapped and fluorescent diffuse in probes the lysosomes. 10 [99] and 11 [100] forAlmost selective at detection the same of HOBr time, in two lysosomes 1,8-naphthalimide-based were described in detail fluorescentby two probes 10 [99] differentand groups11 [(Figure100] for 11). selective The two detectionprobes, containing of HOBr the insame lysosomes morpholine were group described in detail by specificallytwo designed different for groups lysosome (Figure location, 11 ).possessed The two a probes,different containingposition of the the func- same morpholine group tional groupsspecifically of 2-methylthiophenyl designed for lysosomeand amino location,groups. This possessed slight structural a different variation position of the functional between the two compounds did cause a distinctly different response towards HOBr in groups of 2-methylthiophenyl and amino groups. This slight structural variation between their photophysical or sensing properties. Naphthalimide derivative 10 was a two-photonthe fluorescent two compounds (88.8 GM) did probe cause with a distinctly a fluorescence different quantum response yield of towards 0.59 that HOBr in their photo- functionedphysical through or a sensingfluorescence properties. switch-off Naphthalimide manner. In HEPES derivative solution (pH10 was= 7.4, acon- two-photon fluorescent taining 0.1%(88.8 DMSO), GM) probeprobe 10 with displayed a fluorescence a strong absorption quantum peak yield at 437 ofnm 0.59 and a that strong functioned through a fluorescencefluorescence centered at switch-off 540 nm, which manner. was In red-shifted HEPES solutionto 451 nm (pH and = 7.4,obviously containing 0.1% DMSO), quenchedprobe upon10 additiondisplayed of HOBr. a strong This on-o absorptionff fluorescence peak phenomenon at 437 nm and was a attributed strong fluorescence centered to efficientat 540PET nm,process which of the was reaction red-shifted product, towhich 451 nmwas andalso confirmed obviously by quenched density upon addition of functional theory (DFT) calculation. With respect to 11 in PBS buffer-CH3CN solution HOBr. This on-off fluorescence phenomenon was attributed to efficient PET process of the (3:2, v/v, 10 mM, pH = 7.4), the addition of HOBr led to a decrease of the emission of the free probereaction at 555 nm product, and the whichappearance was of also a new confirmed emission bypeak density at 610 nm, functional accompanied theory (DFT) calculation. by a prominentWith respect color transformation to 11 in PBS of buffer-CH the solution3CN from solution yellow to (3:2, red underv/v, 10 365 mM, nm UV pH = 7.4), the addition irradiation.of HOBrThe detection led to alimits decrease of 10 and of the 11 for emission HOBr were of the determined free probe to be at 55533.5 nmnM and the appearance of a new emission peak at 610 nm, accompanied by a prominent color transformation of the solution from yellow to red under 365 nm UV irradiation. The detection limits of 10 and 11 for HOBr were determined to be 33.5 nM and 99 nM, respectively. Both of the two probes showed good lysosome-targeting affinity, low cytotoxicity, fast response time (nearly seconds), high degree of selectivity and excellent persistence of sensing ability for Molecules 2021, 26, x FOR PEER REVIEW 12 of 19

Molecules 2021, 26, 363 11 of 18

and 99 nM, respectively. Both of the two probes showed good lysosome-targeting affinity, low cytotoxicity, fast response time (nearly seconds), high degree of selectivity and HOBrexcellent in a wider persistence pH range, of sensing which ability allowed for HOBr it to in work a wider very pH well range, in which imaging allowed of HOBr it to in HeLa cells.work Moreover, very well probe in imaging10 was of capableHOBr in ofHeLa detecting cells. Moreover, endogenous probe HOBr10 was incapable living of mice due to thedetecting nature endogenous of its two-photon HOBr in properties.living mice due Importantly, to the nature the of distinctits two-photon recognition proper- behavior ties. Importantly, the distinct recognition behavior retrieved from the slight structural retrieveddifference from in the the slightpresent structural two examples difference can be inuseful the presentfor the design two examples of other new can fluo- be useful for the designrescent chemosensors of other new with ﬂuorescent novel sensing chemosensors performance. with novel sensing performance.

Figure 11.FigureChemical 11. Chemical structures structures of two of two lysosome-targeting lysosome-targeting fluorescentfluorescent probes probes (a) (10a) and10 and (b) 11 (b based) 11 based on 1,8-naphthalimide on 1,8-naphthalimide framework for selective detection of HOBr. framework for selective detection of HOBr. 4. Probes Based on Substitution Reactions Caused by HOBr 4. ProbesSelective Based recognition on Substitution and monitoring Reactions of HOBr Caused can be by achieved HOBr through substitution reactions, where HOBr acts as either the reactant or catalyst. In 2018, the first substitution-basedSelective fluorescent recognition probe and 4-methylphenol monitoring of (p HOBr-cresol, can12, λ beex = achieved 260 nm, λ throughem = 305 nm) substitution reactions,was described where HOBrto react acts with as HOBr either (Figure the reactant 12), resulting or catalyst. in formation In 2018, of nonfluorescent the first substitution- basedbromination fluorescent products probe (i.e., 4-methylphenol monobromo-, dibr (pomo-cresols)-cresol, 12, [101].λex = Under 260 nm, the specificλem = 305con- nm) was describedditions, to compound react with 12 HOBrdemonstrated (Figure a 12detection), resulting limit for in formationHOBr down of to nonfluorescent0.37 μM. Intri- bromi- nationguingly, products the biothiols (i.e., monobromo-, (e.g., Cys, GSH, dibromo-cresols) Hcy, etc.) could also [101 react]. wih Under HOBr, the giving specific rise conditions,to the corresponding oxidation product, sulfenic acid. The decreasing fluorescence intensity µ compoundof 12 caused12 demonstrated by this HOBr scavenging a detection antioxidant limit for was HOBr not comparable down to 0.37 to thatM. of Intriguingly,the the biothiolsHOBr by itself. (e.g., As Cys, a result, GSH, the Hcy, competitive etc.) couldreaction also of HOBr react between wih HOBr, 12 and giving biothiols rise to the correspondingalso allowed oxidation the probe to product, determine sulfenic the HOBr acid. scavenging The decreasing activity of fluorescence various biothiols. intensity of 12 caused by this HOBr scavenging antioxidant was not comparable to that of the HOBr by Molecules 2021, 26, x FOR PEER REVIEWitself. As a result, the competitive reaction of HOBr between 12 and biothiols also allowed13 of 19

the probe to determine the HOBr scavenging activity of various biothiols.

Figure 12. The competitive reaction of HOBr between 12 and biothiols.

Given the aggregation-induced emission enhancement (AIEE) properties of BODIPY derivative 13 through J-aggregation [102], Kim and coworkers demonstrated that 13 was also an efficient fluorescent probe for selective recognition of HOBr via dibromo substitution (Figure 13) [103]. In aqueous solution (100 mM acetate buffer, pH = 5.0, containing 0.1% CH3CN), this probe had the absorption and emission maxima centered at 580 and 581 nm, respectively. The addition of HOBr triggered the characteristic absorption and emission bands to decrease markedly and red-shift to 613 and 616 nm (ca. 22-fold fluorescence enhancement at 616 nm), respectively. These spectral changes could be ascribed to the suspended aggregates of the formed dibrominated product, which was confirmed by HPLC-MS analysis. HOBr also induced a naked-eye observable color change of the solution from pink to purple, together with a fluorescence color change from orange to red under UV lamp irradiation (365 nm). This probe showed extremely fast response (<2 s) for HOBr, and maintained its sensing ability in a pH range from 4.0 to 9.0. The emission ratio (F616 nm/F581 nm) of 13 was linearly dependent on the concentration of HOBr in the range of 1.0–5.0 μM, from which the detection limit was deduced to be 3.8 nM. The other ROS, and commonly-seen amino acids, biothiols as well as hydrolytic enzymes (including esterase, trypsin, lipase, lysozyme) did not interfere with the selective recognition of HOBr. Specifically for HOCl, this ROS could also react with 13 to give the dichlorinated product but even at a large excess amount ([HOCl]/[13] ≥ 80). As a result, probe 13 was halogenated with a much higher kinetic selectivity for HOBr over HOCl (≥1200 fold). Benefiting from the obtained advantageous photophysical properties, probe 13 was successfully applied to monitoring EPO activity and fluorescence assays of oxidative stress in cancer cells as well as immune response detection in live mice.

Molecules 2021, 26, x FOR PEER REVIEW 13 of 19

Molecules 2021 26 , , 363 12 of 18 Figure 12. The competitive reaction of HOBr between 12 and biothiols.

Given the aggregation-induced emission enhancementenhancement (AIEE) propertiesproperties of BODIPY derivative 13 through J-aggregation [[102],102], Kim and coworkerscoworkers demonstrated that 13 was also anan efficientefficient fluorescent fluorescent probe probe for for selective selective recognition recognition of HOBr of HOBr viadibromo via dibromo substitution substi- (Figuretution (Figure 13)[103 13)]. In[103]. aqueous In aqueous solution solution (100 mM (100 acetate mM acetate buffer, buffer, pH = 5.0,pH = containing 5.0, containing 0.1% 0.1%CH3CN), CH3CN), this probe this probe had the had absorption the absorption and emission and emission maxima maxima centered centered at 580 and at 580 581 and nm, 581respectively. nm, respectively. The addition The addition of HOBr triggeredof HOBr triggered the characteristic the characteristic absorption absorption and emission and emissionbands to bands decrease to decrease markedly markedly and red-shift and red-shift to 613 and to 616613 nmand (ca.616 nm 22-fold (ca. fluorescence22-fold fluo- rescenceenhancement enhancement at 616 nm), at 616 respectively. nm), respectively These spectral. These spectral changes changes couldbe could ascribed be ascribed to the tosuspended the suspended aggregates aggregates of the of formed the formed dibrominated dibrominated product, product, which which was was confirmed confirmed by byHPLC-MS HPLC-MS analysis. analysis. HOBr HOBr also inducedalso induced a naked-eye a naked-eye observable observable color change color ofchange the solution of the solutionfrom pink from to purple, pink to together purple, withtogether a fluorescence with a fluorescence color change color from change orange from to redorange under to redUV under lamp irradiationUV lamp irradiation (365 nm). (365 This nm). probe This showed probe showed extremely extremely fast response fast response (<2 s) for(<2 s)HOBr, for HOBr, and maintained and maintained its sensing its sensing ability ability in a pHin a range pH range from from 4.0 to 4.0 9.0. to 9.0. The The emission emis- sionratio ratio (F616 (F nm616/F nm581/F581 nm nm) of) of13 13was was linearly linearly dependent dependent on on the theconcentration concentration ofof HOBrHOBr inin thethe range of 1.0–5.0 μµM, from which the detection limit waswas deduced to be 3.8 nM. The other ROS, and commonly-seencommonly-seen aminoamino acids, acids, biothiols biothiols as as well well as as hydrolytic hydrolytic enzymes enzymes (including (includ- ingesterase, esterase, trypsin, trypsin, lipase, lipase, lysozyme) lysozyme) did not did interfere not interfere with thewith selective the selective recognition recognition of HOBr. of HOBr.Specifically Specifically for HOCl, for thisHOCl, ROS this could ROS also could react also with react13 towith give 13 the to give dichlorinated the dichlorinated product productbut even but at a largeeven excessat a large amount excess ([HOCl]/[ amount13 ([HOCl]/[] ≥ 80). As13 a] result,≥ 80). As probe a result,13 was probe halogenated 13 was withhalogenated a much higherwith a kineticmuch higher selectivity kineti forc HOBrselectivity over HOClfor HOBr (≥1200 over fold). HOCl Benefiting (≥1200 fold). from Benefitingthe obtained from advantageous the obtained photophysical advantageous properties, photophysical probe 13properties,was successfully probe 13 applied was suc- to cessfullymonitoring applied EPO activityto monitoring and fluorescence EPO activity assays and of fluorescence oxidative stress assays in cancerof oxidative cells as stress well inas cancer immune cells response as well detection as immune in liveresponse mice. detection in live mice.

Figure 13. BODIPY-based ﬂuorescent probe 13 for selective recognition of HOBr through dibrominated substitution. Reproduced with permission from Reference [103]. Copyright 2018 American Chemical Society.

Zhu and coworkers presented 1,8-naphthalimide derivative 14 for the selective detection of HOBr (Figure 14), whose sensing mechanism was based on intramolecular substitution reaction mediated by HOBr rather than HOBr-acting as the brominating reactant [104]. Upon treatment with HOBr, the absorption band of 14 in H2O-EtOH (9:1, v/v, 20 mM PBS, pH = 7.4) shifted from 438 to 424 nm. Initially, probe 14 itself showed weak ﬂuorescence at 530 nm, which was increased and blue-shifted to 505 nm in the presence of HOBr. Meanwhile, compound 14 exhibited a rapid response toward HOBr with the detection limit of 200 nM and reached its saturation point after about 1 min. This probe demonstrated no obvious interference from other ROS, RNS. and various biology-related metal ions. The obtained recognition behavior and good biocompatibility also allowed the probe to visualize HOBr both in RAW 264.7 cells and in zebraﬁsh. Molecules 2021, 26, x FOR PEER REVIEW 14 of 19

Figure 13. BODIPY-based fluorescent probe 13 for selective recognition of HOBr through dibrominated substitution. Reproduced with permission from Reference [103]. Copyright 2018 American Chemical Society.

Zhu and coworkers presented 1,8-naphthalimide derivative 14 for the selective detection of HOBr (Figure 14), whose sensing mechanism was based on intramolecular substitution reaction mediated by HOBr rather than HOBr-acting as the brominating reactant [104]. Upon treatment with HOBr, the absorption band of 14 in H2O-EtOH (9:1, v/v, 20 mM PBS, pH = 7.4) shifted from 438 to 424 nm. Initially, probe 14 itself showed weak fluorescence at 530 nm, which was increased and blue-shifted to 505 nm in the presence of HOBr. Meanwhile, compound 14 exhibited a rapid response toward HOBr with the detection limit of 200 nM and reached its saturation point after about 1 min. This probe Molecules 2021, 26, 363 demonstrated no obvious interference from other ROS, RNS. and various biology-related 13 of 18 metal ions. The obtained recognition behavior and good biocompatibility also allowed the probe to visualize HOBr both in RAW 264.7 cells and in zebrafish.

FigureFigure 14. 1,8-naphthalimide1,8-naphthalimide derivative derivative 14 for14 selectivefor selective detection detection of HOBr. of HOBr.

5. SummaryTable 1. Summary and Outlook of the fluorescent probes for HOBr.

Signal λex/λem Response Detection Entry Probe Solvent System The design and synthesis of fluorescentApplications probes with specific recognition propertiesRef. are quiteType fascinating (nm) as theseTime molecules are amenable to biological imagingLimit applications owing 445/550; 1 1 0.2 M PBS (pH = 7.4)to ratiometric their remarkable advantages900 s of in HOBr-imaging vivo bioimaging in RAW264.7 analysis cells and real-time n.d. a visualization.[68] 610/632 Selective and sensitive recognition of endogenous HOBr are in urgent demand, which 2 2 0.2 M PBS (pH = 7.4) turn-off 702/755 900 s HOBr-imaging in RAW264.7 cells n.d. a [68] 20 mM PBS can make a better understanding of its roles in numerous physiological and pathological 610/635, 3 3 containing 20% processes.ratiometric In this review,3.0 min the advances HOBr-imaging made so in farRAW264.7 in fluorescent cells probes 50 nM for [69] HOBr have 711 CH3CN (pH = 7.4) been discussed, which is the first comprehensive summary related to this area. The monitoring HOBr in arthritis model mice and 10 mM PBS (pH = recognition properties were outlined in Table1, and particular attention was paid to the 4 4 turn-on 395/460 30 s real-time evaluating the development of 30.6 nM [75] 7.4) design strategies, structural diversity, sensing mechanisms, and their applications. arthritis 10 mM PBS Table 1. Summary of the fluorescentimaging endogenous probes for HOBr HOBr. in HepG2 cells and 5 5 containing 0.5% turn-on 480/525 ca. 3.0 min 17 pM [76] zebrafish 3 Response Detection Entry ProbeCH CN Solvent(pH = 7.4) System Signal Type λ λ (nm) Applications Ref. ex/ em Time Limit 10 mM PBS -CH3CN 6 6 turn-off 488/655 8.0 min monitoring HOBr in MCF-7 cells 660 nM [82] 445/550; HOBr-imaging in 1 1 (3: 2, v0.2/v, MpH PBS = 7.4) (pH = 7.4) ratiometric 900 s n.d. a [68] 610/632 RAW264.7 cells 10 mM PBS-EtOH 460/505, tracking the changes of HOBrHOBr-imaging in RAW 264.7 in 2 7 27 0.2 M PBS (pH =ratiometric 7.4) turn-off50 s 702/755 900 s 92 nMn.d. a [83] [68] (6:4, v/v, pH = 7.4). 545 cells and zebrafishRAW264.7 cells 20 mM PBS containing 20% HOBr-imaging in 3 3 10 mM HEPES ratiometric 610/635, 711 3.0 min 50 nM [69] CH CN (pH = 7.4) imaging native HOBr in mitochondriaRAW264.7 cells of 8 8 containing3 0.3% turn-on 624/663 ca. 3.0 min 20 pM [91] HepG2 cells and zebrafishmonitoring HOBr in arthritis model mice and 4 4 DMSO10 mM(pH PBS= 7.4) (pH = 7.4) turn-on 395/460 30 s 30.6 nM [75] 10 mM PBS real-time evaluating the 405/437, imaging of HOBr in mitochondriadevelopment of arthritis 9 9 containing 0.5% ratiometric 30 s imaging endogenous 18 nM [93] 10 mM PBS containing 0.5% 528 RAW264.7 cells 5 5 DMSO (pH = 7.4) turn-on 480/525 ca. 3.0 min HOBr in HepG2 cells and 17 pM [76] CH3CN (pH = 7.4) 10 10 10 mM HEPES turn-off 430/540 immediately imaging of exogenous and endogenouszebrafish HOBr 33.5 nM [99] 10 mM PBS -CH CN (3: 2, v/v, monitoring HOBr in 6 6 3 turn-off 488/655 8.0 min 660 nM [82] pH = 7.4) MCF-7 cells tracking the changes of 10 mM PBS-EtOH (6:4, v/v, 7 7 ratiometric 460/505, 545 50 s HOBr in RAW 264.7 cells 92 nM [83] pH = 7.4). and zebrafish imaging native HOBr in 10 mM HEPES containing 0.3% 8 8 turn-on 624/663 ca. 3.0 min mitochondria of HepG2 20 pM [91] DMSO (pH = 7.4) cells and zebrafish imaging of HOBr in 10 mM PBS containing 0.5% 9 9 ratiometric 405/437, 528 30 s mitochondria of 18 nM [93] DMSO (pH = 7.4) RAW264.7 cells imaging of exogenous and 10 mM HEPES containing 0.1% 10 10 turn-off 430/540 immediately endogenous HOBr in Hela 33.5 nM [99] DMSO (pH = 7.4) cells and mice imaging of exogenous and 10mM PBS-CH CN (3:2, v/v, 11 11 3 ratiometric 475/555, 610 12 s endogenous HOBr in 99 nM [100] pH = 7.4) HeLa cells determination of the HOBr scavenging activity 12 12 distilled water turn-off 260/305 n.d. a 0.37 µM[101] of biothiols and some pharmaceutical samples monitoring EPO activity and fluorescence assays of oxidative stress in cancer 100 mM acetate buffer containing 13 13 ratiometric 480/581, 616 ≤2 s cells (HCT116 and A549) 3.8 nM [103] 0.1% CH CN (pH = 5.0) 3 as well as immune response detection in live mice. a n.d. means not determined. Molecules 2021, 26, 363 14 of 18

Compared to other ROS, HOBr received relatively little attention, and the research area of fluorescent probes for HOBr has gained slow development since the first case was reported in 2012. Developing HOBr-specific fluorescent probes is an inter-disciplinary effort that requires the combined knowledge of organic chemistry, chemical biology and medicinal chemistry. Collectively, there are extensive challenges and opportunities in this emerging research field. In general, construction of new fluorescent probes containing versatile backbones with distinct recognition properties (e.g., high quantum yield, high photostability, rapid response time, low detection limit, high sensitivity and selectivity, low cytotoxicity, organelle-specificity, etc.) is significant, which will undoubtedly enrich the rare examples of fluorescent probes for HOBr. Specifically, the potential interference from HOCl ought to be taken into account when designing a fluorescent probe for HOBr with novel performance, since this reactive oxygen species has a high similarity to HOBr in both chemical and physical properties. Considering that there is only one reported example of a two-photon excitation probe and no case of second near-infrared biochannel (NIR-II, 1000–1700 nm) fluorescent probes for HOBr, more efforts should be made to conceive and study these luminescence probes. This is because these two types of fluorescent probes are crucial for long-time tracking of tissue, body-imaging, and biological processes due to their high deep penetration and low autofluorescence as well as time-resolved fluorescence imaging. A wide range of other types of probes for HOBr including fluorescent nanoparticles, nanoclusters, quantum dots, polymers, proteins, metal complexes (e.g., platinum, ruthenium, iridium, and rare earth complexes, MOF) have not been achieved, which ought to be the subject of initial studies. In addition, a sensing strategy for HOBr using supramolecular assembly has not been addressed. Needless to say, the research of fluorescent probes for HOBr is just starting and still in its infancy, which leaves a great number of possibilities and opportunities. The review shown here not only provides a comprehensive summation of the construction and applications of fluorescent probes for HOBr, but we hope it will also be helpful for boosting this frontier field.

Funding: Yuyu Fang thanks the National Natural Science Foundation of China (No. 22007007), the fellowship from China Scholarship Council (No. 201908510001) and Xinglin Scholar Talent Research Supporting Program of CDUTCM (No. QNXZ2018012) for financial support. Wim Dehaen acknowledges project financing from KU Leuven, grant number C14/19/78. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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