An Endeavor in the Reaction-Based Approach to Fluorescent

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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An Endeavor in the Reaction-Based Approach to Fluorescent Probes for Biorelevant Analytes: Challenges and Achievements Published as part of the Accounts of Chemical Research special issue “Activity-Based Sensing”. Subhankar Singha,†,‡ Yong Woong Jun,† Sourav Sarkar,† and Kyo Han Ahn*,† †

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Department of Chemistry, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyungbuk 37673, Republic of Korea ‡ Institute of Advanced Studies and Research, JIS University, Kolkata 700091, India CONSPECTUS: The promising features of fluorescence spectroscopy have inspired a quest for fluorescent probes for analysis and monitoring of molecular interactions in biochemical, medical, and environmental sciences. To overcome the competitive supramolecular interactions in aqueous media encountered with conventional molecular-recognitionbased probes, the use of reaction-based probes that involve making or breaking of covalent bonds has emerged as a complementary sensing strategy to realize higher selectivity and sensitivity with larger spectroscopic changes. In spite of the enormous efforts, the development of reaction-based fluorescent probes meets with certain challenges in terms of their practical applications, demanding “intelligent design” of probes with an appropriate fluorophore attached to an efficient reactive moiety at the right place. This Account summarizes the results of our efforts made in the development and fine-tuning of reaction-based fluorescent probes toward those goals, classified by the type of analyte (anions, metal cations, and biomolecules) with notes on the challenges and achievements. The reaction-based approach was demonstrated to be powerful for the selective sensing of anions (cyanide and (amino)carboxylates) for the first time, and later it was extended to develop two-photon probes for bisulfite and fluoride ions. The reaction-based approach also enabled selective sensing of noble metal ions such as silver, gold, and palladium along with toxic (methyl)mercury species and paramagnetic copper ions. Furthermore, microscopic imaging and monitoring of biologically relevant species with reaction-based two-photon probes were explored for hydrogen sulfide, hypochlorous acid, formaldehyde, monoamine oxidase enzyme, and ATP.

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1).3 The reaction-based probes provide higher analyte selectivity and sensitivity with larger spectroscopic changes compared with the probes based on non-covalent interactions in most cases. Meanwhile, the reaction-based probes in most cases rely on irreversible processes, limiting their use to the observation of static changes in the analyte concentration level. In spite of this limitation, to take advantage of the merits, enormous research efforts on the development of reactionbased probes have been sought with certain challenges as follows.4−6 The fundamental issues in developing reaction-based fluorescent probes for biological applications are how to realize smart sensing reactions (for the selective detection of the target analyte under physiologically relevant conditions), fast reaction kinetics (for real-time monitoring of biological processes), signal enhancement (for high sensitivity and low detection limits), and reliable data extraction from the environment-sensitive fluorescence signals.7 In addition, the

INTRODUCTION Molecular sensing, which involves molecular-level detection, measurement, and observation, has witnessed flourishing advancement of fluorescence sensing recently because of its simple, fast, and real-time monitoring capability in vitro as well as in vivo.1,2 The promising features of fluorescence sensing in turn have inspired a quest for fluorescent probes, which as versatile means for monitoring of molecular interactions are widely used in biochemical, medical, and environmental science areas as well as in industry. Fluorescent probes comprise a recognition or reaction site for the target analyte and a signaling fluorophore for readout of the associated emission changes. Compared with the conventional recognition-based approach to fluorescent probes, which relies on weak molecular interactions such as H-bonding between host/ receptor and guest/analyte molecules, recently the reaction- or activity-based approach to fluorescent probes, which involves making or breaking of covalent bonds, has emerged as a complementary sensing strategy to overcome the competitive supramolecular interactions encountered in aqueous media and also synthetic challenges related to receptor molecules (Figure © XXXX American Chemical Society

Received: June 15, 2019

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DOI: 10.1021/acs.accounts.9b00314 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Schematic comparison between recognition-based sensing and reaction-based sensing.

Figure 2. General strategies for molecular sensing with (a) rhodamine and fluorescein4 and (b) the two-photon absorbing acedan and benzocoumarin fluorophores.12

probes. The spirolactam forms of these probes, which are nonfluorescent, undergo analyte-triggered spirolactam ringopening reactions to produce “fully” conjugated products that are highly fluorescent (Figure 2a).9 Similarly, O-protected fluorescein dyes offer turn-on sensing schemes, as analytetriggered deprotection reactions restore their fluorescence. In principle, analyte-specific detection can be realized if an analyte triggers a chemical transformation of a probe along with an emission spectral change. As the emission properties (intensity and wavelength) of fluorophores are highly environmentsensitive, to obtain reliable data, ratiometric signaling needs to be implemented in designing fluorescent probes. By attach-

probes should have cell permeability, organelle specificity (for subcellular investigation), and low cytotoxicity. Furthermore, for in vivo and deep-tissue imaging applications, the probes need to operate in the biological optical window with longer emission wavelengths. The last two decades have witnessed various “intelligent” sensing schemes based on appropriate fluorescent dyes (fluorophores), successfully tackling those issues. Since the pioneering work on the rhodamine-based fluorescent sensing of Cu(II) by Czarnik and co-workers,8 rhodamine dyes have received much attention as versatile sensing platforms for various reaction-based fluorescent B


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Figure 3. First reaction-based fluorescent probes for anions, including cyanide, carboxylates, and aminocarboxylates.

ment of another appropriate fluorophore to those rhodamine or fluorescein probes, ratiometric signaling can be realized through Förster resonance energy transfer (FRET).10 In addition, ratiometric signaling can be achieved by modulating the intramolecular charge transfer (ICT) through chemical transformations.11 To this end, dipolar dyes that have electrondonor and -acceptor groups, such as acedan, naphthalimide, dansyl, rhodol, and (benzo)coumarin fluorophores, have been explored frequently, mostly by analyte-triggered chemical transformations at the donor or acceptor sites (Figure 2b). Besides, such dipolar fluorophores are also two-photonexcitable at low-energy near-infrared wavelengths, providing considerable imaging depth inside intact tissue with high 3D resolution, minimal autofluorescence, and reduced photobleaching and photodamage in fluorescence microscopy imaging.12 Notably, two-photon fluorophores with emission wavelengths above 625 nm are desirable for tissue imaging by two-photon microscopy with minimal autofluorescence interference.13,14 This Account summarizes the progress in our laboratory on the development and tuning of reaction-based fluorescent probes for environmentally and biologically important analytes, classified into three analyte categoriesanions, metal cations, and biomoleculeswith notes on the challenges and achievements.

simple linear-shaped anion, poses a fundamental issue of attaining selectivity over other anions in aqueous media, where H-bonding is very competitive. We demonstrated that the reaction-based approach can solve the selectivity issue. Among different types of reversible covalent bond formation between an ionophore and an anion, the trifluoroacetophenone (TFA)based ionophores, introduced by Herman in 1974,18 are capable of unique molecular interactions. However, TFA-based recognition has not been fully appreciated in the area of molecular recognition, mainly because of its relatively weak binding affinity and hence the unfavorable formation of the tetrahedral adduct. In order to shift the equilibrium toward the adduct, which in turn makes the recognition motif useful for sensing of anions, we introduced an o-carboxamido group into TFA to generate o-carboxamido-TFA (CATFA); the carboxamido group stabilizes the anion−ionophore adduct through intramolecular H-bonding and thus enhances the binding affinity to anions (Figure 3).19 As a result, the CATFA system showed a much higher affinity toward cyanide ion compared with the TFA system. The potential use of CATFA to sense cyanide was initially developed by attaching it to suitable chromophores.20 Later, the reactive moiety was fused with a signaling unit, the cyanodiphenylethylene (CDPE) moiety, to generate the new trifluoroacetophenone derivative 1, which takes part in the binding event to directly transduce to the signaling unit.21 Surprisingly, probe 1 detected cyanide selectively over other anions, but through a turn-off fluorescence sensing mode. The results imply that the stabilized H-bonding in the charged anion adduct induced fluorescence quenching in the CDPE moiety. The first turn-on type fluorescent probe for cyanide was realized with probe 2, where the carboxamide in CATFA was replaced with a dansylamide (Figure 3).22 In the probe, the dansyl sulfonamide proton bestows H-bonding assistance to the anion−TFA adduct, which in turn enhances the fluorescence signal. As a result, a >5-fold fluorescence enhancement was observed upon addition of cyanide to probe 2. Among the other common anions, only acetate (AcO−) and fluoride (F−) showed slight

I. ANION SENSING The special roles of anions in biology15 and industrial pollution prompted us to develop fluorescent probes for environmentally toxic cyanide, biologically important carboxylate and bisulfite, and health-hazardous fluoride ions. We demonstrated that the reaction-based approach is indeed a powerful way to selectively sense anions. Cyanide Sensing

The acute toxic effect of cyanide ions (greater than 3 mg/L cyanide in blood may result in death16) demands readily affordable detection methods for various situations.17 The conventional supramolecular approach to sense cyanide, a C


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Accounts of Chemical Research interference with a weak fluorescence enhancement (about 2fold). The intramolecular H-bonding also had a pronounced effect on the selectivity in polar solvents. Notably, attachment of a fluorophore at the amide site of CATFA led to turn-on sensing of anions, which was further extended to develop the first turn-on sensing system for carboxylates. Carboxylate and Aminocarboxylate Sensing

Carboxylates and aminocarboxylates are important classes of bioorganic compounds.23 Detection of carboxylates in the fluorescence turn-on mode is challenging because of the inherent quenching tendency of anionic species. Our previous probe 2 showed strong fluorescence in the presence of anions like cyanide but provided only a marginal fluorescence enhancement in the presence of carboxylates, possibly because of the formation of deprotonated species due to the acidic sulfonamide proton.22 To suppress the deprotonation, the carboxamide group was introduced into an oligothiophene fluorophore as the recognition motif.24 The resulting terthiophene derivative 3 (Figure 3) turned out to sense carboxylates with large fluorescence enhancement (up to 120-fold) along with improved selectivity over competing anions, except cyanide, which also caused similar fluorescence enhancement. The turn-on fluorescence detection of carboxylates with the CATFA motif inspired us to develop a probe for αaminocarboxylates, anionic forms of α-amino acids. The trifluoroacetyl carbonyl group in a probe can act not only as a binding motif but also as a fluorescence quencher. Upon the formation of anion adducts, however, its quenching property may be modulated by perturbing the frontier orbital energy levels. Thus, two CATFA moieties were introduced at the 9and 10-positions of the anthracene fluorophore to form nonfluorescent probe 4 (ΦF = 0.0078), which selectively detected α-aminocarboxylates in a turn-on sensing mode for the first time (Figure 3).25 The high selectivity toward αaminocarboxylates is due to the cooperative nucleophilic addition at the two sites by the carboxylate and amine groups, forming a 1:1 complex between 4 and, for example, glycinate, with significant fluorescence enhancement (110-fold, ΦF = 0.84) and a large association constant (Kassoc = 1.0 × 107 M−1), whereas simple carboxylates and amines separately showed negligible enhancement. Notably, the distance between the two CATFA moieties of the probe (5.22 Å) was suitable to accommodate α-aminocarboxylates (5.48 Å for the glycinate adduct).

Figure 4. Two-photon probes for bisulfite and fluoride.

competing anions and molecules, including cyanide, cysteine, homocysteine, and glutathione. The excellent sensing property along with two-photon excitability of the probe enabled the detection of endogenous bisulfite in cells as well as in deep tissues of various mouse organs, including the brain, for the first time. For quantitative analysis under biological conditions through ratiometric fluorescence response, we developed a two-photon ratiometric probe for bisulfite.28 Probe 6 (Figure 4), developed by the introduction of aldehyde to benzo[g]coumarin (a twophoton absorbing dye), detected bisulfite through a ratiometric shift of the emission wavelength from 625 to 562 nm (Δ = 63 nm) in buffer media within a minute. The ratiometric sensing behavior of probe 6 enabled quantification of the endogenous bisulfite level in human serum. Fluoride Sensing

The negative impact of fluoride in human physiology prompted the development of fluorescence probes, which was accelerated since the Swager group utilized the fluoridetriggered Si−O bond cleavage reaction for the first time.29 In 2012, we developed 7 (Figure 4), the first reaction-based twophoton probe to observe fluoride levels in tissues.30 Nonfluorescent 7 containing a tert-butyldimethylsilyl ether as the reaction motif underwent fluoride-triggered Si−O bond cleavage followed by cyclization to generate the strongly fluorescent iminobenzocoumarin (IBC), which also showed a strong two-photon absorption property (180 GM). The fluorescence enhancement (at 595 nm) was highly sensitive (detection limit ≤ 4.0 ppm) and selective for fluoride over other anions and biothiols. By taking the advantage of twophoton excitation capability, we applied probe 7 for tissue imaging in live zebrafish (in vivo), showing the distribution of fluoride in living animals for the first time.

Bisulfite Sensing

The physiological roles of endogenously produced sulfur dioxide and its aqueous equilibrium species, bisulfite and sulfite, still remain unclear, mainly because of the lack of suitable analytical tools. In this context, several probes have been developed since the initial report by Mohr group on colorimetric detection of bisulfite (sulfur dioxide species) through the formation of an aldehyde−bisulfite covalent adduct.26 Aiming at practically useful probes with deep-tissue imaging capability, in collaboration with Das group we developed compound 5 as a two-photon probe for bisulfite (Figure 4).27 The benzoxazinone-based probe contains an aldehyde moiety and thus can form a bisulfite adduct with a turn-on fluorescence response in the red wavelength region (∼600 nm). Besides the fast reactivity (4.5 min) and low detection limit (1.86 × 10−6 M; high sensitivity), probe 5 also showed excellent selectivity toward bisulfite/sulfite over

II. METAL CATION SENSING To understand the biology and toxicological effects of metals, the development of fluorescent probes for metal cations with advanced sensing properties has attracted considerable research interest in recent years.31 In this context, our research endeavor was focused on the development of efficient fluorescent probes for noble metals such as silver, gold, and D


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Figure 5. Reaction-based fluorescent probes for noble metal species (silver, gold, and palladium ions), toxic mercury species, and paramagnetic Cu(II).

(detection limit of 14.0 ppb) enabled the quantification of silver species (including silver nanoparticles for the first time) present in consumer products such as fabric softener and hand sanitizer gel. This smart sensing strategy of rhodamine lactam ring opening followed by cyclization has become familiar as an excellent reaction-based sensing platform for selective detection of various other analytes.

palladium as well as toxic mercury species and biologically essential copper ions. Silver Sensing

Reaction-based fluorescence sensing of Ag(I) was initially reported by Czarnik and co-workers, who applied the metalinduced desulfurization reaction.32 However, their probe failed to discriminate Ag(I) from Hg(II). We disclosed the selective sensing of Ag(I) through a reaction-based approach by taking an advantage of Ag(I)-induced alkyl iodide activation.33 Thus, nonfluorescent N-iodoethylrhodamine lactam 8, upon Ag(I) addition, underwent spirolactam ring opening and subsequent oxazoline ring formation to generate a fluorescent ring-opened rhodamine derivative (Figure 5). Complete selectivity for Ag(I) over other metal species along with high sensitivity

Gold Sensing

Gold ions, commonly appearing as Au(III) and Au(I) complexes, show strong alkynophilicity to the alkyne bond, which allows reactions with oxygen, nitrogen, and even carbonbased nucleophiles. Thus, following the similar activation approach to the Ag(I) sensing, an alkyne activation approach was subsequently utilized for the initial reaction-based E


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Accounts of Chemical Research fluorescence sensing of gold complexes by our group34 and concurrently by the Tae group,35 and Kim, Lee and Yoon groups.36 The nonfluorescent N-propargylrhodamine lactam 9 (Figure 5) detected gold species selectively in a turn-on fluorescence mode. This pioneering work using the reactionbased sensing strategy for gold ions through alkyne activation was adopted thereafter by others.37 The subsequent mechanistic investigation performed by us using model compounds elucidated a sensing mechanism including characterization of novel vinylgold intermediates by X-ray crystal structure analyses.38 Unfortunately, such a rhodamine lactam ringopening approach of gold sensing raised issues of low reaction rates (no signal saturation even after 1 h) and the formation of undesired side products depending upon the sensing conditions, which may deteriorate the detection limit and reliability of quantification data. Those issues could be avoided using a different sensing scheme where the signaling unit becomes free from the reaction site upon interaction with the target analyte. Accordingly, mono- and bis(2-ethynyl)benzoyl derivatives of fluorescein (10 and 11, respectively; Figure 5) were developed to sense Au(III) through alkyne activation followed by ester hydrolysis to generate the free fluorophore as the major product, which avoided side reactions and provided a high sensing rate (completed within 1 h).39 However, probes 10 and 11 containing ester linkages would not be stable enough toward ubiquitous cellular esterase activity, which may limit their further use for fluorescence imaging and monitoring of gold species in cells. To overcome the aforementioned issues in gold sensing, we modified the previous rhodamine lactam ring-opening approach by introducing steric strain around the reaction site with an N-(2-ethynylphenyl) moiety,40 which in turn enhanced the reaction rate (∼4.5 times larger pseudo-first-order rate constant than the case of probe 9) as well as suppressed the competing undesired side reactions. As a result, a high turn-on sensitivity with a detection limit of up to 0.5 ppb Au(III) was achieved with probe 12 (Figure 5). The promising sensing behavior of the parent system was extended further to observe a ratiometric detection of gold species through the development of FRET sensing system 13 containing a naphthalimide as a donor fluorophore (Figure 5). Interestingly, the FRET system was highly efficient in sensing even stabilized Au(I) species and enabled ratiometric fluorescence imaging of AuCl3 in cells, promising results for future monitoring of gold species in living systems.

distribution-dependent manner upon exogenous addition of PdCl2. Besides the efficient sensing of palladium species, probe 14 also showed a slight response toward Au(III), which may cause artifacts during analysis of industrial or environmental samples, where Pd(II) usually exists together with other metal ions such as Pt(II), Rh(III), Au(I)/Au(III), or Ag(I). For the selective recovery method with high sensitivity toward palladium species, we further developed a novel fluorogenic detection system based on a metal-catalyzed oxidative insertion reaction.43 Rhodamine spirolactam derivative 15 containing an N-(2-iodophenyl) substituent underwent Pd(0)-catalyzed oxidative insertion followed by spirolactam ring opening and subsequent reductive elimination to generate a fluorescent rhodamine derivative (Figure 5). Although additional reducing agent is necessary to detect Pd(II) species, probe 15 is suitable for analysis of industrial samples, as demonstrated by quantification of the residual palladium content from a Suzuki−Miyaura coupling reaction. Mercury Sensing

In a search of fluorescence detection systems for Hg(II) species, especially focusing on notoriously toxic methylmercury (CH3HgX) species, we developed an aryl vinyl ether-type probe for the first time. Probe 16 underwent mercury-ionpromoted ether hydrolysis to show a turn-on fluorescence signal at 520 nm (Figure 5).44 The sensing mechanism, involving oxymercuration of the vinyl ether to provide a hemiacetal intermediate followed by fragmentation into the alcohol and aldehyde, enabled us to overcome the existing challenges:45 selective detection with high sensitivity (detection limit below 1.0 ppb for HgCl2 under ambient conditions) and, more importantly, the detection of CH3HgX species for the first time. Methylmercury contamination in living systems like cells and zebrafish was efficiently monitored using probe 16 through fluorescence imaging, which also revealed the organ-selective accumulation of methylmercury in zebrafish. The sensing mechanism further prompted us to develop ratiometric probe 17 (Figure 5) by attaching a reactive vinyl ether moiety to the signaling unit of 2-(benzothiazol-2yl)phenol fluorophores, which show blue emission (λ = 420 nm) in the ether form but green emission (λ = 500 nm) through an excited-state intramolecular proton transfer (ESIPT) mechanism upon ether hydrolysis.46 Unfortunately, the ratiometric response of probe 17 was not sufficient for bioimaging because of the low fluorescence quantum yields of the fluorophore and the probe, in addition to the inferior Hg(II) sensitivity compared with the previous probe 16. By aiming at ratiometric sensing along with deep-tissue imaging capability, we developed the first two-photon ratiometric probe for mercury ions based on an acedan analogue.47 The blueemitting probe 18 (λ = 428 nm) contains a dithioacetal moiety that undergoes mercury-ion-promoted selective and fast deprotection to yield the corresponding green-emitting aldehyde (λ = 525 nm) (Figure 5), with an emission shift of 97 nm. The large emission shift is highly beneficial to observe the probe and mercury species in living systems in separate emission channels under microscopic imaging. The Hg(II)promoted hydrolysis mechanism and the fluorescence titration stoichiometry of 18 indicated that it can also be used for the detection of methylmercury species. The probe provided excellent sensitivity toward Hg(II) with a detection limit of 3.0 ppb. Because of the use of a sulfur-containing probe, however, a competitive reaction with Ag(I) was also observed. This

Palladium Sensing

The first reaction-based palladium detection was reported by the Koide group, who utilized a Pd(0)-catalyzed Tsuji−Trost allylic oxidative insertion reaction.41 However, the sensing system required high temperature and additional reagents depending on the oxidation state of palladium, limiting its use for biological applications. Aiming at the detection of palladium species under ambient conditions irrespective of the Pd oxidation state, we explored sensing based on Pdcatalyzed depropargylation for the first time. Probe 14 (Figure 5), a fluorescein-based propargyl ether, underwent hydrolysis catalyzed by palladium species in different oxidation states (Pd(0), Pd(II), and Pd(IV)) to generate the free fluorophore with turn-on fluorescence response.42 The high sensitivity (detection limit of 3.2 ppb for Pd(II)) along with low cytotoxicity of the probe enabled for the first time fluorescent imaging and monitoring of palladium species in vivo (in zebrafish) in a concentration-dependent as well as organF


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Figure 6. Two-photon probes for advanced molecular sensing of various biomolecules, including biothiols,55 hydrogen sulfide,56,60 hypochlorous acid,63 formaldehyde,67 monoamine oxidase70 and amyloid-β plaques,71 and adenosine triphosphate.75

essential biorelevant analytes, such as hydrogen sulfide (a redox-active gasotransmitter), hypochlorous acid (a reactive oxygen species with a protective role), formaldehyde (a gaseous signaling molecule), monoamine oxidase (a neurological disease-associated enzyme), and ATP (an intracellular energy source and signaling molecule).

competition may not be an issue during bioimaging as the presence of Ag(I) in normal living systems is low. Thus, the probe was successfully applied for ratiometric imaging of Hg(II) ions in cells for the first time under both one- and twophoton microscopes. Copper Sensing

The detection of paramagnetic Cu(II) in the fluorescence enhancement mode is highly challenging because of its inherent fluorescence quenching property.48 We disclosed a new sensing scheme involving Cu(II)-promoted enolization of dipicolylamine-substituted acedan 19 and subsequent reduction to produce diamagnetic Cu(I) with ratiometric emission changes (from 540 to 435 nm) (Figure 5).49 The probe showed high sensitivity with a detection limit of 89 nM, enabling the determination of trace amounts of Cu(II) present in environmental samples as well as two-photon microscopy imaging of intracellular Cu(II) ions. The sensing mechanism may be considered for the previous works of fluorescence turnon sensing with probes that involve intramolecular charge transfer upon binding of Cu(II) ions.50

Hydrogen Sulfide Sensing

As an endogenously produced gaseous biothiol, H2S is recognized as the third endogenous gasotransmitter to take part in a series of physiological and pathological processes.52 Consequently, the development of fluorescent probes for H2S has been intensely pursued following the initial sensing strategy of H2S-mediated reduction of an aryl azide developed by the groups of Chang53 and Wang54 concurrently. The major challenges in this endeavor are how to secure high selectivity (against other competing biothiols such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy)), excellent sensitivity (high enough to detect the low levels of endogenous H2S), fast response (to follow the rapid H 2 S fluctuation), and biocompatibility (stable toward hydrolytic enzymes, nontoxic, and cell-permeable) along with favorable photophysical properties for tissue imaging applications. In this context, our initial work on probe 20 (Figure 6), an arylsulfonyl azide that reacts rapidly (within 2 min) and is two-photon-excitable, raised another issue of low selectivity against other biothiols in such electron-deficient azides (especially GSH) at biologically

III. ADVANCED SENSING OF BIOMOLECULES Recently, fluorescence sensing has been advanced toward twophoton microscopic imaging, which enables deep-tissue imaging with reduced photobleaching and photodamage.51 In this context, we have developed two-photon probes for several G


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Accounts of Chemical Research relevant concentrations.55 Later, we addressed all of those required sensing properties by developing two-photon probe 21 containing an electron-rich and sterically demanding Michael acceptor moiety as the H2S-reactive unit (Figure 6).56 Also, we revised the previously proposed sensing mechanism57 by extensive NMR studies. According to the new mechanism, the probe undergoes tandem conjugate addition promoted by initial formation of the sulfide−aldehyde adduct followed by an intramolecular aldol reaction, which accompanies turn-on fluorescence change. The steric and electronic modulation of the reactive moiety endowed the probe with high selectivity as well as fast reactivity (within 5 min) toward H2S. The two-photon excitability of the corresponding acedan fluorophore along with the high sensitivity (detection limit of 50 nM) and high selectivity (particularly over GSH and Cys) of the probe enabled us to image endogenously produced H2S with negligible interference from other biothiols in live cells by two-photon microscopy. The fine-tuning of the selectivity, sensitivity, and reactivity to practically useful levels combined with low cytotoxicity and two-photon bioimaging capability makes the probe a powerful tool for studying the biological roles of H2S. Through collaborative works, the probe was thus applied to observe the regulation of H2S production by the hypothalamic-pituitary axis58 and amino acid restriction-triggered angiogenesis.59 Recently, as a π-extended version of this H2S probe, we developed probe 22 by replacing the acedan fluorophore with the benzocoumarin dye (Figure 6), which is two-photonexcitable at 900 nm and emits in the red wavelength region (above 625 nm),60 which are advantageous features for tissue imaging applications.

Formaldehyde Sensing

Formaldehyde (HCHO), an endogenously produced reactive carbonyl species, takes part in various biological processes, including the storage, preservation, and retrieval of long-term memory. The first reaction-based approach to sense HCHO was reported independently by the groups of Chang65 and Chan.66 To tackle the additional issues in tissue imaging, we developed two-photon probe 24, which has ratiometric imaging capability (Figure 6).67 The probe, composed of a pro-aza-Cope rearrangement moiety (sec-homoallylamine) attached to 1-(naphthalen-2-yl)pyrrolidine (two-photon-absorbing and blue-emissive at 438 nm, ΦF = 0.92), forms an iminium ion in the presence of formaldehyde, which undergoes a cationic 2-aza-Cope rearrangement followed by hydrolysis to produce 6-(pyrrolidin-1-yl)-2-naphthaldehyde (a two-photonabsorbing dye with green emission at 533 nm, ΦF = 0.17). The excellent sensing properties along with the biocompatibility of the probe enabled us to observe various levels of endogenous formaldehyde in cells as well as different organ tissues. An advanced application of the probe for ratiometric imaging of mouse intestinal organoids (villi and crypts) by two-photon ratiometric imaging revealed for the first time a high level of formaldehyde around the Paneth cells of the small intestine, which implicates a possible antimicrobial role of formaldehyde in the animal intestine. Monoamine Oxidases and Amyloid-β Sensing

Monoamine oxidases (MAOs), flavin-dependent enzymes that catalyze the oxidation of a variety of amine neurotransmitters, are associated with the functions of the central nervous system. For the purpose of monitoring and diagnosis of MAOdependent neurodisorders as well as for screening of enzyme inhibitors, the development of practically useful fluorescent probes for MAOs is highly demanded. The first reaction-based fluorescent probe for MAOs, reported by the Silverman group,68 was based on amine oxidation followed by iminium intermediate hydrolysis. This sensing scheme was later modified by the Sames group, who developed a turn-on fluorescent probe that underwent a sequence of oxidation and intramolecular cyclization by MAOs.69 We also adopted the oxidative cleavage chemistry to develop the first two-photon MAO probes, 25 and 26 equipped with a phenyl ether containing propylamine (Figure 6),70 which underwent MAOinduced oxidation followed by iminium ion hydrolysis and βelimination to generate the phenol intermediate, which in turn underwent fast cyclization with the nearby dicyanovinyl moiety to form the highly fluorescent (λem = 585 nm, ΦF = 0.63) and two-photon-absorbing (σ2 = 180 GM) imino-benzocoumarin (IBC) dye. The excellent sensing properties of probe 25 enabled us to visualize MAO activity in cells by two-photon microscopy. We recently applied probe 25 for in vivo monitoring of MAOs activity with respect to the progress of Alzheimer’s disease (AD), a dementia-related neurodisorder, by following the increase in amyloid-β (Aβ) plaques upon aging in ADmodel mice.71 The underlying strategy can be described as follows: probe 25 catalyzes oxidation of MAOs (mostly MAO B in the brain), which produces the fluorescent IBC; IBC molecules present outside Aβ plaques appear as a moderately fluorescent background signal, whereas IBC molecules present inside Aβ plaques, which provide a hydrophobic and highly congested microenvironment, appear as bright fluorescent dots. Thus, by following and quantifying both the background

Hypochlorous Acid Sensing

Hypochlorous acid (HOCl), an endogenously produced reactive oxygen species (ROS), is of special interest because of its role in enhancing immunity through destruction of pathogens in biological systems. The strong oxidizing property, short life, and relatively low biological concentrations of HOCl make it a challenging task to develop the corresponding fluorescent probe for biological application.61 The first twophoton fluorescent probe for HOCl was reported by Chang and co-workers, who utilized oxidative deprotection of an oxathiolane as a new sensing scheme.62 The acedan-based oxathiolane probe showed excellent sensing properties in detecting endogenous HOCl and was used to monitor inflammation-induced HOCl production. As the first version operated only in turn-on signaling mode, in collaboration with Chang’s group we developed its ratiometric version, probe 23 (Figure 6), by attaching the oxathiolane moiety to a benzocoumarin derivative. HOCl-promoted deprotection of the oxathiolane moiety generates the acetylbenzocoumarin, which causes a significant change in ICT and thus ratiometric signaling from yellow (λ = 598 nm) to red (λ = 633 nm) fluorescence.63 Notably, the benzocoumarin containing a proline donor64 showed brighter emission at the longer wavelength, including strong two-photon absorption properties. The probe was highly sensitive toward HOCl because of suppression of the byproduct formation observed with the previous acedan-based probe; hence, it enabled detection of endogenous HOCl without application of any external stimuli in live cells (HeLa cells) as well as in mouse brain tissues through ratiometric imaging by two-photon microscopy. H


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would extend the scope of molecular sensing and imaging to the practically useful level for biomedical applications.

and Aβ plaque signals in mice at different disease states, we were able to correlate the MAO activity with the progress of AD by in vivo two-photon microscopy imaging. It was revealed for the first time that the MAO activity is closely correlated with the progress of AD, which draws our attention to MAOs as potential biomarkers of AD.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ATP Sensing

ORCID

Adenosine triphosphate (ATP), known as an intracellular energy source, is involved in almost all of the fundamental biological activities; hence, maintaining ATP homeostasis in the human body is essential.72 For use in monitoring of ATPassociated biological processes, various fluorescent probes for ATP have been developed, most based on coordinationinduced binding of the ATP triphosphate group with Zn(II)− dipycolylamine (ZnIIDPA) complexes, including the first twophoton turn-on probe based on acedan−ZnIIDPA, which was reported by us.73 Other than the ZnIIDPA coordination, a rhodamine B spirolactam ring-opening process through electrostatic interactions between a polyamine moiety (attached to the rhodamine) and the ATP triphosphate group was explored by the Li and Zhu group to develop a reaction-based turn-on ATP sensing system.74 On the basis of this pioneering work, we developed probe 27 as the first ratiometric two-photon probe for ATP by optimizing the reaction kinetics and emission properties (Figure 6).75 The probe is composed of a rhodamine 6G spirolactam and a twophoton-excitable blue-emitting boron dipyrromethene (BODIPY) moiety linked by a polyamine spacer. Because of the weak binding affinity of the probe toward ATP at neutral pH, the acidic lysosomal environment (pH 4.5−5.5) effected ring opening of the rhodamine 6G spirolactam, making it a lysosomal ATP probe. The two-photon excitation capability of the probe enabled monitoring of lysosomal ATP changes in living cells for the first time through ratiometric imaging by two-photon microscopy. The practical applicability of the probe was further demonstrated through real-time monitoring of lysosomal membrane fusion processes (kiss-and-run and full-collapse) in living cells along with quantitative information on lysosomal ATP changes for the first time.

Subhankar Singha: 0000-0002-8234-4151 Yong Woong Jun: 0000-0001-9359-1265 Kyo Han Ahn: 0000-0001-7192-7215 Notes

The authors declare no competing financial interest. Biographies Subhankar Singha received his Ph.D. degree in 2013 under Prof. Kyo Han Ahn at POSTECH. After working in the same group as a postdoctoral researcher, he was promoted to Research Assistant Professor in 2015. In 2019, he joined the Institute of Advanced Studies and Research at JIS University in Kolkata, India, as an Assistant Professor. His research interests include the development of fluorescent molecules and probes as well as theranostic agents. Yong Woong Jun received his Ph.D. degree in 2018 under Prof. Kyo Han Ahn at POSTECH. He is now working as a postdoctoral researcher at Stanford University. His research interests include fluorescent molecular probes and two-photon microscopic imaging. Sourav Sarkar received his M.S.. degree in 2015 at IIT Kharagpur, India. Currently he is pursuing his Ph.D. degree under Prof. Kyo Han Ahn at POSTECH. Kyo Han Ahn received his Ph.D. degree from KAIST in 1985. He joined the Department of Chemistry at POSTECH in 1986 and was promoted to Full Professor in 2001 and to Distinguished Professor in 2016. He is a fellow of the Korean Academy of Science and Technology. His current research interests include luminescent materials, molecular probes and imaging agents, and bioconjugation.

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ACKNOWLEDGMENTS This work was financially supported by the Global Research Laboratory Program (2014K1A1A2064569) through the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, Republic of Korea. S. Singha acknowledges support from the Basic Science Research Program (2018R1D1A1B07051403) through the NRF of Korea, funded by the Ministry of Education.

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CONCLUDING REMARKS Focusing on the reaction-based approach to molecular sensing of anions, metals cations, and biomolecules, this Account summarizes our continuous efforts on the development of novel reaction-based fluorescent probes with advanced and optimized sensing properties, including two-photon imaging capability for real-time monitoring of biological processes even in subcellular organelles. It is evident that the reaction-based approach to develop fluorescent probes has flourished in the last two decades, providing valuable tools for studying various biological analytes. However, practically useful probes for biomedical applications are still limited in number, demanding further efforts in the field to tackle the challenges and limitations. Considering the highly environment-sensitive nature of the fluorescence intensity, ratiometric probes are essential, particularly those capable of ratiometric imaging in cells and tissues. In addition, probes based on far-red/nearinfrared-emitting fluorophores are in demand for deep-tissue or whole-body imaging. More creative ideas on the reactionbased approach to fluorescent probes along with efforts to develop fluorophores with desirable photophysical properties

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