Fluorescent Chemosensors for Various Analytes Including Reactive Oxygen Species, Biothiol, Metal Ions, and Toxic Gases.

The development of fluorescent chemosensors for various analytes has been actively pursued by chemists. Since their inception, these efforts have led to many new sensors that have found wide applications in the fields of chemistry, biology, environmental science, and physiology. The search for fluorescent chemosensors was initiated by a few pioneering groups in the late 1970s and 1980s and blossomed during the last two decades to include more than hundreds of research groups around the world. The targets for these sensors vary from metal ions, anions, reactive oxygen/nitrogen species, biothiols, and toxic gases. Our group has made contributions to this area in last 18 years. In this perspective, we briefly introduce the history of chemosensors and review studies that we have carried out.

Introduction

Fluorescent chemosensors were defined at that early time as “copan class="Gene">mpounds incorpopan class="Species">rating a binding site, a fluorophore, and a mechanism for communication between the two sites” (Figure ).[1] However, if the fluorescent sensor participates in an irreversible chemical reaction with a specific analyte, then it is generally called a fluorescent chemodosimeter.[2] However, in the last couple of decades, the terms fluorescent chemosensors and fluorescent chemodosimeters have been used interchangeably and fluorescent probes are now more commonly referred to by either name, especially when they are used in biological applications. In this perspective, we use the term “fluorescent chemosensors” to refer for these systems.

Figure 1

Picture of ACS Symposium Series 538 “Fluorescent Chemosensors for Ions and Molecular Recognition” edited by Anthony W. Czarnik.

Picture of pan class="Gene">ACS Sypan class="Gene">mposium Series 538 “Fluorescent Chemosensors for Ions and Molecular Recognition” edited by Anthony W. Czarnik.

During the late 1970s and in 1980s, Sousa,[3] Bousa-Laurent,[4] de Silva,[5] Tsien,[6] Czarnik,[7] and others carried out pioneering investigations focusing on the development of fluorescent chemosensors, in which crown and n class="Chemical">azacrown ethers or other ligands were linked to fluorophores. These chemosensors recognize n>n class="Chemical">metal ions in a selective manner through changes taking place in fluorescence signals. In most cases, methylene bridges were incorporated between the ligands and fluorophores and the fluorescence was modulated by the presence or absence of photoinduced electron-transfer (PET) quenching process involving lone-pair electrons on amine nitrogens or ether oxygens. Typically, fluorescence emission is turned on upon binding of the metal ion as a consequence of blocking PET, referred to as chelation-enhanced fluorescence. As a result, in its early stages, the development of chemosensors was benefited greatly by advances made in host–guest, macrocyclic, and supramolecular chemistry.[8]

The scope of fluorescent chemosensors was expanded in continuing efforts by taking advantage of different or combinations of photophysical mechanisms and new concepts, such as aggregation-induced emission (AIE),[9] two-photon excitation micn class="Chemical">roscopy,[10] and, more recently, high- and super-resolution fluorescence micn>n class="Chemical">roscopy.[11] Probably, the most dramatic advances in this area can be attributed to the development of chemodosimeters that operate on the basis of analyte selective chemical reactions. Even though fluorescent sensors of this type have the clear disadvantage of irreversibility not shared by fluorescent chemosensors based on host–guest chemistry, they often show very high selectivities toward target analytes.[12,13]

In this perspective, we pan class="Chemical">briefly introduce the history of chemosensors and discuss the contributions to this topic we made during last 18 years. The perspective is organized by types of analytes, such as pan class="Chemical">metal ions, anions, reactive oxygen species (ROS), biothiols, and gases. In addition, the concepts employed to design sensors based on analyte-specific binding and reaction will be discussed. Finally, the future directions of research in this area will be commented on briefly.

Fluorescent Chemosensors for Various Analytes

Fluorescent Chemosensors for Metal Ions

pan class="Chemical">Zn2+ is reported to be involved in various physiological processes and diseases, such as n>n class="Disease">ischemic stroke, epilepsy, and Alzheimer’s disease. As a result, we have designed sensors for this metal ion that contain various binding sites or ligands and various fluorophores.[14,15] One example is the Zn2+ chemosensor based on the 7-nitrobenz-2-oxa-1,3-diazole derivative 1, bearing nitrogen binding sites. This substance shows a selective enhancement (5.5-fold) of fluorescence intensity at pH 7.2 upon the addition of Zn2+.[16] In addition, a distinct colorimetric change from red to yellow occurs upon Zn2+, which was attributed to the operation of an internal charge-transfer (ICT) mechanism, as shown in Figure . Pancreatic islets, which play an important role in insulin biosynthesis, contain relatively high Zn2+ concentrations. As a result, the utility of the new probe was demonstrated by its use to detect intrinsic Zn2+ ions present in pancreatic β-cells.

Figure 2

Stepwise binding mechanism of 1 with Zn2+.

Stepwise binding mechanism of 1 with pan class="Chemical">Zn2+.

In another study, the pan class="Chemical">naphthalimide derivative 2, bearing a n>n class="Chemical">DPA group, in aqueous solution was reported to bind Zn2+ in both amide and imidic acid tautomeric forms. For example, 2 binds Cd2+ via an amide tautomeric form (Figure ).[17] The existence of the two different binding modes was supported by nuclear magnetic resonance (NMR) and IR data. Moreover, the addition of Zn2+ induces a 22-fold fluorescent increase in the intensity and a red shift (483 nm to 514 nm) of the emission band of 2. On the contrary, a hypsochromic shift (483–446 nm) takes place upon the addition of Cd2+, which can be attributed to binding via the amide tautomeric form. The respective dissociation constants of 2 for Zn2+ and Cd2+ were calculated to be 5.7 and 48.5 nM. 2 displays green emission in response to Zn2+ and blue emission for Cd2+ in live cells. As shown in Figure , intrinsic Zn2+ ions can be imaged during the development of live zebrafish embryos using 2.

Figure 3

Proposed binding of 2 with metal ions via amide and imidic acid tautomeric forms.

Figure 4

Images of zebrafish pretreated with 5 μM probe 2 for 1 h. (a) Images of 19 h-old, (b) 36 h-old, and (c) 48 h-old zebrafish. (d) Image of 54 h-old zebrafish incubated with 2 for 1 h and (e) image of 54 h-old zebrafish after pretreatment with TPEN (100 μM) for 1 h and subsequent treatment of washed zebrafish with 2 for 1 h (ref (17)).

Proposed binding of 2 with pan class="Chemical">metal ions via n>n class="Chemical">amide and imidic acid tautomeric forms.

Images of pan class="Species">zebrafish pretreated with 5 μM probe 2 for 1 h. (a) Images of 19 h-old, (b) 36 h-old, and (c) 48 h-old pan class="Species">zebrafish. (d) Image of 54 h-old zebrafish incubated with 2 for 1 h and (e) image of 54 h-old zebrafish after pretreatment with TPEN (100 μM) for 1 h and subsequent treatment of washed zebrafish with 2 for 1 h (ref (17)).

Near-infrared (NIR) probes for pan class="Chemical">metal ions, which emit light in the range of 650–900 nm, have strong advantages, such as relatively deep tissue penetration and minimal background interference and photodamage.[18] As a result, we designed and prepared the cyanine derivative 3, bearing a tris(2-pyridylmethyl)amine moiety, as a Zn2+ selective NIR probe.[19,20] This cyanine derivative contains a fully extended polymethine chain that leads to an absorption maximum at 670 nm and an emission maximum at 730 nm. Addition of Zn2+ induces significant hypsochromic shifts in the absorption of 3 to 510 nm and in its emission to 590 nm, which were attributed to the disruption of conjugation by forming the less delocalized diamino-tetraene chromophore, as shown in Figure . The dissociation constant of 3 with Zn2+ was calculated to be 1.2 nM. Cyanine derivative 3 can be utilized to image Zn2+ released during apoptosis and endogenous Zn2+ in zebrafish, as shown in Figure . Especially interesting, a strong red emission was observed in zebrafish neuromasts, in which expressed metallothioneins play an important role in Zn2+ homeostasis.

Figure 5

Proposed binding mechanism for unique hypsochromic shift of 3 upon the addition of Zn2+.

Figure 6

Fluorescence detection of intrinsic Zn2+ ions in zebrafish using probe 3 (a) 24, (b) 36, (c) 48, (d) 72, and (e) 96 h-old zebrafish incubated with 3 for 1 h (Reprinted from ref (18)).

Proposed binding mechanism for unique pan class="Disease">hypsochromic shift of 3 upon the addition of n>n class="Chemical">Zn2+.

Fluorescence detection of intrinsic pan class="Chemical">Zn2+ ions in n>n class="Species">zebrafish using probe 3 (a) 24, (b) 36, (c) 48, (d) 72, and (e) 96 h-old zebrafish incubated with 3 for 1 h (Reprinted from ref (18)).

pan class="Chemical">Rhodamines are known to undergo a distinct colorimetric change from colorless to dark pink along with off–on fluorescent enhancement when their n>n class="Chemical">spirolactone or spirolactam rings suffer cleavage.[21] In the pioneering work in 1997, Czarnik showed that a chemodosimeter based on rhodamine serves as a Cu2+ selective fluorescent and colorimetric chemosensor.[22] In 2005, a reversible fluorescent chemosensor based on rhodamine 4 bearing a polydiacetylene (PDA) ligand was developed by our group.[23] In the report of this effort, the first X-ray crystal structure of a rhodamine lactam derivative was reported, which clearly shows that the spirolactam ring and xanthene core are oriented perpendicular to each other. As shown in Figure , Pb2+ binding to the DPA ligand as well as carbonyl oxygen in 4 induces a ring-opening process, resulting in a large, completely reversible fluorescent enhancement (100-fold) and a colorimetric change.

Figure 7

Proposed binding mode of 4 with Pb2+ via a spirolactam ring-opening process.

Proposed binding mode of 4 with pan class="Chemical">Pb2+ via a n>n class="Chemical">spirolactam ring-opening process.

This concept was then applied to the design of pan class="Gene">Cu2+ and n>n class="Chemical">Hg2+ selective reversible rhodamine-based chemosensors that contain boronic acid binding sites.[24] The design also combines the reaction-based chemodosimeter concept to the rhodamine ring-opening process. Accordingly, the selenolactone rhodamine derivative 5 was synthesized and explored as a chemodosimeter for highly toxic mercury and methylmercury (Figure ).[25] At pH 7.4, seleno-rhodamine derivative 5 shows a highly selective fluorescent enhancement and distinct color change upon the addition of mercury species, caused by a deselenation reaction. Fluorescent chemodosimeter 5 was also applied to image Hg2+ and methylmercury in zebrafish as an animal model. A strong red emission was observed in the fin, eggs, and gallbladder, which was attributed to the presence of Hg2+ and methylmercury at these locations.

Figure 8

Deselenation reaction of 5 with Hg2+ and CH3Hg+.

Deselenation reaction of 5 with pan class="Chemical">Hg2+ and CH3Hg+.

As shown in Figure , the pan class="Chemical">rhodamine-alkyne derivative 6 selectively reacts with n>n class="Chemical">Au3+ over various metal ions at pH 7.4 to form an oxazolecarbaldehyde product. This process induces a ring-opening reaction, resulting in a 250-fold fluorescent enhancement and distinct color change.[26] The observed rate constant for the process was calculated to be 4.5 (±0.20) × 10–4 s–1, and 6 has a Au3+ detection limit of 320 nM. Also, the rhodamine-alkyne derivative 6 was used to image Au3+ in the live cells.

Figure 9

Proposed reaction of 6 with Au3+ to induce spirolactam ring opening and the structure of 7.

Proposed reaction of 6 with pan class="Chemical">Au3+ to induce pan class="Chemical">spirolactam ring opening and the structure of 7.

The pan class="Chemical">1,8-naphthalimide-n>n class="Chemical">alkyene derivative 7 was also developed for use to selectively detect Au3+ in lipid droplets in cells.[27] When Au3+ is added, a large hypsochromic shift (∼56 nm) takes place in the emission spectrum of 7 along with a colorimetric change from yellow to light pink. The detection limit was reported to be 8.44 μM. An enhancement in the rate of reaction of 7 with Au3+ is promoted by surfactants and occurs in differentiated adipocytes compared to that in HeLa cells. 1,8-Naphthalimide-alkyene derivative 7 can be employed to image Au3+ in lipid droplets in cells.

Fluorescent Chemosensors for Anions Using Imidazoliums

Because pan class="Chemical">imidazoliums act as [C–H]+-anion ionic n>n class="Chemical">hydrogen-bonding donors, they have been utilized as anionic receptors and fluorescent chemosensors.[28] The anthracene derivative 8, a sensor of this type, was developed in our laboratory (Figure ). In this sensor, two imidazolium groups are linked to the 1,8 positions of the anthracene ring system.[29] The fluorescence of chemosensor 8 in acetonitrile is efficiently and selectively quenched by H2PO4– and F–, which was attributed to the operation of a PET process. The 1,8-positions of anthracene provide a preorganized binding site, and at the same time, the anthracene ring acts as a fluorophore. The dimeric derivative 9 contains a more organized binding site for anions, and as a result, it displays an enhanced selectivity for H2PO4– over F– as reflected in an enhanced fluorescent quenching efficiency.[30] Moreover, the water-soluble analogue 10, bearing additional ammonium moieties, was used as GTP and ATP selective fluorescent chemosensors at pH 7.4 in pure aqueous solution.[31] A large fluorescent quenching effect was observed upon the addition of GTP with an association constant of 8.7 × 104 M–1, and a moderate fluorescent enhancement was observed upon the addition of ATP with a 1.5 × 104 M–1 association constant.

Figure 10

Structures of anthracene–imidazolium derivatives 8–10.

Structures of pan class="Chemical">anthracene–n>n class="Chemical">imidazolium derivatives 8–10.

pan class="Chemical">Naphthoimidazoliums can serve dual functions as fluorophores and strong n>n class="Chemical">hydrogen-bonding donors. Moreover, electron-deficient aromatic rings are used for anion−π interaction-driven recognition. Our group and one directed by Kim reported that naphthoimidazolium cage compounds bearing electron-rich alkylbenzene rings bind F– inside their cavities through an anion−π interaction.[32] As shown in Figure , F– is encapsulated into the cavity of host 12, which was confirmed by 1H NMR and 19F NMR. A ratiometric fluorescent change of host 12 was observed to take place upon the addition of F–. The 19F NMR spectrum of the complex contains a quartet (J = 84 Hz) attributed to coupling between F– and naphthoimidazolium C2–H. Also, a doublet (J = 84 Hz) is seen in the 1H NMR spectrum of the complex. Finally, we concluded that F– interacts with the naphthoimidazolium moieties of host 11 outside of the cavity.

Figure 11

Different binding modes of naphthoimidazolium-based cage compounds 11 and 12 with F–.

Different binding modes of pan class="Chemical">naphthoimidazolium-based cage copan class="Gene">mpounds 11 and 12 with F–.

We observed that owing to the presence of the pan class="Chemical">benzobisimidazolium group, the rigid cyclic fluorescent chemosensor 13 displaying an enhanced C–H n>n class="Chemical">hydrogen bonding with anions (Figure ).[33] In CH3CN and CH3CN-buffer (pH 7.4) (9:1, v/v), 13 undergoes a selective enhancement in its emission upon the addition of HSO4–. On the contrary, no significant change was observed following the addition of other anions such as F–, Cl–, Br–, I–, H2PO4–, CH3CO2–, CN–, and NO3–. The significant downfield shift of Ha from 7.56 to 8.10 ppm in the 1H NMR spectrum promoted by the addition of HSO4– confirms the existence of C–H hydrogen-bonding interactions with HSO4–. The effects of charge, preorganization, and multiple C–H hydrogen bonding are responsible for the selectivity of this rigid cyclic chemosensor.

Figure 12

Structure of cyclic chemosensor 13.

It is quite challenging to design sensors that recognize specific pan class="Chemical">nucleoside triphosphates in mixtures containing similar nucleoside triphosphates, such as adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), cytidine 5′-triphosphate (CTP), uridine 5′-triphosphate (UTP), and TTP.[34] In studies focusing on this issue, we observed that imidazolium receptor 14, bearing two pyrene groups, displays a unique selectivity for ATP over other nucleoside triphosphates.[35] Fluorescent chemosensor 14 displays strong excimer emission at 487 nm with relatively weak monomer emission at 375 nm. The selectivity of 14 was proposed to be associated with different binding modes of ATP compared to that of other nucleoside triphosphates. As shown in Figure , adenine of ATP prefers to be bound between two pyrene rings, whereas GTP, CTP, UTP, and TTP bases prefer to be located on the outside of two pyrene rings as a consequence of greater H−π interactions. Accordingly, a highly selective ratiometric change (I375/I487) occurs in the emission spectrum of 14 upon the addition of ATP. This fluorescent chemosensor was applied to image ATP in the live cells.

Figure 13

Different binding modes for imidazolium–pyrene derivative 14 with ATP and GTP to induce ratiometric changes of pyrenes.

Different binding modes for pan class="Chemical">imidazolium–n>n class="Chemical">pyrene derivative 14 with ATP and GTP to induce ratiometric changes of pyrenes.

Fluorescent Chemosensors for Anions Using Metal Ion Binding Sites or Metal-Displacement Approaches

Interactions with pan class="Chemical">metal ions are effective in promoting strong binding of anions in an aqueous solution. This is especially true for a fluorescent chemosensor containing n>n class="Chemical">Zn2+, which we have actively investigated for detecting phosphate species, such as pyrophosphate (PPi) and ATP.[36] A Zn2+ complex of naphthaldimide derivative 15, bearing two DPA ligands, was found to selectively recognize PPi in 100% aqueous solution.[37] An excimer emission at 490 nm was observed to arise upon the addition of PPi to 15 at pH 7.4. As shown in Figure , as a result of favorable interactions between two Zn2+ sites and phosphate anions as well as π–π interaction, 2 + 2 type excimer formation occurs between 15 in the presence of PPi.

Figure 14

2 + 2 Type excimer formation of the Zn2+–15 complex with PPi.

2 + 2 Type excimer formation of the pan class="Chemical">Zn2+–15 complex with PPi.

A preorganized binding pocket containing two pan class="Chemical">Zn2+ sites was created using n>n class="Chemical">fluorescein[38] and acridine[39] as fluorophores (Figure ). The Zn complex with fluorescein derivative 16, bearing two DPA ligands, showed a moderate fluorescent enhancement (∼150%) as well as color change to pink upon the addition of PPi at pH 7.4.[38] The association constant of the formed complex was calculated to be 9.8 × 104 M–1. On the contrary, no significant change was observed upon the addition of phosphate (Pi). Fluorescence of the Zn complex of acridine–DPA derivative 17 is quenched by PPi, and a moderate fluorescent enhancement is promoted by Pi at pH 7.4 (Figure ).[39] The association constant for PPi was calculated to be 4.85 × 107 M–1 and that of Pi was found to be 9.36 × 104 M–1. The presence of additional hydrogen-bonding interactions between acridine nitrogen and OH of Pi was suggested to be responsible for the moderate fluorescence enhancement.

Figure 15

Structures of the Zn complexes 16 and 17.

Structures of the pan class="Chemical">Zn complexes 16 and 17.

In contrast, the pan class="Chemical">Zn complex of perylene–n>n class="Chemical">DPA 18 shows large fluorescent enhancements upon the addition of UTP and UDP at pH 7.4. Other phosphate derivatives, such as ATP, GTP, CTP, and TTP, and PPi and Pi do not induce any significant emission changes.[40] As shown in Figure , the fluorescent chemosensor 18 binds to UTP or UDP via Zn interactions with the uridine base and phosphate group. The chemosensor was applied to distinguish between the activities of the two related enzymes, UDP–glucose pyrophosphorylase and β-1,4-galactosyltransferase (β-1,4-GalT). A fluorescence decrease was observed in the presence of UDP–glucose pyrophosphorylase because UTP and glucose-phosphate are transformed to uridine 5′-diphosphate-glycoside and PPi by this enzyme. On the other hand, a fluorescence enhancement occurs in the presence of β-1,4-GalT.

Figure 16

Structure of perylene–Zn complex 18 and its proposed binding mode with UTP and UDP with two Zn sites.

Structure of perylene–pan class="Chemical">Zn complex 18 and its proposed binding mode with n>n class="Chemical">UTP and UDP with two Zn sites.

Displacement mechanism of fluorescent chemosensor 19–pan class="Gene">Cu2+ with n>n class="Chemical">cyanide to revive the NIR emission.

Fluorescent chemosensors for pan class="Chemical">cyanide have been actively investigated using various design stpan class="Species">rategies.[41] The fact that cyanide forms a very stable [Cu(CN)] species with Cu2+ was utilized to design a Cu2+ displacement-based NIR probe.[42] Cyanine derivative 19 shows NIR emission at 748 nm (Figure ). When Cu2+ is added to a solution of 19 at pH 7.4, almost complete quenching of the fluorescence of 19 occurs in association with a red shift of its absorption maximum from 718 to 743 nm. Addition of cyanide anion revives the emission at 748 nm, which is attributed to the formation of [Cu(CN)] species. Pseudomonas aeruginosa (PA) is known to produce toxic HCN, which is related to the pathogenesis of CF lung disease. To explore this issue, a green fluorescent protein (GFP)-labeled PA14 strain was incubated with the nematode Caenorhabditis elegans (Figure ). Chemosensor 19 was successfully employed to image HCN produced in the nematode by PA14. When the β-lactam antibiotic, ceftazidime, is then preadministered to the PA14 treated nematode, the green emission from GFP and NIR fluorescence of 19 was significantly reduced.

Figure 17

Displacement mechanism of fluorescent chemosensor 19–Cu2+ with cyanide to revive the NIR emission.

Figure 18

Fluorescence images of C. elegans, infected by P. aeruginosa and treated with the antibiotic ceftazidime. The nematodes were fed with GFP-labeled P. aerugionosa (PA14) for 2 d followed by incubation with ceftazidime (200 μg/mL) for 2 h. The scale bars represent 20 μm (Reprinted from ref (42)).

Fluorescence images of pan class="Species">C. elegans, infected by n>n class="Species">P. aeruginosa and treated with the antibiotic ceftazidime. The nematodes were fed with GFP-labeled P. aerugionosa (PA14) for 2 d followed by incubation with ceftazidime (200 μg/mL) for 2 h. The scale bars represent 20 μm (Reprinted from ref (42)).

ROS and Nitrogen Species

pan class="Chemical">ROS and n>n class="Chemical">reactive nitrogen species (RNS) play key roles in physiological processes and various diseases, such as neurodegenerative disorders, cancer, and so forth. Fluorescent chemosensors for ROS and RNS have been designed based on specific reactions they promote.[43] Hypochlorous acid (HOCl) is a class of ROS, which is produced from hydrogen peroxide (H2O2) and Cl– by myeloperoxidase (MPO).[44] HOCl is generated in the phagocytic cells as a defense system to kill pathogens. On the other hand, excess HOCl can cause chronic inflammatory, cardiovascular and kidney diseases.[45]

Among the various pan class="Chemical">ROS such as n>n class="Chemical">H2O2, NO•, •OH, ROO•, ONOO–, 1O2, HOCl, and •O2–, HOCl at pH 5.5 can be selectively detected by using fluorescent enhancements of the thiolactone and selenolactone rhodamine derivatives 20 and 21.[46] As shown in Figure , HOCl promoted ring-opening caused by sulfur oxidation is the reason for the large fluorescent enhancement (∼20-fold) of 20. The HOCl detection limits of 20 and 21 were reported to be 0.4 and 0.6 μM, respectively. Fluorescent chemosensor 20 was successfully applied to image microbiocidal HOCl produced in the mucosal epithelia of fruit fly upon bacterial infection. It is also known that the enzyme, NADPH oxidase 2 (NOX2), plays a key role in the production of HOCl by neutrophils.[47] Upon bacterial infection, endogenous HOCl, generated in bone marrow-derived neutrophils (BMDNs) extracted from WT mice (NOX2+/+) or NOX2-deficient mice (NOX2–/–), can be clearly detected by using 20.[48] Strong emission of 20 was observed in BMDNs extracted from WT mice (NOX2+/+), whereas no significant fluorescence is seen in the case of NOX2-deficient mice (NOX2–/–).

Figure 19

Structures of HOCl selective fluorescent probes 20–21 and the ring-opening reaction of 20 with HOCl via sulfur oxidation.

Structures of n class="Chemical">HOCl selective fluorescent probes 20–21 and the n>n class="Disease">ring-opening reaction of 20 with HOCl via sulfur oxidation.

Oxidative cleavage of C–B bonds, especially in the pan class="Chemical">borono-Dakin oxidation of n>n class="Chemical">arylboronic acids and their esters, has been widely utilized for the design of fluorescent chemosensors for hydrogen peroxide (H2O2).[49] Our previous thiolactone strategy was utilized in conjunction with borono-Dakin oxidation of arylboronic ester, to design the so-called “dual-lock” system in 22, which serves as a selective fluorescent chemosensor for HOCl (Figure ).[50]22 contains an arylboronic ester and a thiolactone group. As shown in Figure , H2O2, ONOO–, and HOCl convert the arylboronic ester in nonfluorescent 22 to a phenol (22-I), which is still nonfluorescent. On the other hand, only HOCl oxidizes the thiolactone to form the fluorescein derivative 22-II, resulting in strong green emission (∼120-fold enhancement) due to the occurrence of a ring-opening process. 22 operates in the relatively large pH window of 5.5 to 9.3, which means it can be used to image HOCl in the rather acidic lysosome environment. 22 was also used to detect bacteria-induced HOCl production in the mucosal epithelia of fruit fly.

Figure 20

Borono-Dakin oxidation of 22 with OCl–, H2O2, and ONOO– and selective oxidation of thiolactone moiety with HOCl to induce strong green fluorescence.

pan class="Chemical">Borono-Dakin oxidation of 22 with n>n class="Chemical">OCl–, H2O2, and ONOO– and selective oxidation of thiolactone moiety with HOCl to induce strong green fluorescence.

pan class="Chemical">Imidazoline-2-thione moiety was also utilized as a reactive center in the n>n class="Chemical">HOCl selective fluorescent chemosensor. For example, substances containing this group, such as 23 and 24, are converted to imidazolium products, which are fluorescent (Figure ).[51] Upon the addition of HOCl to a solution of 23, a new peak at 378 nm appears in the absorption spectrum with the decrease of the peak at 420 nm. Twelvefold fluorescent enhancement at 505 nm was also observed upon the addition of HOCl to 23. The imidazoline-2-thione containing sensor 24 displays changes in its absorption and fluorescence spectra that are similar to those of 23. 22 was applied to detect endogenous HOCl produced in RAW 264.7 macrophages, which are activated by lipopolysaccharides (LPS) (Figure ). A strong green emission in the live cells and tissues was observed by using two-photon microscopy (TPM). Pretreatment with the known MPO inhibitors, 4-aminobenzoicacid hydrazide (ABAH) and flufenamic acid, causes a dramatic fluorescence quenching effect. Furthermore, RAW 264.7 macrophages in a cocultured system can be distinguished from HeLa cells by green emission of 23 resulting from the production of HOCl.

Figure 21

Structures of 23, 24, and 25 and reaction of 23 with HOCl to form fluorescent imidazolium product 26.

Figure 22

TPM images of (a–e) 23 and (f) 26 (10 μM, ρDMF = 0.5%) labeled RAW 264.7 cells. (a) Control image. (b) Cells pretreated with NaOCl (200 μM) for 30 min and then incubated with 23. (c) Cells pretreated with LPS (100 ng/mL) for 16 h, interferon gamma (IFN-γ) (400 U/mL) for 4 h, and PMA (10 nM) for 30 min and then with 23. (d) Cells pretreated with LPS, IFN-γ, and 4-ABAH (50 μM) for 4 h and then incubated with 23. (e) Cells pretreated with LPS, IFN-γ, and FAA (50 μM) for 4 h and then with 23. (g) Average TPEF intensities in (a–f), n = 5. Scale bar: 20 μm (Reprinted from ref (51)).

Structures of 23, 24, and 25 and reaction of 23 with pan class="Chemical">HOCl to form fluorescent pan class="Chemical">imidazolium product 26.

pan class="Chemical">Imidazoline-2-thione derivative, bearing n>n class="Chemical">triphenylphosphine as a mitochondria directing group, was employed to detect HOCl in mitochondria. Imidazoline-2-thione derivative 25 can also be utilized to image HOCl in live cells and tissues using the TPM technique (Figure ).[52] Pearson’s colocalization coefficient of this substance using Mito Tracker Red as a reference was found to be 0.83.

TPM images of (a–e) 23 and (f) 26 (10 μM, ρDMF = 0.5%) labeled pan class="CellLine">RAW 264.7 cells. (a) Control image. (b) Cells pretreated with n>n class="Chemical">NaOCl (200 μM) for 30 min and then incubated with 23. (c) Cells pretreated with LPS (100 ng/mL) for 16 h, interferon gamma (IFN-γ) (400 U/mL) for 4 h, and PMA (10 nM) for 30 min and then with 23. (d) Cells pretreated with LPS, IFN-γ, and 4-ABAH (50 μM) for 4 h and then incubated with 23. (e) Cells pretreated with LPS, IFN-γ, and FAA (50 μM) for 4 h and then with 23. (g) Average TPEF intensities in (a–f), n = 5. Scale bar: 20 μm (Reprinted from ref (51)).

The pan class="Chemical">pyrene containing n>n class="Disease">N-heterocyclic carbene (NHC) borane complex 27 was developed in our laboratory as the first sensor of HOCl (Figure ).[53] We proposed that NHC borane reacts selectively with HOCl over other ROS via an electrophilic type oxidation pathway, which stands in contrast with nucleophilic oxidations of arylboronic acids or esters promoted by ROS. As shown in Figure , NHC borane 27 displays an aggregated excimer emission and undergoes oxidative cleavage of the B–C bond to afford an imidazolium product, which shows a strong monomeric emission. Accordingly, ratiometric changes in the green (477 nm) and blue (374 nm) channels were observed. TPM was utilized along with 27 to carry out ratiometric detection of endogenous HOCl in live cells and tissues.

Figure 23

Structure of NHC borane derivative 27 and its reaction with HOCl. Excimer formation of 27 and preferred monomer formation of imidazolium product after the reaction with HOCl.

Structure of pan class="Chemical">NHC borane derivative 27 and its reaction with n>n class="Chemical">HOCl. Excimer formation of 27 and preferred monomer formation of imidazolium product after the reaction with HOCl.

The pan class="Chemical">naphthalimide derivative 28, containing n>n class="Chemical">boronate and morpholine groups, was designated by us to be a fluorescent chemosensor for H2O2.[54] As shown in Figure , the p-dihydroxyborylbenzyloxycarbonyl group in 28 acts as a selective site for reaction with H2O2 at pH 7.4. Fluorescence emission at 528 nm is selectively promoted by reaction of 28 with H2O2 over various ROS and RNS. Because 28 contains a morpholine group, which is known to target lysosomes, it can be employed to detect endogenous H2O2 in the lysosome of the RAW 264.7 cells.

Figure 24

H2O2 selective fluorescent chemosensor 28 and its reaction with H2O2 to form fluorescent product 28-I.

pan class="Chemical">H2O2 selective fluorescent chemosensor 28 and its reaction with n>n class="Chemical">H2O2 to form fluorescent product 28-I.

The pan class="Chemical">ROS n>n class="Chemical">ONOO– also plays important roles in signal transduction, even though ONOO– shows antibacterial activity through its ability to damage DNA and proteins owing to its high oxidizing and nitrating capacities.[55] The coumarin–hemicyanine derivative 29 showed ratiometric changes in its emission along with distinct colorimetric changes when it is in the presence of peroxynitrite (ONOO–).[56] Upon the addition of ONOO– to 29, ratiometric fluorescent changes (F515nm/F635nm) were clearly observed. As shown in Figure , 1,3,3-trimethyloxindole and Coum-CHO are produced by the reaction of this sensor with ONOO–, which is responsible for the increase of green emission and decrease of red emission. Coumarin–hemicyanine derivative 29 can be used to image the production of ONOO– in RAW 264.7 cells, which are treated with LPS and IFN-g and stimulated by PMA. Pretreatment of the RAW 264.7 cells with aminoguanidine, a NO synthase inhibitor, or 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), a superoxide scavenger, causes no significant change in fluorescence emission upon the addition of 29 (Figure ).

Figure 25

Reaction of coumarin–hemicyanine derivative 29 with ONOO– to show distinct ratiometric change.

Figure 26

Confocal fluorescence images of endogenous ONOO in RAW 264.7 cells during the phagocytic immune response. The cells were stained with 5 μM 29 for 30 min and then washed with DPBS before imaging. (a) Control; (e) LPS (1 μg/mL) for 16 h, interferon-γ (50 ng/mL) for 4 h, PMA (10 nM) for 30 min; (i) LPS (1 μg/mL) for 16 h, interferon-γ (50 ng/mL) for 4 h, PMA (10 nM) for 30 min, and then AG (1 mM) for 16 h; (m) LPS (1 μg/mL) or 16 h, interferon-γ (50 ng/mL) for 4 h, PMA (10 nM) for 30 min, and then TEMPO (100 μM) for 16 h. The green channel (a,e,i,m) represents fluorescence at 490–540 nm with an excitation wavelength at 473 nm, the red channel (b,f,j,n) represents fluorescence at 575–675 nm with an excitation wavelength at 559 nm, images (c,g,k,o) represent DIC channels (differential interference contrast), and images (d,h,I,p) represent merged images of red and green channels (Reprinted from ref (56)).

Reaction of pan class="Chemical">coumarin–n>n class="Chemical">hemicyanine derivative 29 with ONOO– to show distinct ratiometric change.

Confocal fluorescence images of endogenous pan class="Chemical">ONOO in n>n class="CellLine">RAW 264.7 cells during the phagocytic immune response. The cells were stained with 5 μM 29 for 30 min and then washed with DPBS before imaging. (a) Control; (e) LPS (1 μg/mL) for 16 h, interferon-γ (50 ng/mL) for 4 h, PMA (10 nM) for 30 min; (i) LPS (1 μg/mL) for 16 h, interferon-γ (50 ng/mL) for 4 h, PMA (10 nM) for 30 min, and then AG (1 mM) for 16 h; (m) LPS (1 μg/mL) or 16 h, interferon-γ (50 ng/mL) for 4 h, PMA (10 nM) for 30 min, and then TEMPO (100 μM) for 16 h. The green channel (a,e,i,m) represents fluorescence at 490–540 nm with an excitation wavelength at 473 nm, the red channel (b,f,j,n) represents fluorescence at 575–675 nm with an excitation wavelength at 559 nm, images (c,g,k,o) represent DIC channels (differential interference contrast), and images (d,h,I,p) represent merged images of red and green channels (Reprinted from ref (56)).

More recently, a N-dearylation reaction promoted by pan class="Chemical">ONOO– was utilized as the basis for our design of the two-photon n>n class="Chemical">ONOO– probe 30.[57] In PBS buffer, chemosensor 30 shows selective fluorescent enhancement (14-fold) at 501 nm upon the addition of ONOO–. The emission of the naphthalene fluorophore in 30 is quenched by electron transfer from the electron-rich aniline group. Reaction of 30 with ONOO– generates 30-I that has enhanced fluorescence emission and two-photon absorption properties (Figure ). The detection limit of 30 for ONOO– was calculated to be 35 nM. Chemosensor 30 was applied to image endogenous ONOO– in RAW 264.7 cells and to detect endogenous ONOO– in rat hippocampal tissues. It is worth mentioning that 30 has excellent two-photon properties associated with a high two-photon cross-sectional value (δ ≈ 100 GM) and a deep light penetration depth of 120 μm.

Figure 27

Reaction of 30 with ONOO– to form a fluorescent product 30-I.

Reaction of 30 with pan class="Chemical">ONOO– to form a fluorescent product 30-I.

pan class="Chemical">Rhodamine derivative 31, bearing a n>n class="Chemical">hydrazide group, was explored as an ONOO– selective fluorescent chemosensor (Figure ).[58] Addition of ONOO– to a solution of 31 induces a strong far-red emission at 638 nm. The detection limit of 1 for ONOO– was calculated to be 45 nM. Both exogenous and endogenous ONOO– in RAW 264.7 and HeLa cells can be detected using 31. 31 was further applied to detect endogenous ONOO– in mouse BMDNs, which are infected by using GFP-tagged P. aeruginosa (PAO1). As shown in Figure , different fluorescence emissions were observed in BMDNs extracted from wild-type (Nox2+/+) and Nox2-deficient (Nox2–/–) mice, which can be attributed to the presence of different ONOO– concentrations.

Figure 28

Structure of 31 and confocal microscopy images of GFP-tagged PAO1 and 31 in BMDNs. Images of neutrophils derived from Nox2+/+ (top) or Nox2–/– (bottom) mice treated with GFP-tagged PAO1 and 31 for 1 h. Images of GFP-tagged PAO1 (green) were obtained at 488 nm using 500–550 nm excitation. Images of 31 (red) were obtained at 561 nm using 630–660 nm excitation. The scale bars are 20 μm.

Structure of 31 and confocal micpan class="Chemical">roscopy images of GFP-tagged n>n class="Species">PAO1 and 31 in BMDNs. Images of neutrophils derived from Nox2+/+ (top) or Nox2–/– (bottom) mice treated with GFP-tagged PAO1 and 31 for 1 h. Images of GFP-tagged PAO1 (green) were obtained at 488 nm using 500–550 nm excitation. Images of 31 (red) were obtained at 561 nm using 630–660 nm excitation. The scale bars are 20 μm.

The fluorescent chemosensor 32 was found by us to be an excited-state intramolecular proton transfer (ESIPT) fluorophore that responds to NO (Figure ).[59] At pH = 7.4, 32 displays blue emission at 470 nm which is attributed to the opepan class="Species">ration of an ESIPT mechanism. Upon the addition of NO, the intensity of the emission band at 560 nm increases with a decrease of the original emission band at 470 nm. In addition, the emission color clearly changes from blue to yellow, which can be detected by using the naked eyes. A linear relationship was observed between the emission intensity at 560 nm and the concentpan class="Species">ration of NO in the range of 0–10 μM with a detection limit of 17 nM. This chemosensor was also applied to image NO in living cells.

Figure 29

Proposed reaction of the ESIPT fluorescent chemosensor 32 with NO.

Fluorescent Chemosensors for Biothiols and H2S

pan class="Chemical">Cysteine (n>n class="Chemical">Cys), homocysteine (Hcy), and glutathione (GSH) are the three main biothiols involved in human physiology. Abnormal levels of Cys, a precursor of GSH, are related to various human diseases, such as liver damage, edema, and loss of muscle and fat.[60] The total Hcy level in plasma is related to cognitive impairment in the elderly and birth defects.[61] On the other hand, the most abundant nonprotein thiol, GSH, plays various important roles, such as intracellular signal transduction and intracellular redox control agent, and it is related to critical diseases including Alzheimer’s disease and cancer.[62] In mammalian cells, the concentrations of these biothiols are in the range of 1–10 mM for GSH, 30–200 μM for Cys, and 5–15 μM for Hcy. In last two decades, fluorescent chemosensors for these biothiols have been actively investigated by various groups worldwide.[63,64]

Our first contribution to this area was reported in 2010.[65] The pan class="Chemical">fluorescein derivative 33 is reported to undergo Michael addition reactions with n>n class="Chemical">GSH, Cys, and Hcy followed by spirocyclic lactone ring opening, as shown in Figure . This induces an “off–on” fluorescent enhancement at 520 nm at pH 7.4. This probe can be used to image GSH selectively in live cells and organisms owing to the much higher cellular concentrations of GSH compared to those of Cys and Hcy. The fluorescein derivative 33 can also be utilized to image biothiols in murine P19 embryonic carcinoma cells and zebrafish. When the cells are pretreated with N-methylmaleimide, a Michael acceptor of thiols, almost no fluorescence is observed.

Figure 30

Proposed reaction of chemosensor 33 with biothiols to form fluorescent spiroring-opened product.

Proposed reaction of chemosensor 33 with pan class="Chemical">biothiols to form fluorescent spiroring-opened product.

Strongin’s group described 34, the first example of a pan class="Chemical">Cys selective fluorescent chemosensor that relies on a kinetically favored seven-membered ring formation promoted by n>n class="Chemical">Cys.[66]Figure shows the unique modulation of polymethine π-electron cyanine system in probe 34 caused by reaction with Cys. This process induces large shifts in the absorption and emission spectra of 34. Hydroxy cyanine 34 displays maximum absorption at 710 nm and emission at 730 nm. On the other hand, its keto form 34-I has a dramatic hypsochromic shift to 535 nm in its absorption and 625 nm for its emission maxima. The related cyanine derivative 35, bearing an acrylate moiety, was reported to be a Cys selective NIR chemosensor.[67] As shown in Figure , Cys undergoes an addition reaction with 35 followed by kinetically favored seven-membered ring formation to form 34-I. As a result, similar distinct changes occur in the absorption (from 770 to 515 nm) and emission (from 780 to 570 nm) spectra. It is known that the intracellular Cys level is significantly increased during glucose deprivation. Cyanine derivative 35 can be employed to selectively detect this increase in Cys level in MCF-7 cells by a distinct emission enhancement at 590 nm and a decrease in the NIR emission.

Figure 31

Structures of 34 and 34-I and reaction of 35 with Cys via Michael-type addition followed by intramolecular rearrangement.

Structures of 34 and 34-I and reaction of 35 with pan class="Chemical">Cys via Michael-type addition followed by intramolecular rearrangement.

The pan class="Chemical">salicyladazine derivative 36, bearing an n>n class="Chemical">acrylate moiety, serves as a Cys chemosensor that operates in a kinetically selective manner.[68] When 36 is treated with Cys for 15 min, a selective fluorescent enhancement (∼10-fold) at 505 nm occurs. On the other hand, both Cys and GSH induce a fluorescence increase of 36 after 40 min. Similar results were obtained in studies of cell imaging. As shown in Figure , the reaction responsible for these changes involves Michael-type addition followed by cyclization to afford the salicylaldazine dye. The hydroxyl group in the salicylaldazine dye forms an intermolecular hydrogen bond, which was attributed to the emission at 505 nm.

Figure 32

Proposed reaction mechanism of 36 with Cys via Michael-type addition followed by intramolecular rearrangement to form AIE and ESIPT active products.

Proposed reaction mechanism of 36 with pan class="Chemical">Cys via Michael-type addition followed by intramolecular rearrangement to form AIE and ESIPT active products.

The pan class="Chemical">pyrene derivatives, 37 and 38, display selective fluorescence enhancements at 450 nm in the presence of n>n class="Chemical">Hcy at pH 7.4 (Figure ).[69] The formation of thiazinane heterocyclic rings from the reaction of Hcy with the aldehyde groups in these substances was attributed to the selectivity toward Hcy. The detection limits of 37 and 38 were reported to be 1.94 and 0.14 μM, respectively. Pyrene derivatives, 37 and 38, can be employed to image Hcy in mammalian cells.

Figure 33

Structures of pyrene derivatives 37 and 38.

Structures of pan class="Chemical">pyrene derivatives 37 and 38.

The pan class="Chemical">aryl-thioether-n>n class="Chemical">substituted nitrobenzothiadiazole derivative, 38, was investigated as a Cys and Hcy selective fluorescent chemosensor that acts via an aryl substitution reaction by the thiol moieties in these analytes followed by intramolecular rearrangement, as shown in Figure .[70] Treatment of 38 with Cys and Hcy at pH 7.4 induces a 20-fold fluorescent enhancement at 535 nm. More importantly, at acidic pH such as 6.0, only Cys induces this fluorescence enhancement, the selectivity being a consequence of the higher acidity of the thiol in Cys (pKa 8.53) compared to that of Hcy (pKa 10.00). Strong green emission was also used for detection by 38 of Cys and Hcy in the live HeLa cells.

Figure 34

Proposed reactions of 38 with Cys and Hcy to form fluorescent amino products via an aryl substitution reaction followed by intramolecular rearrangement.

Proposed reactions of 38 with pan class="Chemical">Cys and pan class="Chemical">Hcy to form fluorescent amino products via an aryl substitution reaction followed by intramolecular rearrangement.

Two pan class="Chemical">cyanine derivatives, 39 bearing a 2,4-dinitrobenzene n>n class="Chemical">sulfonamide group and 40 containing a 5-dimethylaminonaphthyl sulfonamide group, were investigated in our laboratory as NIR fluorescent chemosensors for biothiols at pH 7.4 (Figure ).[71] Addition of GSH, Cys and Hcy to 39 generates the fluorescent product 41, whereas cyanine derivative 40 reacts selectively with GSH to cause this fluorescent enhancement. It is known that overdose of the painkiller acetaminophen can damage the liver and kidney cells and lower GSH levels. We confirmed these phenomena using a mouse model and the GSH fluorescent chemosensor 40. More recently, in collaboration with Yin and Tan, it is reported that 42 serves as a dual-channel fluorescent chemosensor for GSH (Figure ).[72] Probe 42 is composed of cyanine IR-780 and 1,8-naphthalimide, which are linked by a thiol-reactive sulfonamide group. Reaction with GSH promotes large synchronous enhancements of the visible emission (495 nm) and NIR emission (795 nm) bands. In addition, 42 shows an excellent targeting ability for mitochondria.

Figure 35

Reactions of 39, 40, and 42 with biothiols to form fluorescent product 41.

Reactions of 39, 40, and 42 with pan class="Chemical">biothiols to form fluorescent product 41.

The two pan class="Chemical">GSH NIR fluorescent chemosensors, 43 and 44, were also developed by us (Figure ).[73] Addition of n>n class="Chemical">GSH to both of these nonfluorescent compounds induces dramatic enhancements (∼50-fold for 43 and ∼100-fold for 44) in their emission, whereas only a relatively small enhancement occurs upon the addition of Hcy. The detection limits of 43 and 44 for GSH were reported to be 6.3 and 3.3 μM, respectively. At pH 7.4, chemosensor 43, which has an absorption maximum at 660 nm, is not fluorescent. Upon the addition of GSH, strong fluorescence emission at 805 nm arises corresponding to an absorption maximum at 780 nm. The detailed mechanisms for reactions of 43 and 44 with biothiols are given in Figure . Both 43 and 44 can be utilized to image GSH in the live cells with strong fluorescence being observed in the tumors in tumor-bearing mice in which GSH is overexpressed.

Figure 36

Replacement reactions of 43 and 44 with GSH and subsequent rearrangement reactions with Cys and Hcy.

Replacement reactions of 43 and 44 with pan class="Chemical">GSH and subsequent rearrangement reactions with n>n class="Chemical">Cys and Hcy.

A displacement approach was recently reported to create the pan class="Chemical">GSH sensor, n>n class="Chemical">bis-pyrene-Cu2+45 (Figure ).[74] This derivative displays excimer emission at 450 nm and relatively weak monomer emission at 400 nm at pH 7.4. Addition of Cu2+ induces selective and efficient fluorescence quenching. The addition of biothiols to the Cu2+–45 ensemble revives the emission. The biothiol detection limit was calculated to be 1.7 μM. Endogenous GSH in cells and in tissues was successfully imaged by using 45 and TPM.

Figure 37

Structure of bis-pyrene derivative 45.

Structure of pan class="Chemical">bis-pyrene derivative 45.

The pan class="Chemical">naphthalene dialdehyde derivatives, 46–48, were reported to be n>n class="Chemical">GSH selective fluorescent chemosensors (Figure ).[75] Addition of GSH to these substances causes a fluorescence enhancements (100-fold for 46, 80-fold for 47, and 36-fold for 48) at ∼531 nm upon excitation at 450 nm. All three derivatives can be used to image intracellular GSH in HeLa cells. The detection limits of 46, 47, and 48 were calculated to be 64 nM, 68 nM, and 1.3 μM, respectively. The proposed mechanism for the reaction of these sensors with GSH is shown in Figure . Importantly, 47, (MNDA) bearing an electron-donating group, can be used to image GSH in live cells by using TPM with excitation at 900 nm. Importantly, 46 and 48 were applied to predict the mortality of patients with sepsis (Figure ), which means that these chemosensors can be used for medical diagnosis.

Figure 38

Simultaneous sensing of biothiols based on reactions with dialdehyde 46–48.

Figure 39

Diagram for use of 46 and 48 to predict the mortality of patients with sepsis (Copyright from ref (75)).

Simultaneous sensing of pan class="Chemical">biothiols based on reactions with n>n class="Chemical">dialdehyde 46–48.

Diagram for use of 46 and 48 to predict the mortality of pan class="Species">patients with n>n class="Disease">sepsis (Copyright from ref (75)).

Because the intracellular pan class="Chemical">GSH concentration in cells is in the range of 1–10 mM, the development of reversible GSH chemosensor with desirable dissociation constants is important. The fluorescent chemosensor 49 is reported to display ratiometric changes in its absorption upon the addition of GSH (Figure ).[76] For example, GSH causes the formation of a new absorption peak at 409 nm and a decrease in the peak at 474 nm. Similar ratiometric changes were also observed in the fluorescence spectrum, which displays an increase at 488 nm and decrease at 560 nm upon the addition of GSH. Dilution and addition of NEM or H2O2 induce the reverse reaction and associated photophysical changes. The dissociation constant of 49 with GSH of 2.59 mM and a fast response time (t1/2 = 5.82 s) are close to ideal. The GSH concentration in live HeLa cells was calculated to be 5.40 ± 0.87 mM based on the calibration curve obtained from ratiometric cell imaging data using 49, which is very close to the reported value.

Figure 40

Reversible reaction of 49 with GSH to show a ratiometric fluorescent change.

Reversible reaction of 49 with pan class="Chemical">GSH to show a pan class="Species">ratiometric fluorescent change.

The concentpan class="Species">ration of n>n class="Chemical">H2S in the central nervous system is reported to be between 50 and 160 μM 45, and the sulfide level in blood plasma is between 10 and 100 μM.[77] In studies designed to develop sensors that detect H2S at these levels, we found that the naphthalimide derivative 50 serves as a H2S selective fluorescent chemosensor (Figure ).[78] A 68-fold fluorescence enhancement was observed to occur at 532 nm upon the addition of H2S to a solution of 50 as a consequence of a reaction (Figure ) that produces the piperazine–naphthalimide product 50-I. Because it contains a triphenylphosphonium group, 50 locates mainly in mitochondria. The H2S detection limit of 50 was reported to be 2.46 μM.

Figure 41

Reaction of 50 with H2S to form quencher-free product 50-I.

Reaction of 50 with pan class="Chemical">H2S to form quencher-free product 50-I.

The 2-(2’-hydroxyphenyl)benzothiazole derivative, 51, was designed as a pan class="Chemical">H2S selective chemosensor,[79] operating by the reaction shown in Figure . ESIPT emission at 480 nm takes place upon the addition of H2S to 51. On the other hand, AIE at 540 nm was observed in higher vol % fractions of PBS. Probe 51 can be used to image H2S in live cells using one-photon and TPM.

Figure 42

Reaction of 51 with H2S to form ESIPT activated product 51-I.

Reaction of 51 with pan class="Chemical">H2S to form ESIPT activated product 51-I.

The pan class="Chemical">phenolphthalein derivative 52 was developed as a n>n class="Chemical">thiophenol (PhSH)-selective chemosensor (Figure ).[80] Thiophenol reacts with 52 to form phenolphthalein quantitatively, which turns pink (547 nm) when the pH is increased. Probe 52 was also applied to a filter paper, which undergoes a distinct color change to pink upon the addition of thiophenol at pH 10.5. The detection limit of 52 for thiophenol was found to be 6.15 μM. An AND logic gate was constructed using PhSH and base as inputs and color change of 52 viewed by the naked eye as an output.

Figure 43

Reaction of 52 with PhSH to form phenolphthalein.

Reaction of 52 with PhSH to form pan class="Chemical">phenolphthalein.

Gas Selective Fluorescent Chemosensors

Our group reported several examples of fluorescent chemosensors for pan class="Chemical">CO2 and toxic gases, such as n>n class="Chemical">phosgene and nerve gas mimics.[81,82] In 2002, in collaboration with Sessler, Lee, and Bielawski, we designed an “anion-activated CO2 recognition” strategy using benzobisimidazolium salts 53 (Figure ).[83] Addition of F– induces efficient fluorescent quenching of 53 and the appearance in its absorption spectrum of an ICT band centered at 344 nm as a result of initial formation of (C–H)+···F– ionic H-bonding and eventually NHCs. Addition of CO2 to F– treated 53 revived the original absorption at 290 nm and fluorescence emission. The CO2 detection limit of this system was reported to be 30 ppm. The formation of the NHC carbene in this process was confirmed by isolation of the product formed using CS2 instead of CO2.

Figure 44

Proposed reaction of 53 with F– followed by the addition of CO2 or CS2.

Proposed reaction of 53 with F– followed by the addition of pan class="Chemical">CO2 or pan class="Gene">CS2.

The anion-activated stpan class="Species">rategy to sense n>n class="Chemical">CO2 was utilized by us to develop a sol–gel-based system.[84] Naphthalimide derivative 54, utilized in this system, contains a cholesterol and carbamate ester moiety (Figure ) and undergoes optimal gelation in DMSO among other solvents. The critical gelation concentration was found to be 0.18 wt %. In the sol or gel state, 54 has a green color and blue emission. A distinct color change to orange as well as an emission color change to orange occurs upon the addition of F–. The green color and blue emission are revived when CO2 gas is introduced. When this system is heated to 80 °C under N2, CO2 is released and the orange color and orange emission were regenerated. As shown in Figure , F– induces deprotonation of the amide NH moiety in 54, which results in the distinct color and emission change owing to ICT.

Figure 45

Reaction of 54 with F– followed by the addition of CO2.

Reaction of 54 with F– followed by the addition of pan class="Chemical">CO2.

pan class="Chemical">PDAs were utilized as colorimetric and fluorescent chemosensors for n>n class="Chemical">CO2. PDAs are unique conjugated polymers, which have blue color, and undergo a distinct colorimetric change to red and a fluorescence enhancement upon exposure to various stimuli.[85,86] A PDA, derived from diacteylene monomer containing a imidazolium head group and the other primary amine, was constructed for the detection of CO2.[87] As shown in Figure , CO2 reacts with primary amine moiety in the PDA to form a carbamate, which then interacts with imidazolium moiety resulting in the creation of stress in the conjugated backbone and a consequent color transition from blue (623 nm) to red (540 nm). A fluorescent enhancement also accompanies addition of CO2. We used this system to create PDA nanofibers, which can be used to detect CO2 gas in the presence of triethylamine vapor.

Figure 46

Reaction of PDA-based chemosensor with CO2 to form carbamate moiety.

Reaction of pan class="Chemical">PDA-based chemosensor with n>n class="Chemical">CO2 to form carbamate moiety.

Several chemosensors were recently reported for sensing the toxic gas, pan class="Chemical">phosgene. The first is the fluorescent and colorimetric chemosensor 55, which is based on cyclization reaction of a n>n class="Chemical">o-phenylenediamine moiety.[88] Fluorescent chemosensor 55 undergoes a selective colorimetric change from light yellow to dark pink along with the development of a strong red emission upon the addition of phosgene (Figure ). On the other hand, the nerve gas mimic, diethyl chlorophosphate (DCP), induces formation of green fluorescence. Phosgene reacts with 55 to form a benzimidazolone-containing product, whose ICT character is dramatically altered because the electron-donating amine groups are converted to an electron-withdrawing urea group. 55 was applied to a polyethylene oxide membrane, which can be employed to detect phosgene and DCP vapor by induction of different color and fluorescent changes.

Figure 47

Proposed reaction of 55 with phosgene to form benzimidazolone-containing product, which can block PET process with a huge alteration of ICT property.

Proposed reaction of 55 with pan class="Chemical">phosgene to form n>n class="Chemical">benzimidazolone-containing product, which can block PET process with a huge alteration of ICT property.

Same stpan class="Species">rategy was also applied to the design of n>n class="Chemical">4-chloro-7-nitrobenzo[c]-[1,2,5]oxadiazole 56-, rhodamine 57-, and naphthalimide 58-based chemosensors (Figure ).[89] The structures of these chemosensors and their products of reactions with phosgene are shown in Figure . A distinct color change from dark orange to pale yellow occurs when 56 is subjected phosgene. On the other hand, rhodamine derivative 57 shows a large fluorescent enhancement (∼60-fold) at 575 nm with a color change from colorless to pink when exposed to this nerve agent. The detection limit of 58 for phosgene was found to be 2.8 ppb. Nanofibers containing 56 and 57 display clear color and fluorescent changes when exposed to phosgene.

Figure 48

Proposed reactions of 56–58 with phosgene to form benzimidazolone-containing products.

Proposed reactions of 56–58 with pan class="Chemical">phosgene to form n>n class="Chemical">benzimidazolone-containing products.

A so-called “second-genepan class="Species">ration sensor” for n>n class="Chemical">phosgene was developed in our laboratory. The new rhodamine derivative 59 shows a distinct color change to dark pink and a 3.7-fold fluorescence enhancement at 578 nm when treated with phosgene.[90] The detection limit was calculated to be 3.2 ppb. Most importantly, unlike previously developed chemosensors, 55–58, 59 reaction with phosgene does not produce HCl as a byproduct. The mechanism of this process, shown in Figure , was confirmed by using 1H and 13C NMR as well as mass spectroscopy. As shown in Figure , chemosensor 59 embedded in nanofibers displays distinct colorimetric and fluorescent changes in response to phosgene gas.

Figure 49

Reaction of rhodamine derivative 59 with triphosgene to induce spriolactam ring-opening process.

Figure 50

Colorimetric and fluorescent changes of chemosensor 59 in a poly(ethylene oxide) nanofiber upon exposure to phosgene (0.8 mg/L phosgene gas).

Reaction of pan class="Chemical">rhodamine derivative 59 with trin>n class="Chemical">phosgene to induce spriolactam ring-opening process.

Colorimetric and fluorescent changes of chemosensor 59 in a pan class="Chemical">poly(ethylene oxide) nanofiber upon exposure to n>n class="Chemical">phosgene (0.8 mg/L phosgene gas).

pan class="Chemical">2-(2-Aminophenyl)benzothiazole 60 was also synthesized, which serves as an ESIPT-based fluorescent chemosensor. This substance reacts with n>n class="Chemical">phosgene in the presence of triethylamine to produce color and fluorescence changes (Figure ).[91] Chemosensor 60 displays blue emission at 445 nm, which upon exposure to phosgene changes to an emission at 495 nm in conjunction with a decrease in original blue emission. In addition, a color change from colorless to yellow occurs upon exposure to phosgene. The detection limit of 60 for phosgene was found to be 0.14 ppm with a linear range from 0–3.0 μM.

Figure 51

Reaction mechanism of 60 with phosgene to form tetracyclic product 60-I bearing strong electron-withdrawing group.

Reaction mechanism of 60 with pan class="Chemical">phosgene to form tetracyclic product 60-I bearing strong electron-withdrawing group.

Another ESIPT-based chemosensor, 61, shows selective fluorescence and colorimetric changes in response to the nerve gas mimic, pan class="Chemical">diethyl cyanophosphonate (n>n class="Chemical">DECP), both in solution and the gas phase.[92] As shown in Figure , the salicylaldehyde oxime moiety in 61 is converted to a nitrile in association with a large fluorescent enhancement (∼60-fold) at 480 nm. The detection limit was calculated to be 1.3 nM. Probe 61 (0.45 w/w %) was incorporated in electrospunfibers, using polyvinylpyrrolidone as the matrix, which display clear colorimetric and fluorescent changes upon exposure to DECP vapor.

Figure 52

Stepwise reaction of 61 and DECP to form fluorescent nitrile derivative 61-III.

Stepwise reaction of 61 and pan class="Chemical">DECP to form fluorescent pan class="Chemical">nitrile derivative 61-III.

Future Outlook

Since the pioneering work in the late 1970s and 1980s, fluorescent chemosensors have been the subject of intense studies. Advances made in molecular recognition and supramolecular chemistry greatly influenced the early development of these chemosensors. After the initial phase of studies in this area, the recognition of saccharides by boronic acid groups was utilized by the James group.[93] Also, Lippard groups reported the development of pan class="Chemical">Zn2+ selective fluorescent chemosensors that have biological applications.[14] The Nagano group also reported sensors for enzymes[94] and BODIPY-based fluorescent chemosensors, and their applications to molecular logic gates were developed by Akkaya.[95]

A key contribution was made in 1997 by Czarnik’s group, which developed a pan class="Chemical">rhodamine derivative bearing n>n class="Chemical">hydrazine moiety that binds to Cu2+. These workers found that hydrolysis of the Cu2+complex induces ring-opening, resulting in a large fluorescence enhancement.[22] This can be considered as the starting point from which organic chemists began the study of chemodosimeters, in which various organic reactions promote fluorescence changes. For example, Koide reported several examples of reaction-based chemosensors for Hg2+ and Pd2+.[13] In addition, Chang reported pioneering work on the development of H2O2 selective chemosensors based on boronic acid/ester oxidation process.[12] Since that time, a variety of signaling units and the new mechanism of AIE were actively studied by Tang.[9] TPM has become a powerful tool especially for tissue imaging because of its advantage of deep penetration.[96]

We believe the previous and current advances made in developing fluorescent chemosensors will stimulate further activity aimed at devising approaches to new target analytes or for disease diagnosis.[97] Even though designing new chemosensors is a challenging process, we believe that interdisciplinary collabon class="Species">rations between chemists and biologists will facilitate the progress in this area. New fluorophores need to be synthesized by organic chemists and material chemists, which can show superior photophysical properties, such as excellent quantum yield, high photostability, desirable solubility in aqueous solution, NIR emission for deep penetn>n class="Species">ration, and so forth. A new binding site as well as a new reaction site for specific analyte will be an important aspect for the development of new fluorescent chemosensors. In addition, fluorescent chemosensors for biological species can be combined with research on drug delivery[98,99] and photodynamic therapy.[100] Trigger groups previously utilized for fluorescent chemosensors are currently utilized for activatable drug delivery and activatable photosensitizers. Recently, “one for all” concept was reported for the phthalocyanine-based activatable photosensitizers, in which fluorescence and singlet oxygen generation can be controlled.[101] Moreover, fluorescent chemosensors can be applied to solve environmental problems.

Consequently, we feel that fluorescent chemosensors will play key roles in future studies in the areas of chemistry, biology, chemical biology, and medical and materials sciences.