A Unique “Integration” Strategy for the Rational ... - ACS Publications

May 11, 2017 - DOI: 10.1021/acs.accounts.7b00087 ... His research interests focus on the development of fluorescent dyes and their biological applicat...
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A Unique “Integration” Strategy for the Rational Design of Optically Tunable Near-Infrared Fluorophores Hua Chen,‡,§ Baoli Dong,†,§ Yonghe Tang,† and Weiying Lin*,†,‡ †

Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China ‡ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, P. R. China S Supporting Information *

CONSPECTUS: Fluorescence imaging is a rapidly growing technique for noninvasive imaging of biological molecules and processes with high spatial and temporal resolution. For effective biological imaging, it is essential and important to develop robust fluorescent dyes, in particular, near-infrared (NIR) fluorescent dyes with favorable optical properties. Compared with the visible light emitting dyes, NIR dyes have relatively longer emission wavelengths (650−900 nm) with lower energy and are advantageous as imaging agents owing to the minimum photodamage of NIR light to biological samples, deep penetration into tissues, and low interference from autofluorescence of biomolecules. Although great efforts have been devoted to engineer NIR fluorophores, it is still very challenging to regulate their photophysical properties as they often lack optically tunable mechanisms, and this shortcoming considerably restricts the realization of their full potential. Consequently, the rational design of small-molecule optically tunable NIR fluorophores is of high priority and great value. In general, two key characteristics are indispensable for designing excellent optically tunable NIR fluorescent dyes. First, NIR fluorescent dyes should display the maximal absorption and emission located in the NIR region and also have the prominent properties including excellent fluorescence quantum yields, large Stokes shifts, good chemical stability and photostability, low cytotoxicity, and desirable compatibility with biological systems. Second, in principle, functional NIR dyes should also possess optically tunable groups, which can be easily modified to afford responsive sites for the targets of interest. With these considerations in mind, in this Account, we described a unique “integration” strategy for judicious design of the optically tunable NIR fluorophores, which are an intuitive combination of the traditional NIR dyes and the optically tunable mechanisms in the visible light emissive dyes. Thus, the versatile strategy may allow not only retention of the NIR emission properties of NIR dyes but also inheritance of the optically tunable mechanisms from the visible light emissive dyes. By the unique integration strategy, a built-in optically tunable group is strategically installed into the traditional NIR fluorescent dyes to directly tune their optical properties. Herein, we present a concise review of the rational design strategy and biological applications of small-molecule optically tunable NIR fluorescent dyes via the unique integration strategy, and we focused mainly on our work and some representative examples from other groups based on our NIR platforms. This Account includes the detailed integration strategy of each class of the NIR fluorescent dyes, the development of their derivatives, and their imaging applications in living systems. Currently, employment of NIR dyes to construct fluorescent sensors for in vivo imaging has made significant progress; however, it is still far from reaching full potential. The oft-used NIR dyes are largely limited to a few traditional NIR fluorophores, such as cyanines, squaraines, and bodipy derivatives.4 However, these dyes generally have no optically available tunable groups, rending them often favorable as simple fluorescent tags for biospecies but not as the platforms for developing targetsensitive fluorescent sensors. To address this problem, several strategies have been formulated. For example, the photoinduced

1. INTRODUCTION Near-infrared (NIR) fluorescent dyes are generally defined as materials that emit NIR fluorescence (650−900 nm). To date, applications of the NIR fluorescent dyes have emerged in broad fields including night-vision technologies, fluorescence imaging, biochemical assays, heat-blocking coating, and medical diagnosis.1,2 In particular, fluorescence imaging is a rapidly growing technique for noninvasive visualizing of biological molecules and processes with high spatial resolution. Because of minimum tissue absorption, low interference from autofluorescence of biomolecules, minimal photodamage of the NIR light to biological samples, and deep tissue penetration, NIR dyes have attracted great attention in the area of in vivo imaging.3 © 2017 American Chemical Society

Received: February 13, 2017 Published: May 11, 2017 1410

DOI: 10.1021/acs.accounts.7b00087 Acc. Chem. Res. 2017, 50, 1410−1422

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Figure 1. Illustration of the unique “integration” strategy for the design of novel NIR functional dyes with optically tunable mechanisms.

Figure 2. (A) Design of the CS NIR functional dyes based on the integration of rhodamine with merocyanine A. (B) Representative structures of the CS NIR dyes.

on fluorescence of the fluorescent sensors based on cyanines.6 Nagano et al. constructed a Si-rhodamine-based NIR hypochlorous acid sensor, and it acts via the characteristic spirocyclization-based

electron transfer (PET) mechanism has been employed for construction of some cyanine-based NIR fluorescent sensors.5 Alternatively, Murthy et al. depicted a concise strategy to switch 1411

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Figure 3. Comparison of the fluorescence on−off switching mechanisms of rhodamine dyes and CS dyes. (A) Spirocyclization of rhodamine and rhodamine amide derivatives. (B) Spirocyclization of CS and CS amide derivatives. Reproduced with permission from ref 13. Copyright 2012 American Chemical Society.

Figure 4. (A) Structures of CSA1−6. Reproduced with permission from ref 14. Copyright 2013 Elsevier Ltd. (B) Structures of ACA1−6. Reproduced with permission from ref 15. Copyright 2015 Royal Society of Chemistry.

extensively applied as the platforms to construct fluorescent sensors. In brief, the versatility of the optically tunable mechanisms of visible light emissive dyes makes them highly desirable for constructing fluorescent sensors. Inspired by the versatile optically tunable mechanisms in the visible light emissive dyes, we introduced a unique “integration” strategy to address the issue that the traditional NIR dyes generally lack optically tunable mechanisms (Figure 1). The robust integration strategy is an intuitive combination of the traditional NIR dyes and the optically tunable mechanisms in the visible light emissive dyes. Thus, the effective strategy allows not only retention of the NIR emission properties of NIR dyes but also inheritance of the optically tunable mechanisms from the visible light emissive dyes. By the integration strategy, a built-in optically tunable group is strategically installed into the traditional NIR dyes to directly tune their optical properties. The integration strategy may offer a convenient and rational

fluorescence on−off switching mechanism.7 Shabat et al. presented a graceful way to control the fluorescence of cyaninebased sensors by a protection−deprotection mechanism.8−10 Overall, the effective optically tunable mechanisms for design of NIR sensors are scarce, which essentially limits the widespread development of the NIR sensors. Compared with NIR dyes, visible light emissive dyes have been extensively employed to construct fluorescent sensors as a result of their versatile optically tunable mechanisms. For example, the traditional 7-hydroxycoumarin derivatives contain a key hydroxyl group that can be easily amended to tune their photophysical profiles, and this makes 7-hydroxycoumarin derivatives extensively utilized in optical imaging. For other classic dyes such as fluorescein and rhodamine B, their fluorescence can be conveniently tuned by intramolecular cyclization.11 The ringopen form is fluorescent, while the spirocyclic form is nonfluorescent. Rhodamine and fluorescein derivatives have been 1412

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Accounts of Chemical Research method for engineering new functional NIR dyes with desirable optically tunable mechanisms. Herein, we focus on the discussion of the rational design and biological applications of the optically tunable small-molecule NIR dyes based on the integration strategy, using illustrative examples mainly from our studies, including CS, CyBX, HD, and CHMC. The following content regarding the optically tunable NIR dyes is divided into four parts, exemplifying integration of rhodamine with merocyanine dyes, integration of rhodamine with cyanine dyes, integration of hydroxycoumarins/fluorescein with merocyanine dyes, and the second-generation of optically tunable NIR dyes.

2. INTEGRATION OF RHODAMINE WITH MEROCYANINE DYES 2.1. Design of CS NIR Dyes

Controlling the optical properties of merocyanine A, a NIR fluorescent dye, is challenging as it has no appropriate fluorescence switching mechanism (Figure 2A). However, we envisioned that if an effective fluorescence mechanism was judiciously installed, merocyanine A could be employed as a vigorous NIR fluorescent platform with optically tunable properties. Rhodamine dyes possess excellent photochemical properties including high fluorescence quantum yields and excellent photostability. Importantly, rhodamine dyes can undergo intramolecular cyclization to regulate their fluorescence profiles. The open-ring form is fluorescent, while the spirocyclic form is nonfluorescent. By exploiting the effective fluorescence ON−OFF switching mechanism, various fluorescent sensors based on the rhodamine platform have been constructed.12 However, these fluorescent sensors generally have visible absorption and emission. This renders them unfit for in vivo imaging. Thus, it is important to construct rhodamine-based dyes with NIR absorption and emission. According to the integration strategy, we have engineered unprecedented NIR functional dyes, named as CS dyes,13 which are essentially the integration of merocyanine A and rhodamine dyes (Figure 2). In general, CS dyes show NIR optical properties like merocyanine A. Moreover, the carboxylic acid group may regulate their fluorescence via spirocyclization. Thus, CS dyes could possess the powerful fluorescence ON−OFF switching mechanisms like rhodamine dyes and also have the advantages of NIR optical properties of merocyanine A. As designed, the optical profiles of CS1−6 resemble those of the traditional cyanine-based NIR dyes. Significantly, similar to rhodamine dyes, CS1−6 also essentially hold a carboxylic acidbased fluorescence on−off switching mechanism due to their desirable electron-pulling systems for spirocyclization (Figure 3). Notably, CS1−6 dyes are superior to the classic rhodamine dyes because their absorption and emission both locate in the NIR region.

Figure 5. (A) Structure of CS-HClO. (B) Fluorescence response of CS-HClO to HClO. (C) Fluorescence images of endogenous HClO from the mice with CS-HClO. Reproduced with permission from ref 13. Copyright 2012 American Chemical Society.

(Figure 4A).14 The Stokes shifts of CSA1−6 range from 84 to 178 nm in MeOH, significantly larger than those of the classic rhodamines and CS NIR dyes (Table S1). This valuable feature of CSA may be rationalized by the excited-state intramolecular charge transfer between the electron donor and acceptor. Although CSA dyes have large Stokes shifts, their fluorescence quantum yields are no more than 12.3% and are much smaller than those of CS NIR dyes (typically more than 29%). In other words, the large Stokes shifts of CSA dyes are achieved by the sacrifice of their fluorescence quantum yields. Thus, we intended to reach a balanced state between large Stokes shifts and high fluorescence quantum yields for CS derivatives. With this in mind, in 2015, we developed another type of CS analog, abbreviated as ACA NIR dyes (Figure 4B).15 The ACA dyes showed favorable Stokes shifts (43−70 nm), which are larger than those of CS NIR dyes (Table S1). Significantly, the fluorescence quantum yields of ACA (up to 40.1%) are generally higher than those of CSA. 2.3. Biological Applications of CS Dyes and Their Analogs

The NIR functional fluorescent dyes CS and their analogs possess excellent photochemical properties such as high fluorescence quantum yields, excellent chemical stability and photostability, and NIR absorption and emission. More importantly, a carboxylic acid-based fluorescence on−off switching mechanism is integrated. These favorable features may make these NIR fluorescent dyes robust for biological imaging applications. Recently, a variety of fluorescent sensors based on CS and their analog NIR platforms have been constructed (Table S2). Due to the space limitation, we will only highlight some representative examples in the following section. 2.3.1. Application of CS Dyes as a HClO Sensor. To demonstrate the capability of CS derivatives as NIR fluorescent sensors for in vivo imaging applications, we engineered a NIR sensor CS-HClO by exploiting the HClO-triggered reaction of thiosemicarbazides with 1,3,4-oxadiazoles.13 CS-HClO displayed almost no fluorescence, because it exists as a spirocyclic form. However, addition of HClO induced a significant variation in the emission profile owing to the formation of the open form fluorescent product. CS-HClO was further successfully

2.2. Development of CS Analogues

The Stokes shifts of typical rhodamine dyes are only 20 nm, which can result in self-quenching and fluorescence detection errors owing to excitation backscattering effects. Accordingly, development of novel fluorophores with better photophysical profiles still attracts high attention. Like rhodamine fluorophores, CS dyes have relatively small Stokes shifts (generally less than 35 nm). Therefore, it is critical to construct CS analogues with improved Stokes shifts. With this in mind, we have engineered CSA, a type of CS analogues with large Stokes shifts 1413

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Figure 6. (A) Proposed sensing mechanism of SA-pNIR to pH. (B) Fluorescence response of SA-pNIR to pH. (C) Time-lapse illumination of subcutaneous tumors with SA-pNIR. (D) Biodistribution of SA-pNIR in the tumor-bearing mice. Reproduced with permission from ref 16. Copyright 2015 Royal Society of Chemistry.

Figure 7. (A) Structure of FUC-1. (B) Luminescence (a) and FUCL (b) images of HeLa cells treated with FUC-1 to monitor MeHg+. (C) Ex vivo luminescence and FUCL images of the mice incubated with FUC-1in the absence (a) and presence (b) of MeHg+. Reproduced with permission from ref 17. Copyright 2016 WILEY-VCH Verlag GmbH & Co. (D) Structure of NRh. (E) FUCL response of NRh to Cu2+. (F) In vivo FUCL images of normal mouse (left) and Wilson disease mouse (right) injected with NRh. Reproduced with permission from ref 18. Copyright 2016 WILEY-VCH Verlag GmbH & Co.

2.3.3. Application of CS Dyes As Upconversion Luminescent Sensors. Interestingly, Li et al. demonstrated that CS NIR dyes also could display favorable upconversion luminescent properties, and they further constructed some CS-based upconversion luminescent sensors (Figure 7).17,18 For instance, based on the CS platform, a NIR upconversion luminescent MeHg+ sensor FUC-1 was developed.17 FUC-1 exhibited good upconversion luminescent character, desirable NIR fluorescence increase, and low limit of detection for MeHg+. In addition, the sensor was used for detecting MeHg+ ex vivo and in vivo via Stokes luminescence and frequency upconversion luminescence (FUCL) bioimaging. By a similar approach, an upconversion luminescent sensor, NRh, for Cu2+ via the CS platform was engineered.18 The sensor NRh was shown to have a

employed to detect endogenous HClO in living macrophage cells and mice (Figure 5). 2.3.2. Application of CS Dyes As Tumor Detection and Imaging Agents. By exploitation of the desirable NIR properties of CS dyes, a targetable sialic acid-based CS sensor (SA-pNIR, Figure 6) was constructed for tumor detection by Han’s group.16 SA-pNIR contains a sialic acid unit for tumor targeting and a ring-closed CS moiety, which can respond to lysosomal aciditytriggered isomerization to give off a fluorescence signal. The favovable characters of SA-pNIR include desirable tumor-to-normal tissue signal contrast, sufficient retention in tumors, and minimal systemic toxicity. Furthermore, SA-pNIR could also transform NIR light into cytotoxic heat in the cells, implying the perspective for tumor photothermal therapy. 1414

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Figure 8. Design of CyBX NIR dyes based on the integration of rhodamine with cyanine dyes.

Figure 9. (A) Responsive mechanism of the sensor CyBN to pH. (B) pH-dependent fluorescence spectra (a) and photographs (b) of CyBN. (C) Fluorescence images of EC109 cells stained with CyBN and 10 mM NH4Cl. (D) Proposed sensing mechanism of CyBS with Hg2+. (E) Illustrative fluorescence images of the mice incubated with (a) PBS, (b) CyBS, or (c) CyBS + Hg2+. Reproduced with permission from ref 20. Copyright 2015 Royal Society of Chemistry.

low detection limit and be suitable for detecting Cu2+ex vivo and in vivo by FUCL imaging. Finally, the sensor was applied to detect Cu2+ in a Wilson disease mouse model. 2.3.4. Application of CS Dyes and Their Analogues As Sensing Agents for Other Targets. In addition to the above illustrative examples, CS dyes have also been extensively employed as a versatile NIR platform for developing fluorescent sensors to various targets of interest such as Cu2+, Hg2+, HClO, pH, H2O2, etc. (Table S2).

extensively applied NIR dye, the carboxylic acid group is tactically incorporated into the central carbon of the cyanine backbone to give CyBX dyes (Figure 8).20 We speculated that CyBX might be converted into the spiro-form (Spiro-CyBX) just like rhodamine dyes. As CyBX still keeps the intrinsic framework of heptamethine cyanine, it may display NIR absorption and emission. Nevertheless, the classic “push−pull” feature of cyanines is disturbed in their spiro-form by the spirocyclization-based pathway. Thus, we envisioned that SpiroCyBX could display essentially no NIR absorption and fluorescence as the carboxylic acid moiety could act as an integral mechanism to control the optical properties of heptamethinecyanine in a way similar to classic rhodamine dyes. As designed, indeed CyBX dyes have NIR absorption and emission, which are in good agreement with those of heptamethinecyanine. Notably, the class of CyBX dyes is considerably different from the type of CS dyes, as unlike the latter, the former still maintains the intact cyanine backbone.

3. INTEGRATION OF RHODAMINE WITH CYANINE DYES 3.1. Design of CyBX NIR Dyes

Heptamethinecyanines are desirable for biological imaging applications in living systems due to their NIR absorption and emission properties.4 Many cyanine-based NIR fluorescent sensors have been engineered, a few of which are operated by the PET mechanism.3,19 However, quenching the fluorescence of cyanines by the PET mechanism is very difficult because of their relatively high-lying occupied molecular orbital energy levels. Based on the integration strategy, we installed the optically tunable mechanisms of the visible light emissive dyes into NIR dyes. Exemplified by heptamethine cyanine (Cy7), which is an

3.2. Biological Applications of CyBX Dyes

CyBX dyes not only have NIR absorption and emission but also possess the intrinsic spiro-cyclization-based fluorescence switcher. These desirable features suggest that CyBx could operate as the robust platforms for constructing NIR fluorescent sensors. 1415

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agreement with the response mechanism described for benzothioate-based fluorescent Hg2+ sensors. Importantly, we also established that CyBS could image Hg2+ in living cells and animals.

4. INTEGRATION OF FLUORESCEIN OR 7-HYDROXYCOUMARIN WITH CYANINE DYES 4.1. Design of HD Dyes

Cyanine dyes are the most commonly used NIR fluorescent dyes for bioapplications.4 As aforementioned, the regulation of the cyanine fluorescence by a PET mechanism is difficult via the Rehm−Weller equation. By contrast, the traditional 7-hydroxycoumarins feature a hydroxyl group, which may be readily modified to tune their optical profiles. This character renders 7-hydroxycoumarins broadly employed in biological imaging. However, 7-hydroxycoumarin-based dyes generally show short absorption and emission wavelengths. Fluorescein displays a similar shortcoming. Therefore, they are not applicable for in vivo imaging, in which NIR absorption and emission are prerequisite. Inspired by the handy regulation mechanism of the key hydroxyl group in 7-hydroxycoumarins and fluorescein, we decided to transplant the key hydroxyl group into cyanines by the integration strategy. Toward this end, we developed a novel type of NIR dyes named as HD NIR fluorophores (Figure 10).21 Significantly, HD dyes are advantageous over the conventional 7-hydroxycoumarins and fluorescein with the NIR absorption and emission, while preserving an optically tunable hydroxyl group. This characteristic can be extensively exploited to design NIR sensors for in vivo imaging in living animals.

Figure 10. (A) Design of HD NIR functional dyes based on the integration of fluorescein or 7-hydroxycoumarin dyes with cyanines. (B) Photophysical properties of 7-hydroxycoumarin, fluorescein, and HD dyes. Reproduced with permission from ref 21.Copyright 2012 American Chemical Society.

We first evaluated the feasibility of CyBN (CyBX, X = N) as a NIR pH sensor (Figure 9A−C).20 As designed, CyBN exhibited strong fluorescence at pH 4.5. However, the emission decreased gradually with increasing pH. The pH-dependent emission profiles could be explained by the fluorescent ring-open form of CyBN dominating under acidic conditions while the nonfluorescent spirolactam form dominated under basic conditions. CyBN was further exploited to detect pH variations in living cells and animals. To further examine the feasibility of CyBX as NIR sensors in living animals, we also engineered CyBS (CyBX, X = S) as a fluorescent Hg2+ sensor (Figure 9D,E). CyBS showed nearly no fluorescence in PBS solution. Nevertheless, addition of Hg2+ elicited a significant effect on both the absorption and fluorescence profiles, as the sensor coordinated with Hg2+ to afford a fluorescent ring-open form of CyBS−Hg−CyBS, in

4.2. Development of HD Analogues

Organic dyes with indolium units that could be attacked by strong nucleophiles thus improving the chemical stability of HD dyes is highly desirable.22 In 2015, our group introduced a type of HD analogues, XC dyes (Figure 11).23 XC dyes have both absorption and emission in the NIR region, and their optical profiles can be regulated by modifications on the critical hydroxyl group. Importantly, the comparison of the optical response to the strong nucleophiles demonstrated that XC dyes are generally superior to the previously developed HD dyes in their chemical stability against strong nucleophiles.23

Figure 11. Structures of the HD analogs: XC, HD-NH2, and HD-NHCOCH3 dyes. 1416

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Figure 12. (A) Structure of NIR-Thiol. (B) Fluorescence response of NIR-Thiol to cysteine (Cys). (C) Fluorescence images for the mice treated with a different amounts [(a) 0, (b) 20, (c) 40, or (d) 160 nmol] of NIR-Thiol. Reproduced with permission from ref 21.Copyright 2012 American Chemical Society. (D) Structure of XC-H2S. (E) Fluorescence response of XC-H2S to H2S. (F) Illustrative fluorescence images of the mice incubated with (a) only XC-H2S, (b) XC-H2S + NaHS, or (c) only XC5. Reproduced with permission from ref 23. Copyright 2015 WILEY-VCH Verlag GmbH & Co.

Figure 13. (A) Structure of NIR-H2O2. (B) Fluorescence response of NIR-H2O2 to H2O2. (C) Fluorescence images of endogenous H2O2 with NIR-H2O2in living mice: (a) neither LPS nor NIR-H2O2 was injected; (b) saline was injected, followed by injection of NIR-H2O2; (c) LPS was injected, followed by injection of NIR-H2O2; (d) quantified emission intensity from the abdominal area of the mice of groups a−c. Reproduced with permission from ref 21.Copyright 2012 American Chemical Society. (D) Structures of DHX1 and CuDHX1. (E) Fluorescence response of CuDHX1 to AS. (F) (a) Bright field image; (b) blue channel showing nuclei; (c, d) NIR channel before and after addition of AS, respectively. Reproduced with permission from ref 26. Copyright 2014 American Chemical Society.

synthetic route to HD dyes, which is valuable for further applications of HD and their analogues.

Like the hydroxyl group, the amine group may be readily modified to tune the fluorescence of HD dyes. Thus, we further engineered HD-NH2 as another class of HD analogues (Figure 11).24 HD-NH2 exhibited intense fluorescence with a emission peak at 720 nm; by contrast, the control HD-NHCOCH3 with no electron-donating group displayed very faint emission. These data suggest that the optical profiles of HD-NH2 can be tuned by modifications on the crucial amine group. Interestingly, in 2015, Richard reported a flexible de novo synthesis of HD fluorophores in 60−70% overallyields,25 providing an efficient

4.3. Biological Applications of HD Dyes and Their Analogs

4.3.1. Application of HD Dyes and Their Analogs as Thiol or H2S Sensors. The optically tunable group makes HD dyes and their derivatives suitable for engineering diverse NIR sensors (Table S3). On the basis of the HD NIR platform, our group developed the NIR fluorescent sensor NIR-Thiol for thiols (Figure 12A).21 NIR-Thiol was constructed by modifying 1417

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Figure 14. (A) Sensing mechanism of NIR-Pd with Pd2+. (B) Fluorescence response of NIR-Pd to Pd2+. (C) Brightfield and fluorescence images of HeLa cells with NIR-Pd and Hoechst 33258 before (a−d) and after (e−h) the addition of PdCl2. Reproduced with permission from ref 27. Copyright 2013 Royal Society of Chemistry.

Figure 15. (A) Structure of NIR-NTR. (B) Variations in fluorescence spectra of NIR-NTR in the presence of nitroreductase. (C) Images of nitroreductase in living zebrafish incubated with (a, b) and without (c, d) NIR-NTR. Reproduced with permission from ref 28. Copyright 2014 Elsevier Ltd. (D) Structure of NIR-lactamase. (E) Fluorescence images of various S. aureus strains treated with NIR-lactamase: (a) ATCC BAA44; (b) ATCC 11632; (c) ATCC 29213; (d) relative pixel intensity of the corresponding fluorescence images. Reproduced with permission from ref 29. Copyright 2014 American Chemical Society.

Moreover, by feat of the favorable properties of XC dyes, we have also designed a NIR fluorescent H2S sensor, XC-H2S (Figure 12D).23 As expected, the free sensor displayed essentially no fluorescence; however, addition of H2S elicited drastic variations in the fluorescence profiles with a big turn-on signal at 725 nm (Figure 12E). The sensor XC-H2S was further

the hydroxyl group with 2,4-dinitrobenzenesulfonate moiety. The free sensor is almost nonfluorescent. Nevertheless, a significant fluorescence turn-on response (up to 180-fold) was noted upon the addition of thiols (Figure 12B). Significantly, the sensor could detect endogenous thiols in living cells and animal (Figure 12C). 1418

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Figure 16. (A) Structure of Lyso-pH. (B) Plot of I670/I708 versus pH values in the range pH 2.6−9.2. (C) Colocalization images of Lyso-pH with DND-99 (a) or rhodamine 123 (b) in MCF-7 cells. (D) Relationship between lysosomal pH and heat shock in HeLa cells (a) and MCF-7 cells (b) studied by Lyso-pH. Reproduced with permission from ref 30. Copyright 2014 WILEY-VCH Verlag GmbH & Co.

designing NIR sensors, we devised a NIR fluorescent sensor, NIR-Pd, on the basis of HD fluorophore and deprotection mechanism of propargylether by Pd2+(Figure 14).27 The NIR sensor showed both a significantly enhanced and ratiometric fluorescence response to Pd2+. Furthermore, the sensor exhibited excellent selectivity for Pd2+ and was employed for detecting Pd2+ in live cells. 4.3.4. Application of HD Dyes and Their Analogs as Assaying Agents for Enzymes. In 2015, a NIR fluorescence sensor, NIR-NTR, was developed for visualizing nitroreductase in zebrafish by Ma’s group (Figure 15A−C).28 The sensor was engineered by attaching 4-nitrobenzyl as a quenching and interactive unit to HD platform. The fluorescence sensing of NIR-NTR to nitroreductase was based on the enzyme-catalyzed reduction of 4-nitrobenzyl moiety, followed by the 1,6-rearrangement-elimination. Compared with the existing fluorescent assaying agents for nitroreductase, NIR-NTR displayed advantageous optical properties with NIR fluorescence emission over 700 nm, and it was further applied to monitor nitroreductase in living zebrafish. Ma et al. also engineered another NIR sensor, NIR-lactamase, for detecting β-lactamase (Figure 15D,E).29 The sensor was constructed by transplanting cephalosporin into the HD platform. NIR-lactamase displayed very faint fluorescence owing to the

demonstrated to be capable of monitoring H2S in living animals (Figure 12F). 4.3.2. Application of HD Dyes and Their Analogs as ROS or RNS Sensors. The excellent chemical stability of HD NIR fluorescent dyes made them favorable for judicious design of ROS or RNS fluorescent sensors for biological imaging in living animals. Via the HD dyes, a NIR fluorescent sensor NIR-H2O2 for H2O2 was constructed by our group (Figure 13A−C).21 Compared with the traditional sensors for H2O2 based on 7-hydroxycoumarin/fluorescein, the major advantage of NIR-H2O2 is that it has NIR absorption and emission. Our studies suggest that the sensor is applicable for imaging endogenous H2O2 in living cells and animals. By the unique character of HD fluorescent dyes, a NIR fluorescent sensor for detecting nitroxyl (HNO) was constructed by Lippard et al. (Figure 13D−F).26 This copper-based sensor, CuDHX1, possesses a HD fluorophore and a cyclam group as the Cu(II) binding site. Upon treatment with Angeli’s salt (AS), CuDHX1 displayed a 5-fold fluorescence enhanced response and good selectivity for HNO. They further showed that CuDHX1 can be applied for monitoring HNO in live cells. 4.3.3. Application of HD Dyes and Their Analogs as Sensors for Metal Ions. In order to further demonstrate the diversity of HD dyes and their analogs as the platforms for 1419

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Figure 17. (A) Design of the optically tunable NIR dye (CHMC). (B) Proposed sensing mechanism of CHMC-thiol with Cys. (C) Bright-field and fluorescence images of HeLa cells: (a−d) CHMC-thiol + NEM; (e−h) only CHMC-thiol; (i−l) CHMC-thiol + Cys. (D) Fuorescence images of thiols with CHMC-thiol in the mice: (a, d) the mice were given an injection of N-ethylmaleimide (NEM) followed by injection with CHMC-thiol; (b, e) only CHMC-thiol was injected; (c, f) the mice were injected with Cys and CHMC-thiol. Reproduced with permission from ref 31.Copyright 2016 Royal Society of Chemistry.

alkylation of the hydroxyl group of the HD fluorophore. However, β-lactamase could respond to the β-lactam ring in the sensor, thus inducing fragmentation, which further elicited the release of the HD dye to provide a large fluorescence response. Finally, the sensor was utilized to visualize β-lactamase in various S. aureus strains. 4.3.5. Application of HD Dyes and Their Analogs as pH Sensors. By incorporating a morpholine group into the HD platform, Ma’s group constructed a lysosome-targeting NIR ratiometric pH sensor, Lyso-pH (Figure 16).30 Upon excitation at 635 nm, the NIR emission of Lyso-pH at 670 nm decreased mildly with pH values changing from 4.0 to 7.4. The variations in the intensities were accompanied by a significant enhancement of the emission at 708 nm. The sensor had a pKa of 5.00 and a fluorescence quantum yield of 0.16 at pH 5.0, and showed a favorable I670/I708 linearity in the pH range 4.0−6.0. In addition, the sensor also displayed desirable reversibility between pH 4.0 and 8.0. By using Lyso-pH, they demonstrated that the lysosomal pH values increased during the heatshock process (Figure 16). 4.3.6. Application of HD Dyes as Sensing Agents for Other Targets. Owing to their favorable NIR photochemical properties, the HD dyes and their analogues have been extensively utilized as powerful platforms for the construction of NIR sensors. For example, NIR sensors for a variety of targets, such as hydrazine, selenol, H2O2, HNO, H2Se, tyrosinase, and γ-glutamyltranspeptidase have been constructed by exploiting the optically tunable hydroxyl group of the HD platforms (Table S3).

5. THE SECOND-GENERATION OF OPTICALLY TUNABLE NIR DYES BASED ON THE INTEGRATION STRATEGY It is very challenging to develop a single NIR fluorescent sensor for monitoring various concentrations of an analyte with different fluorescence readouts. We envisioned that this type of sensor should bear at least two interaction sites. However, HD dyes contain only one chemical handle that can be converted into a response site. To address this problem, we decided to integrate a HD NIR dye with chloro-substituted cyanine to provide a unique NIR dye, CHMC (chloro-hydroxyl merocyanine), which holds, notably, an important chloro unit and a key hydroxyl group that can be converted into two response sites for an analyte (Figure 17).31 In this Account, we called this class of optically tunable NIR dyes with at least two interaction sites as the secondgeneration optically tunable NIR platforms constructed based on the integration strategy. The new CHMC dye was applied as a robust platform to engineer a NIR sensor, CHMC-thiol, for monitoring concentration range-dependent Cys (Figure 17). The 2,4-dinitrobenzenesulfonate unit of the sensor was designed as a high reactive site for thiols, while the chloro group was employed as a low sensitivity site for thiols. As designed, CHMC-thiol exhibited almost no fluorescence, while treatment with a low concentration level of Cys (0−50 μM) can elicit marked variations in the fluorescence spectra at 680 nm with excitation at 550 nm. However, after addition of a high concentration level of Cys (50−500 μM) to CHMC-thiol, the emission peak at 680 nm 1420

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Accounts of Chemical Research gradually decreased while a new emission band at 625 nm appeared with excitation at 550 nm. Thus, fascinatingly, the sensor can show a turn-on readout to the low concentration level of thiols but a ratiometric fluorescence signal to the high concentration level of thiols. Furthermore, the sensor was applied to detect the concentration variations of thiols with different modes of fluorescence signals in living cells and animals.

Weiying Lin is a Professor of the Institute of Fluorescent Probes for Biological Imaging at the University of Jinan. His research interests cover the interdisciplinary areas of molecular recognition, photochemistry, materials science, analytical chemistry, and chemical biology.

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6. CONCLUDING REMARKS AND PERSPECTIVES In this Account, we have described a unique integration strategy for the rational design of optically tunable NIR fluorophores. By this integration strategy, combining the traditional NIR dyes with the optically tunable mechanisms in the visible light emissive dyes has afforded several types of optically tunable NIR dyes such as CS, CyBX, HD, and CHMC dyes. Significantly, this strategy can allow NIR dyes to possess two critical features: (1) NIR emission properties; (2) optically tunable mechanisms. We have highlighted the design principle and biological applications of some representative examples of the optically tunable NIR dyes mainly from our work. We expect that this robust integration strategy could be expanded for the design of a diverse array of functional NIR fluorescent dyes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.accounts.7b00087. Photophysical data of the NIR dyes CS in EtOH, CSA1−6 in MeOH and ACA1−6 in EtOH, representative examples of NIR fluorescent sensors based on CS dyes and their analog NIR platforms, and representative examples of NIR fluorescent sensors based on HD dyes and their analogues (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Weiying Lin., E-mail: [email protected]. ORCID

Baoli Dong: 0000-0002-6173-9218 Weiying Lin: 0000-0001-8080-4102 Author Contributions §

H.C. and B.D. contributed equally to this work.

Funding

Funding was partially provided by NSFC (21672083, 21472067), Taishan Scholar Foundation (TS 201511041), and the startup fund of University of Jinan. Notes

The authors declare no competing financial interest. Biographies Hua Chen is a doctoral student under the supervision of Professor Weiying Lin at the Hunan University. His research interests focus on the development of fluorescent dyes and their biological applications. Baoli Dong is a lecturer in the group of Professor Weiying Lin at the University of Jinan, and his research interests focus on the design and synthesis of fluorescent sensors for biological species. Yonghe Tang is a doctoral student under the supervision of Professor Weiying Lin at the University of Jinan. His research interests include the development of fluorescent sensors. 1421

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