Chinese Chemical Letters  2019, Vol. 30 Issue (10): 1799-1808   PDF    
New trends of molecular probes based on the fluorophore 4-amino-1, 8-naphthalimide
Lin Zhoua, Lijuan Xieb,*, Chuanhao Liub, Yi Xiaoa,*     
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China;
b Institute of Molecular Medicine & School of Biomedical Sciences, Huaqiao University, Quanzhou 362021, China
Abstract: 4-Amino-1, 8-naphthalimide (ANI) represents a valuable fluorophore from which a large number of probes have been derived in order to meet the requirements from the fields of biological sensing and imaging. In this review, the major progresses of ANI-based fluorescent probes in the past decade have been highlighted and categorized into three trends. The future development of ANI probes is also expected. This review provides a great deal of references and illuminating comments which will be helpful for the researchers designing and using fluorescent probes.
Keywords: 4-Amino-1, 8-naphthalimide     Two-photon     Subcellular organelle     Enzyme     Hybrid    
1. Introduction

Fluorescent probes have attracted more and more attention for their widespread applications in the visualization of the dynamical changes of biological microenvironment and the monitoring of diverse species in subcellular organelles [1]. In the development of fluorescent probes, it is critical to choose a fluorophore that, to a large extent, determines the applicability of the probe. Among many fluorophores, 4-amino-1, 8-naphthylimide (ANI) is a very popular precursor to construct of various probes [2-4]. Firstly, the synthesis and modifications of ANI derivatives are relatively easy for their simple structures. Secondly, the excellent photophysical properties of ANI derivatives are well known. They emit strong yellow-green fluorescence with high fluorescence quantum yields, large Stokes shifts and considerable sensitivity toward some environmental factors. This class of compounds also have good photostability and thermal stability as well as multiple derived sites [5]. Moreover, a few recent studies have revealed their excellent two-photon excited fluorescence activity, which further promotes the expansion of ANI's application territory [6-12].

ANI should be classified as a conventional dye, as it has been playing a valuable role in the field of fluorescence sensing and imaging for a long time. Before 2010, ANI-based probes were generally not targeted toward any subcellular organelle and they were just dispersed unevenly in cytoplasm after entering the cells [13-15], so that it was impossible for them to detect microenvironment factors in specific subcellular organelles. However, the situation changed afterwards, and inspiring research results on ANI-based targetable probes constantly emerged in the past several years. Some of works on ANI are pioneering, even for the whole field of fluorescence sensing and imaging. Therefore, it is very necessary to summarize the major advances in the design and application of ANI probes from 2010 to 2019. In brief, there are three new trends in ANI probes that are different from the conventional ones. The first trend is to develop the targetable probes suitable for applications in subcellular organelles, rather than nonspecific probes. Second trend is to design ANI probes as substrates of special enzymes, in order to sensitively determine the enzymes' activities. The third one is ANI-based specific labels to protein tags as hybrid sensors to monitor the proteins of interest. Interestingly, parallel to ANI, similar design concepts are also frequently utilized on classical fluorophores, e.g., rhodamine, BODIPY. Thus, we believe this review should be helpful for the researchers to grasp the recent trends in the development of fluorescent probes.

2. Subcellular organelle-targeted fluorescent probes

The subcellular organelles are essential components of the cells and key parts for maintaining the normal physiological function of the cells. When a subcellular organelle in a cell fails or loses its function, it can cause disease. Therefore, research on subcellular organelles is particularly needed. At present, scientists have designed a large number of fluorescent probes for the study of subcellular organelles. All of these molecules are designed based on the same idea, linking different targeted groups to 4-amino-1, 8-naphthalimide (ANI) for targeting to different subcellular organelles or internal organs [16]. The morpholine is introduced into the ANI to enter the lysosome [17-24], the positively charged triphenylphosphine or other positively charged small molecule can be used as a targeting group to enter the mitochondria [25-31], the Hoechst can be used as a targeting group to enter the nucleus [32].

The main structure of the current ANI-based fluorescent probe with targeting function is to introduce a small molecular targeting group or a positive charge at one site of the ANI fluorophore entry into specific subcellular organelles and other tissue cells by targeting groups or positive charges, including lysosome [33-36], mitochondria [26, 37-39] and nucleus [32] (Scheme 1). These probes all have access to a targeting group and a recognition group on the amide nitrogen atom of the ANI or at the 4- or 5-position. Upon entry into the cell, the recognition moiety of the probe interacts with the analyte or the recognition group leaves, thereby altering the fluorescence intensity of the probe precursor. In this way, it is possible to visualize living cells and provide a real-time monitoring method for physiological changes of cells.

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Scheme 1. Representative subcellular organelle-targetable probe.

As a weakly acidic organelle in cells, lysosomes are the main site for the transport of biomacromolecules. Its main role is to break down the nucleic acids, carbohydrates and lipids in the cells. The normal functioning of lysosomal function is affected by many factors. Therefore, the detection of the internal environment of lysosomes is of great significance. It is precisely because of these small molecular targeting groups that the probe can be accurately positioned and tracked. It is well known that morpholine is a widely recognized lysosomal localization group, and probes targeting lysosomes with morpholine as a localization group have been reported in large numbers.

In 2012, Xiao and co-workers reported a ANI-based two-photon fluorescent probe (Lyso-NINO) with lysosomal targeting for NO detection [18] (Fig. 1). The probe uses ANI as the acceptor fluorophore and o-phenylenediamine as the electron donor and NO trap. The acidic morpholino group enables the probe to be accurately localized to lysosomes, and for the first time, twophoton fluorescence microscopy and flow cytometry are used to capture NO in macrophage lysosomes, and have high selectivity and sensitivity to NO. When NO is not captured, the fluorescence of the probe is quenched due to the PET effect, whereas after capturing NO, the PETaction is blocked and the probe emits intense fluorescence. MTT experiments have shown that it has lower cytotoxicity than commercial lysosomal targeted NO probes (Fig. 2). The biggest advantage of this probe is that it does not release any other toxic factors after capturing NO, and will not cause secondary damage to the cells. The characteristics of twophoton excited fluorescence make Lyso-NINO promising as a probe capable of monitoring NO in living samples.

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Fig. 1. Structure of the NO probe Lyso-NINO and its sensing mechanism. Copied with permission [18]. Copyright 2012, American Chemical Society.

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Fig. 2. Lyso-NINO co-localizes to lysosomes in MCF-7 cells. Copied with permission [18]. Copyright 2012, American Chemical Society.

Similar to the above-mentioned report, another two-photon thiol probe 10 with lysosomal targeting function based on ANI has been reported [17]. The two ends of ANI are linked to 2, 4-dinitrobenzenesulfonyl chloride and morpholine as a recognition group and a targeting group respectively (Fig. 3). The response to various amino acids showed that the probe has high selectivity to thiols and can be used for the detection of thiols in lysosomes. The probe has a large two-photon absorption cross section and a high reactivity with thiols. Due to the strong charge transfer in the molecule, the probe fluorescence is quenched. When it reacts with the thiol, the 2, 4-dinitrophenyl group leaves and the probe fluorescence recovers. Co-localization experiments showed that the probe has a good lysosomal localization function (Fig. 4). However, the reaction of this probe with a thiol releases a small molecule of 2, 4-dinitrobenzenesulfonyl chloride, which may cause some damage to the cells, so there is a further improvement in the design of probe molecules in the future.

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Fig. 3. Structure of the thiol probe 10 and its sensing mechanism. Reproduced with permission [17]. Copyright 2016, Springer Nature.

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Fig. 4. Co-localization experiments using 10 to lysosomes in HeLa cells. Cells were stained with (A) 10 and (B) Lyso-Tracker Red. (C) Overlay of (A, B). (D) Intensity correlation plot of stain 10 and Lyso-Tracker Red. (E) Intensity profile of regions of interest (ROI) across HeLa cells. Reproduced with permission [17]. Copyright 2016, Springer Nature.

Mitochondria are one of the vital organelles in many eukaryotes. They are closely related to the body's energy supply, signal transduction, cell differentiation, cell death, and cell growth cycle. Once any malfunction of mitochondria can cause disease. Therefore, the exploration of the internal environmental factors of mitochondria is particularly important.

Up to now, a great number of fluorescent probes with mitochondrial targeting functions have been reported. Most probes targeting mitochondria usually carry a cationic charge, which is designed according to the membrane structure of the mitochondria. The negative charge inside the mitochondria interacts with the positive charge carried by the fluorescent probe, allowing the probe to target the mitochondria.

In 2014, Sessler and co-workers reported a mitochondriatargeted probe for quantitative measuring pH in living cell (probe 11, Fig. 5) [27]. The probe consisted of triphenylphosphonium, benzyl chloride moieties and piperazine-linked ANI fluorescence off-on signaling unit. Triphenylphosphine targets the probe to mitochondria and benzyl chloride immobilizes the probe to the mitochondria. Co-localization experiments with MitoTracker Red (MTR) showed that the probe can accurately localize in mitochondria (Fig. 6). The fluorescence of the probe is quenched due to the PET effect from piperazine to ANI. When in an acidic environment, the PET effect is inhibited and the fluorescence is turned on.

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Fig. 5. Structure of the probe 11 and its sensing mechanism. Reproduced with permission [27]. Copyright 2014, American Chemical Society.

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Fig. 6. Colocalization experiments involving probe 11 and MitoTracker Red (MTR) in HeLa cells. Copied with permission [27]. Copyright 2014, American Chemical Society.

The authors found that by treating cells with carbonyl cyanide m-chlorophenyl hydrazine (CCCP) to lower the membrane potential, the probe remains stable on the mitochondria. In addition, the probe accurately measures the pH of normal and starved cells, as well as real-time monitoring of pH changes associated with mitochondrial acidification and fusion that occur during mitophagy resulting from nutrient deprivation. The design of the probe provides a tool for the dynamic study of pH in mitochondria and also provides a strategy for designing new probes in the future.

The similar design was reported by Yoon et al. in 2016 [29]. Yoon and his co-workers designed a mitochondria-targetable hydrogen sulfide probe 12 (Fig. 7). The probe consists of a central fluorophore ANI, a mitochondrial targeting group a triphenylphosphine cation, and a hydrogen sulfide recognition group 7-nitro-1, 2, 3-benzooxadiazole (NBD). NBD is a hydrogen sulfide selective cleavage group with a significant off-on fluorescence response for hydrogen sulfide. The probe successfully achieves the detection of hydrogen sulfide in vitro and in vivo, exhibiting excellent properties with 68-fold fluorescence enhancement, with high sensitivity, low detection limit and low cytotoxicity. Confocal microscopy imaging showed that the probe was able to accurately localize to the mitochondria (Fig. 8). Intracellular imaging indicates that the probe can be used to further investigate the pathological effects of H2S on living organisms.

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Fig. 7. Chemical structure of probe 12 and its reaction with H2S. Copied with permission [29]. Copyright 2016, American Chemical Society.

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Fig. 8. Probe 12 was constrained with Mitotracker Deep Red and fluorescence images acquired by confocal microscopy. Copied with permission [29]. Copyright 2016, American Chemical Society.

The nucleus is ubiquitous in eukaryotic cells. Its main structure is a bilayer membrane-nuclear membrane which completely encapsulates the nucleus and contains a large amount of genetic material DNA. As an important part of chromosomes, DNA stores a large amount of genetic information internally. Once the DNA is damaged, it may cause genetic mutation or other pathological changes [40]. In recent years, research on DNA damage has become a hot topic for scientists [41].

It is well known that Hoechst is a nuclear targeting unit [42-46]. In 2017, Xiao Group reported a nuclear-specific dual-emission sensor Hoe-NI for ratio imaging of DNA damage processes [32]. The sensor attaches Hoechst to ANI by "click chemistry", and Hoe-NI can be introduced into the nucleus by Hoechst-linked ANI (Fig. 9). Hoe-NI has dual emission characteristics of ANI and Hoechst at the same wavelength excitation.

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Fig. 9. Structure of Hoe-NI and principles of fluorescent labeling of DNA. Copied with permission [32]. Copyright 2017, Wiley Publishing Group.

In vitro DNA titration experiments showed that the fluorescence of the two groups of dyes increased significantly with the increase of DNA concentration. This was due to the separation of Hoechst and ANI units after the binding of Hoe-NI with DNA, which inhibited the fluorescence quenching, and the fluorescence enhancement of ANI was more obvious than that of Hoechst due to the transfer of fluorescence resonance energy (Fig. 9).

Confocal imaging showed that the dye could be specifically labeled into the nucleus, and the experimental results proved that Hoe-NI has universal application in nuclear labeling (Fig. 10). MTT experiments have demonstrated that Hoe-NI has a relatively low cytotoxicity. In addition, Xiao et al. induced DNA breakage in cancer cells with an anticancer drug etoposide, and performed imaging with Hoe-NI. The results indicate that Hoe-NI is a potential indicator of DNA damage in anti-tumor therapy assays.

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Fig. 10. Confocal fluorescence imaging of cells stained with Hoe-NI and cell viability. (a–d) L02 cells. (e–h) SMMC-7721 cells. (i) Cell viability of different concentrations of Hoe-NI (1 mmol/L, 5 mmol/L and 10 mmol/L) at 24 h. Copied with permission [32]. Copyright 2017, Wiley Publishing Group.

The experiment of DNA damage in living cells induced by hydroxyl radicals produced by Fe2+ and hydrogen peroxide showed that the fluorescence intensity of the dye decreased with the increase of DNA damage (Fig. 11), which further indicated that Hoe-NI is suitable for detecting DNA damage caused by hydroxyl radicals in living cells.

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Fig. 11. The effect of ·OH on fluorescence ratio changes of Hoe-NI with ctDNA and cells labeling. (a) Fluorescence spectra of Hoe-NI and different concentration of H2O2. (b) Ratio (F505/F450) changes of Hoe-NI with ctDNA upon addition of Fe2+ and different concentration of H2O2. Reproduced with permission [32], Copyright 2017, Wiley Publishing Group.

Subunit-targeted fluorescent probes based on ANI have been synthesized in large quantities and have achieved outstanding effects, especially in detecting intracellular environmental factors. Scientists have used these probes to visualize cells and have made major breakthroughs in treating diseases. However, these probes still have some drawbacks, such as a shorter emission wavelength and are susceptible to autofluorescence interference after entering the cell. Therefore, researchers can consider extending the conjugated system of naphthalimide to increase its emission wavelength to the near-infrared region to eliminate background noise.

3. Enzyme probes based on 1, 8-naphthalimide

In addition to fluorescent probes for detecting small molecule active species, there is a class of probes for detecting the activity of enzymes in living cells [47]. Enzymes play important roles in human physiological activities, and many cancers are associated with abnormal activities of enzymes [48]. Prevention of early cancer is usually achieved by detecting the activity of the enzyme. Determining the expression of certain enzymes in cells is an effective means of preventing and treating cancer. Therefore, the detection of enzyme activity has become a hot topic in current research.

Tissue hypoxia is usually accompanied by disease. It is well known that the environment surrounding cancer cells in living organisms is usually hypoxic, because the reducing substances around the cancer cells are elevated, resulting in reducing stress in the body. This reducing stress is often associated with many diseases, including solid tumors [49], heart diseases [50] and Alzheimer's disease [51]. Nitroreductases (NTRs) are significantly higher in hypoxic tissues than in normal tissues. Therefore, in addition to using oxygen sensors to detect hypoxia around cancer cells, the detection of reductase is also an effective method. In recent years, the detection of nitroreductase has been reported (Scheme 2) [52-54].

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Scheme 2. Examples of nitroreductases probes.

In 2011, Qian et al. reported an ANI-based fluorescent probe 13 for detecting cellular hypoxia [52] (Fig. 12). The probe is formed by linking a nitrobenzyl group and an ANI via a carbamate group. The electron-withdrawing urethane group weakens the ICT effect and causes a blue shift in the fluorescence emission wavelength. Upon interaction with the enzyme, the fluorophore ANI is released and the fluorescence emission wavelength is greatly red shifted. In addition, the authors used probes to image the hypoxia in the cells and compared them to images under normal oxygen levels (Fig. 13). The results indicate that the probe is capable of visualizing hypoxia in solid tumors. Cytotoxicity experiments indicate that the probe is not toxic to cells. The report of this probe provides a new basis for the design of the subsequent nitroreductase probes.

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Fig. 12. Sensing mechanism of probe 13 on nitroreductase. Copied with permission [52]. Copyright 2011, American Chemical Society.

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Fig. 13. Fluorescence microphotographs of A549 cells incubated at aerobic condition (a–c) and hypoxic conditions (d–f). Copied with permission [52]. Copyright 2011, American Chemical Society.

In 2014, the Fang team reported the first probe TRFS-green for the assessment of mammalian thioredoxin reductase (TrxR) activity, which consists of ANI chromophore and 1, 2-dithiopentyl ring with fluorescence quenching (Fig. 14) [55]. This probe exhibits significant fluorescence enhancement induced by TrxR to disulfide bond cleavage and subsequent intramolecular ring and releases free ANI fluorophore. In vitro, TRFS-green was shown to be more selective for TrxR than other related enzymes and various small molecule thiols and bioreductive molecules. Fang et al. also successfully applied TrxR-green to TrxR active imaging in living cells (Fig. 15). The cells were treated by the addition of a TrxR inhibitor and the addition of a TrxR inhibitor and the fluorescence signal of TRFS-green in the cells was continuously attenuated.

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Fig. 14. Structure of TRFS-green and proposed mechanism of the fluorescence turn on of TRFS-Green switched by TrxR/NADPH. Copied with permission [55]. Copyright 2014, American Chemical Society.

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Fig. 15. Fluorescence changes of TRFS-green in living Hep G2 cells. (a) Hep G2 cells only (left), Hep G2 treated with TRFS-green (middle), and Hep G2 treated with DNCB followed by further treated with TRFS (right). (b) Relative fluorescence intensity in single cell in (a) was quantified by the software of Leica Qwin accompanied with the Leica microscope. (c) Quantification of fluorescence intensity of TRFS-green in Hep G2 cells. Copied with permission [55]. Copyright 2014, American Chemical Society.

After two years, Fang Group introduced mitochondria-targeted triphenylphosphine cations into the probe TRFS-green to obtain Mito-TRFS, which enabled visualization of TrxR2 in mitochondria (Fig. 16) [56]. In the cellular model of PD, staining cells by MitoTRFS indicated a dramatic decrease in TrxR2 activity, providing a mechanism for TrxR2 dysfunction to be associated with the etiology of PD.

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Fig. 16. Structure of the Mito-TRFS and its sensing mechanism. Copied with permission [56]. Copyright 2015, Royal Society of Chemistr.

Recently, Zhang et al. designed a glycosylated ANI-based activatable fluorescent probe 16 [57]. The probe consists of a hexosaminidase responsive group N-acetyl-β-D-glucosaminide and an ANI fluorophore modified with an ethylene glycol unit (Fig. 17). When the probe 16 is reacted with hexosaminidase, the glucoside of the linked ANI is rapidly cleaved, while the color (from colorless to yellow) and fluorescence (from blue to green) of the probe change significantly.

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Fig. 17. Structure of the probe 16 and its sensing mechanism. Copied with permission [57]. Copyright 2019, American Chemical Society.

The probe has better water solubility, fluorescence properties and affinity for hexosaminidases than 4-methylumbelliferyl N-acetyl-β-D-glucosaminide. The response mechanism of the probe to hexosaminidase was evaluated by HPLC analysis and TD DFT calculation. Cell imaging experiments have shown that the probe has low cytotoxicity and can be used as a tool for detecting intracellular hexosaminidases activity (Fig. 18).

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Fig. 18. Fluorescent images of MDBK and SW480 cells incubated with probe 16 for 1 h. (a–c) MDBK cells. (d–f) SW480 cells. (g–i) SW480 cells pretreated with PUGNAc for 1 h. Copied with permission [57]. Copyright 2019, American Chemical Society.

In 2015, Cui and co-workers designed a two-photon ratiometric fluorescent probe NCEN based on ANI for sensing human carboxylesterase 2 (hCE2) (Fig. 19) [58]. Upon stimulation with hCE2, the probe hydrolyzed to release ANI and its fluorescence spectrum was greatly red-shifted (90 nm). Using this probe, the authors first tested the high selectivity of hCE2 in living cells and tissues. This probe has good specificity for hCE2 and has been successfully used to detect the true activity of hCE2 in complex biological samples such as cell and tissue preparations.

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Fig. 19. Schematic illustration of NCEN and its fluorescence response toward hCE2. Copied with permission [58]. Copyright 2015, American Chemical Society.

Both NCEN and its hydrolysate, NAH, exhibit excellent twophoton properties with absorption cross sections of 512 GM and 542 GM, respectively. Tests on the sensing ability of CE2 in fresh mouse liver samples showed that the addition of LPA (a selective inhibitor of CE2) inhibited the hydrolysis of NCEN in mouse liver preparations (Fig. 20). These results indicate that NCEN can be used as a two-photon ratio fluorescent probe for imaging endogenous hCE2 in living specimens with high resolution and high sensitivity. The successful biological application of NCEN in living cells and tissues suggests that this probe has the potential to be a tool for endogenous hCE2 detection in other complex biological samples and provides new avenues for the prevention and treatment of related diseases caused by hCE2.

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Fig. 20. Two-photon confocal fluorescent images of endogenous CE2 in a mouse liver slice stained with NCEN (20 mmol/L). Copied with permission [58]. Copyright 2015, American Chemical Society.

4. Hybrid sensors and other probes

The function of traditional small molecule fluorescent probes is often simple, and they are unable to accurately and rapidly locate and detect intracellular factors for complex intracellular environments. In order to solve this problem, it is necessary to develop some multifunctional hybrid probes whose functions are not limited to simple detection or labeling. The so-called hybrid probe is to integrate two or more single-function small molecule dyes or functional groups, and achieve multiple targets at the same time.

The method of encoding a fusion protein tag enables stable covalent labeling of proteins by small molecules. SNAP-tag-based H2O2 fluorescent probe [59], Halo-tag-based NO fluorescent probe [60] and CLIP-tag-based H2S fluorescent probe [61] have been reported.

In 2017, Xiao Group reported three SNAP-tag-based ANI fluorescent dyes TNI-BG, QNI-BG and ONIBG with subcellular protein labeling [62]. The design is to link the O6-benzylguanine (BG) derivative to the two-photon fluorophore ANI by a "click" reaction, and has been used for specific labeling of subcellular proteins and fluorescent imaging through the SNAP-tag (Fig. 21).

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Fig. 21. Strategy for protein labeling and two-photon fluorescent imaging with ANIs. Copied with permission [62]. Copyright 2017, Wiley Publishing Group.

Different end groups were introduced into the 4-position amino group of the ANI ring to adjust solubility and dispersion, and to optimize labeling efficiency and specificity. TNI-BG, QNI-BG and ONI-BG all showed high labeling efficiency of SNAP-tag in solution and bacteria. The difference is that only ONI-BG has high labeling efficiency for mammalian cells (Fig. 22), and protein-labeled ANIs exhibit a high two-photon absorption cross section. These experimental results indicate that the method can be used to label and study other proteins of interest even in tissue and in vivo.

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Fig. 22. One-photon fluorescent imaging of subcellular SNAP-tag protein labeled with ANI BG derivatives. Cells express nucleus H2B targeted (a–c) or diffused (d) SNAP-tag stained with 5 mmol/L TNI-BG (a), QNI-BG (b) and ONI-BG (c, d). Copied with permission [62] Copyright 2017, Wiley Publishing Group.

Xu Group designed an ANI-derived fluorescent probe BGAN-2C for labeling SNAP-tag fusion proteins in living cells (Fig. 23) [63]. The probe is capable of rapid labeling onto the SNAP-tag fusion protein. The environmental sensitivity of ANI and the inhibition of photoinduced electron transfer from O6-benzylguanine to ANI resulted in a 36-fold increase in the fluorescence intensity of the probe. The high signal-to-noise ratio and fast response rate ensure that the probe can be labeled into living cells without washing (Fig. 24). In addition, Xu et al. synthesized a series of compounds (BGAN-DM, BGAN-2C, BGAN-8C, BGAN-12C) to confirm that the short linker between BG group and ANI fluorescence group brought ANI into hydrophobic environment, and inhibited BGinduced fluorescence quenching. In vitro tests showed that guanine could quench the fluorescence of compounds BGAN-DM, BGAN-2C, BGAN-8C and BGAN-12C, and the fluorescence of the four compounds was enhanced after labeling.

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Fig. 23. Structure of compounds BGAN-R and the reaction with the SNAP-tag. Copied with permission [63]. Copyright 2017, Royal Society of Chemistry.

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Fig. 24. No-wash HEK 293 live-cell imaging of SNAP-tagged proteins labeled with BGAN-2C. Copied with permission [63]. Copyright 2017, Royal Society of Chemistry.

After that, another fluorescent probe BGAN-Amino with a ANI fluorophore was reported by this group, which can be rapidly labeled with SNAP-tag [64]. Based on these properties, the authors used this probe to image proteins in mitochondria and nucleus in living cells, and achieved the effect of wash-free.

In addition to the introduction of small molecule protein tags to fluorophores, there is also a class of fluorescent probes based on trivalent arsenic receptor binding [65-69]. Xu and co-workers reported a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells [70]. The probe connects coumarin and ANI by arsenic, which is used to selectively interact with VDP, to form an energy transfer system (from coumarin to ANI) (Fig. 25). When not bound to the protein, the energy of the coumarin is transferred to the ANI due to the FRET effect, at which time only the ANI emits fluorescence. After entering the cell, since trivalent arsenic can selectively interact with the protein vicinal dithiols, the vicinal dithiols on the VDP is selectively exchanged with the 2, 3-dimercaptopropanol of VTAF to make the two fluorophores far away, thereby eliminating the FRET effect, and the VDP will also be specifically labeled by the coumarin moiety.

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Fig. 25. Chemical structures of VTAF and design strategy of VTAF for selective and ratiometric measurements of VDPs. Copied with permission [70]. Copyright 2014, American Chemical Society.

The probe VTAF achieves a quantitative determination of vicinal dithiols of VDP by a ratiometric fluorescence signal, and VTAF is used for specific labeling to achieve a no-wash effect, which is very advantageous for in situ capture of endogenous VDP. When cells are stimulated with different redox reagents, VTA can track the dynamic changes of vicinal dithiols in VDP cells. The subcellular distribution of endogenous VDP was clearly observed in living cells by colocalization experiments and ratio images (Fig. 26). These results are very helpful in further studying the important role of protein vicinal dithiols in cellular redox homeostasis and live cell protein function. In this way, it is expected that some drugs for treating redox-related diseases will be developed in the future.

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Fig. 26. Top: Colocalization of VTAF with Mito-Tracker Deep Red in live MCF-7 cells. Bottom: Enlarged images of single cell stained with VTAF and Mito-Tracker Deep Red. Copied with permission [70]. Copyright 2014, American Chemical Society.

5. Summary and perspective

Fluorescent probes based on 4-amino-1, 8-naphthalimide (ANI) fluorophore have made considerable headway in the past decade. Firstly, many ANI probes specifically targeting different kinds of subcellular organelles have been developed and successfully applied to visualize and sense important species and environmental factors. Secondly, quite a few ANI probes as enzyme substrates have been designed to determine the enzyme catalytic activity in live cells. Thirdly, hybrid sensors based on protein tags labelled with ANI fluorophores have also been constructed, which is an important improvement to make it feasible to sense and image on protein level.

ANI had been found very promising two-photon fluorophore. And several teams, including our group, designed a few organelles targetable ANI probes and apply them in two-photon microscopy imaging or two-photon fluorescence lifetime imaging, considering the advantages of two-photon technique over one-photon. We believe that, in the near future, many other ANI probes will also find extensively applications in two-photon imaging.

Although several cases of ANI-based enzyme substrates have been reported, there remains very large scope to develop enzyme activity probes. Actually, there are many valuable enzymes calling for specific probes to determine the catalytic efficiency within live cells. ANI with a conjugated structure simpler than most other fluorophores is relatively easy to modify into enzyme substrates. Therefore, it can be predicated that ANI will be one of the most popular fluorophore candidates for the development enzyme probes.

As for the development of hybrid sensors, there are also good opportunities left for ANI. To this date, different fluorophores have been designed as specific labels toward different protein tags. However, these fluorophores just light up the proteins of interest but they are not sensitive to a certain species or an environmental factor. In another word, they are actually markers, rather than sensors. Although very few attempts to design the hybrid sensors based on the combination of protein tags and small-molecule probes have been made, this situation will be change soon. And without any doubt, ANI will catch up this critic trend.

Acknowledgment

This work is supported by the National Natural Science Foundation of China (Nos. 21421005, 21576040 and 21776037).

References
[1]
H. Zhu, J. Fan, J. Du, et al., Acc. Chem. Res. 49 (2016) 2115-2126. DOI:10.1021/acs.accounts.6b00292
[2]
Z. Chen, Y. Xu, X. Qian, Chin. Chem. Lett. 29 (2018) 1741-1756. DOI:10.1016/j.cclet.2018.09.020
[3]
W. Xu, Z. Zeng, J. Jiang, et al., Angew. Chem. Int. Ed. 55 (2016) 13658-13699. DOI:10.1002/anie.201510721
[4]
R.M. Duke, E.B. Veale, F.M. Pfeffer, et al., Chem. Soc. Rev. 39 (2010) 3936-3953. DOI:10.1039/b910560n
[5]
S. Banerjee, E.B. Veale, C.M. Phelan, et al., Chem. Soc. Rev. 42 (2013) 1601-1618. DOI:10.1039/c2cs35467e
[6]
L. Rong, C. Zhang, Q. Lei, et al., Regen. Biomater. 3 (2016) 217-222. DOI:10.1093/rb/rbw022
[7]
Z. Zhang, J. Wu, Z. Shang, et al., Anal. Chem. 88 (2016) 7274-7280. DOI:10.1021/acs.analchem.6b01603
[8]
Y. Tang, X. Kong, A. Xu, et al., Angew. Chem. Int. Ed. 55 (2016) 3356-3359. DOI:10.1002/anie.201510373
[9]
S.K. Yao, Y. Qian, Sens. Actuators B:Chem. 252 (2017) 877-885. DOI:10.1016/j.snb.2017.06.091
[10]
Y. Liu, Y. Liu, W. Liu, et al., Spectrochim. Acta A 137 (2015) 509-515. DOI:10.1016/j.saa.2014.08.072
[11]
G.J. Mao, T.T. Wei, X.X. Wang, et al., Anal. Chem. 85 (2013) 7875-7881. DOI:10.1021/ac401518e
[12]
X. Chen, C.S. Lim, D. Lee, et al., Biosens. Bioelectron. 91 (2017) 770-779. DOI:10.1016/j.bios.2017.01.042
[13]
D. Cui, X. Qian, F. Liu, et al., Org. Lett. 6 (2004) 2757-2760. DOI:10.1021/ol049005h
[14]
D. Srikun, E.W. Miller, D.W. Domaille, et al., J. Am. Chem. Soc. 130 (2008) 4596-4597. DOI:10.1021/ja711480f
[15]
B. Zhu, X. Zhang, Y. Li, et al., Chem. Commun. 46 (2010) 5710-5712. DOI:10.1039/c0cc00477d
[16]
M.H. Lee, J.H. Han, P.S. Kwon, et al., J. Am. Chem. Soc. 134 (2012) 1316-1322. DOI:10.1021/ja210065g
[17]
J. Fan, Z. Han, Y. Kang, et al., Sci. Rep. 6 (2016) 19562. DOI:10.1038/srep19562
[18]
H. Yu, Y. Xiao, L. Jin, J. Am. Chem. Soc. 134 (2012) 17486-17489. DOI:10.1021/ja308967u
[19]
Q. Qiao, M. Zhao, H. Lang, et al., RSC Adv. 4 (2014) 25790-25794. DOI:10.1039/C4RA03725A
[20]
X. Xie, F. Tang, X. Shangguan, et al., Chem. Commun. 53 (2017) 6520-6523. DOI:10.1039/C7CC03050A
[21]
D. Kim, G. Kim, S.J. Nam, et al., Sci. Rep. 5 (2015) 8488. DOI:10.1038/srep08488
[22]
B. Zhang, X. Yang, R. Zhang, et al., Anal. Chem. 89 (2017) 10384-10390. DOI:10.1021/acs.analchem.7b02361
[23]
W. Feng, Q.L. Qiao, S. Leng, et al., Chin. Chem. Lett. 27 (2016) 1554-1558. DOI:10.1016/j.cclet.2016.06.016
[24]
S.I. Reja, M. Gupta, N. Gupta, et al., Chem. Commun. 53 (2017) 3701-3704. DOI:10.1039/C6CC09127J
[25]
Y. Dai, B.K. Lv, X.F. Zhang, et al., Chin. Chem. Lett. 25 (2014) 1001-1005. DOI:10.1016/j.cclet.2014.05.020
[26]
S. Huang, R. Han, Q. Zhuang, et al., Biosens. Bioelectron. 71 (2015) 313-321. DOI:10.1016/j.bios.2015.04.056
[27]
M.H. Lee, N. Park, C. Yi, et al., J. Am. Chem. Soc. 136 (2014) 14136-14142. DOI:10.1021/ja506301n
[28]
B. Deng, M. Ren, J.Y. Wang, et al., Sens. Actuators B:Chem. 248 (2017) 50-56. DOI:10.1016/j.snb.2017.03.135
[29]
Y.L. Pak, J. Li, K.C. Ko, et al., Anal. Chem. 88 (2016) 5476-5481. DOI:10.1021/acs.analchem.6b00956
[30]
B. Wang, X. Zhang, C. Wang, et al., Analyst 140 (2015) 5488-5494. DOI:10.1039/C5AN01063B
[31]
X. Zhang, Q. Sun, Z. Huang, et al., J. Mater. Chem. B 7 (2019) 2749-2758. DOI:10.1039/C9TB00043G
[32]
F. Yang, C. Wang, L. Wang, et al., Chin. Chem. Lett. 28 (2017) 2019-2022. DOI:10.1016/j.cclet.2017.07.030
[33]
Z. Qu, J. Ding, M. Zhao, et al., J. Photochem. Photobiol. A:Chem. 299 (2015) 1-8. DOI:10.1016/j.jphotochem.2014.10.015
[34]
L. Chen, J. Li, Z. Liu, et al., RSC Adv. 3 (2013) 13412-13416. DOI:10.1039/c3ra41898g
[35]
B. Liang, B. Wang, Q. Ma, et al., Spectrochim. Acta A 192 (2018) 67-74. DOI:10.1016/j.saa.2017.10.044
[36]
B. Guo, J. Jing, L. Nie, et al., J. Mater. Chem. B 6 (2018) 580-585. DOI:10.1039/C7TB02615C
[37]
M.H. Lee, J.H. Han, J.H. Lee, et al., J. Am. Chem. Soc. 134 (2012) 17314-17319. DOI:10.1021/ja308446y
[38]
H.W. Liu, S. Xu, P. Wang, et al., Chem. Commun. 52 (2016) 12330-12333. DOI:10.1039/C6CC05880A
[39]
Y. Chen, J. Qi, J. Huang, et al., Spectrochim. Acta A 189 (2018) 634-641. DOI:10.1016/j.saa.2017.08.063
[40]
S.P. Jackson, J. Bartek, Nature 461 (2009) 1071-1078. DOI:10.1038/nature08467
[41]
G. Hewitt, D. Jurk, F.D. Marques, et al., Nat. Commun. 3 (2012) 708. DOI:10.1038/ncomms1708
[42]
G. Lukinavicius, C. Blaukopf, E. Pershagen, et al., Nat. Commun. 6 (2015) 8497. DOI:10.1038/ncomms9497
[43]
A. Nakamura, K. Takigawa, Y. Kurishita, et al., Chem. Commun. 50 (2014) 6149-6152. DOI:10.1039/C4CC01753F
[44]
A. Nakamura, S. Tsukiji, Bioorg. Med. Chem. Lett. 27 (2017) 3127-3130. DOI:10.1016/j.bmcl.2017.05.036
[45]
A.T. Szczurek, K. Prakash, H.K. Lee, et al., Nucleus 5 (2014) 331-340. DOI:10.4161/nucl.29564
[46]
X. Zhang, Z. Ye, X. Zhang, et al., Chem. Commun. 55 (2019) 1951-1954. DOI:10.1039/C8CC08575G
[47]
W. Chyan, R.T. Raines, ACS Chem. Biol. 13 (2018) 1810-1823. DOI:10.1021/acschembio.8b00371
[48]
H.W. Liu, L. Chen, C. Xu, et al., Chem. Soc. Rev. 47 (2018) 7140-7180. DOI:10.1039/C7CS00862G
[49]
R. Pio, L. Corrales, J.D. Lambris, The Role of Complement in Tumor Growth, Springer New York, New York, 2014, pp. 229-262. https://www.ncbi.nlm.nih.gov/pubmed/24272362
[50]
R.M. Touyz, A. Anagnostopoulou, L. de Lucca Camargo, et al., Circ. Res. 119 (2016) 969-971. DOI:10.1161/CIRCRESAHA.116.309854
[51]
A. Lloret, T. Fuchsberger, E. Giraldo, et al., Curr.AlzheimerRes. 13 (2016) 206-211. DOI:10.2174/1567205012666150921101430
[52]
L. Cui, Y. Zhong, W. Zhu, et al., Org. Lett. 13 (2011) 928-931. DOI:10.1021/ol102975t
[53]
X. Ao, S.A. Bright, N.C. Taylor, et al., Org. Biomol. Chem. 15 (2017) 6104-6108. DOI:10.1039/C7OB01406F
[54]
Z. He, Y. Chou, H. Zhou, et al., Org. Biomol. Chem. 16 (2018) 3266-3272. DOI:10.1039/C8OB00670A
[55]
L. Zhang, D. Duan, Y. Liu, et al., J. Am. Chem. Soc. 136 (2014) 226-233. DOI:10.1021/ja408792k
[56]
Y. Liu, H. Ma, L. Zhang, et al., Chem. Commun. 52 (2016) 2296-2299. DOI:10.1039/C5CC09998F
[57]
L. Dong, S. Shen, H. Lu, et al., ACS Sens. 4 (2019) 1222-1229. DOI:10.1021/acssensors.8b01617
[58]
Q. Jin, L. Feng, D.D. Wang, et al., ACSAppl.Mater.Interfaces 7 (2015) 28474-28481. DOI:10.1021/acsami.5b09573
[59]
M. Abo, R. Minakami, K. Miyano, et al., Anal. Chem. 86 (2014) 5983-5990. DOI:10.1021/ac501041w
[60]
J. Wang, Y. Zhao, C. Wang, et al., PLoS One 10 (2015) e0123986. DOI:10.1371/journal.pone.0123986
[61]
J. Chen, M. Zhao, X. Jiang, et al., Analyst 141 (2016) 1209-1213. DOI:10.1039/C5AN02497H
[62]
C. Wang, X. Song, Y. Xiao, ChemBioChem 18 (2017) 1762-1769. DOI:10.1002/cbic.201700161
[63]
S. Leng, Q. Qiao, L. Miao, et al., Chem. Commun. 53 (2017) 6448-6451. DOI:10.1039/C7CC01483J
[64]
Q. Qiao, W. Liu, J. Chen, et al., Dye. Pigment. 147 (2017) 327-333. DOI:10.1016/j.dyepig.2017.08.032
[65]
C. Huang, Q. Yin, W. Zhu, et al., Angew. Chem. Int. Ed. 50 (2011) 7551-7556. DOI:10.1002/anie.201101317
[66]
A.L. Femia, C.F. Temprana, J. Santos, et al., Protein J. 31 (2012) 656-666. DOI:10.1007/s10930-012-9441-6
[67]
S. Shen, X.F. Li, W.R. Cullen, et al., Chem. Rev. 113 (2013) 7769-7792. DOI:10.1021/cr300015c
[68]
B. Chen, Q. Liu, A. Popowich, et al., Metallomics 7 (2015) 39-55. DOI:10.1039/C4MT00222A
[69]
N. Kotera, E. Dubost, G. Milanole, et al., Chem. Commun. 51 (2015) 11482-11484. DOI:10.1039/C5CC04721H
[70]
C. Huang, T. Jia, M. Tang, et al., J. Am. Chem. Soc. 136 (2014) 14237-14244. DOI:10.1021/ja5079656