Recognition Moieties of Small Molecular Fluorescent Probes for

Jun 20, 2019 - It is our wish that this Account will promote the appearance of more specific .... This probe can be employed to discriminate the tyros...
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Article Cite This: Acc. Chem. Res. 2019, 52, 1892−1904

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Recognition Moieties of Small Molecular Fluorescent Probes for Bioimaging of Enzymes Xiaofeng Wu, Wen Shi, Xiaohua Li, and Huimin Ma*

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Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China CONSPECTUS: Enzymes are a class of important substances for life, and their abnormal levels are associated with many diseases. Thus, great progress has been made in the past decade in detecting and imaging enzymes in living biosystems, and in this respect fluorescent probes combined with confocal microscopy have attracted much attention because of their high sensitivity and unrivaled spatiotemporal resolution. Fluorescent probes are usually composed of three moieties: a signal or fluorophore moiety, a recognition or labeling moiety, and an appropriate linker to connect the two aforementioned moieties. At present, however, research and reviews on enzymatic probes mostly focus on fluorophores and/or linkers, whereas those on the recognition moiety are relatively few. Moreover, current enzymatic probes with some recognition moieties have drawbacks such as poor selectivity, high background fluorescence, or/and low sensitivity and are unsatisfactory for practical applications. Thus, developing new recognition moieties with higher specificity or/and sensitivity to the enzyme of interest is very desirable but still challenging. In this Account, we introduce the recognition moieties of fluorescent probes for several enzymes, including tyrosinase, monoamine oxidase A (MAO-A), nitroreductase (NTR), and aminopeptidases. Highlights are given on how new specific recognition moieties of tyrosinase and MAO-A were designed to eliminate the interference by reactive oxygen species (ROS) and MAO-B, respectively. Here we present four recent examples in which designed fluorescent probes are employed to image enzymes in living biosystems. The first example shows that 3-hydroxyphenyl can serve as a new and more specific recognition moiety than the traditional 4-hydroxyphenyl group for tyrosinase, enabling the development of a highly selective fluorescent probe for imaging of tyrosinase without interference by ROS. The second presents a general design strategy for fluorescent probes specific for an enzyme, which involves combining the characteristic structure of an inhibitor of the target enzyme along with its traditional reactive group as a new recognition moiety, and successfully demonstrates it by selective detection of MAO-A in the presence of its isomeric MAO-B. The third mainly illustrates that 5-nitrothiophen-2-yl alcohol with a stronger electron-donating S atom is a better fluorescence quenching and recognition moiety than 5-nitrofuran-2-yl alcohol for NTR, leading to the development of a highly sensitive method for NTR assay. Lastly, on the basis of known observations, we show that besides the specific interaction with the target, another function of some recognition moieties may be responsible for tuning the fluorescence signal, which is exemplified by the linking of several aminopeptidases’ recognition moieties to the free hydroxyl or amino group of different fluorophores. It is our wish that this Account will promote the appearance of more specific recognition moieties and fluorescent probes with excellent properties and that new biofunctions of the enzymes will be uncovered.

1. INTRODUCTION There are six major classes of enzymes, namely, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases, all of which play vital and different roles in biosystems.1−3 Among oxidoreductases, for example, tyrosinase is found to be related to skin diseases and melanoma cancer, whereas monoamine oxidase (MAO) is involved in neuropsychiatric and depressive disorders.3−5 These enzymes are also widely distributed in subcellular organelles.1,6 Thus, it is of great significance to develop excellent analytical methods for elucidating the locations and biofunctions of enzymes in living organisms such as cells and zebrafish.7,8 In this respect, fluorescent probes combined with confocal microscopy have © 2019 American Chemical Society

shown distinct overall advantages in terms of simplicity and high sensitivity and spatiotemporal resolution.9,10 Fluorescent probes usually consist of three moieties: (1) a signal/fluorophore moiety, whose change in spectroscopic property should be as large as possible upon reaction with the analyte of interest; (2) a recognition/labeling moiety, which is responsible for the specific interaction with the target; and (3) an appropriate linker to connect the two aforementioned moieties (in some cases the two moieties are directly integrated without any linker).9 Currently, most research and Received: April 28, 2019 Published: June 20, 2019 1892

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Accounts of Chemical Research reviews on enzymatic probes focus on fluorophores and/or linkers,10−14 whereas few deal with recognition moieties.15 The recognition moiety is actually responsible for not only the specific interaction with the target but also modulation of the fluorescence signal. Unfortunately, some enzymatic recognition moieties still suffer from poor selectivity or weak signal modulation ability. For example, 4-hydroxyphenyl, a classical recognition moiety for tyrosinase, reacts with both reactive oxygen species (ROS) and tyrosinase in most cases; this crossinterference may lead to false-positive signals and inaccurate results. In this Account, we present the recognition moieties of fluorescent probes for several enzymes that we have studied, including tyrosinase, MAO-A, nitroreductase (NTR), and aminopeptidases. The structures of the recognition moieties, reaction mechanisms, and bioimaging applications are discussed, with a special focus on how new recognition moieties for tyrosinase and MAO-A were engineered to overcome the interference of ROS and MAO-B, respectively. We hope that this Account will be helpful in the development of excellent recognition moieties for better understanding of the biofunctions of enzymes in living biosystems.

via protection−deprotection or substitution of the free hydroxyl or amino group of some fluorophores by the enzyme’s recognition moiety. Similarly, Figure 1B displays the use of a self-immolative linker (e.g., a 4-hydroxybenzyl alcohol unit) to connect fluorophore and recognition moiety for developing fluorescent off−on probes. This measure sometimes has the superiority of diminishing the hindrance between the enzyme’s active site and the bulky fluorophore, thus enhancing the signal response. These off−on probes have low background fluorescence; upon activation by an enzyme, the recognition moiety or linker is cleaved or released, leading to the recovery of strong fluorescence.3,9,10,14 A ratiometric probe may be designed by linking a ratiometric fluorophore (e.g., a 1,8-naphthalimide skeleton in most cases) to the recognition moiety (Figure 1C). This kind of probe produces a shift in its emission or excitation wavelength upon reaction, and on the basis of such a shift a division manipulation can be performed by calculating the ratio of the fluorescence intensities at the two wavelengths, which eliminates the influences from many factors (e.g., probe bleaching, instrumental conditions, etc.). On the basis of our long-term research, we find that protection−deprotection or substitution of a free hydroxyl or amino group of some fluorophores usually results in either complete fluorescence quenching (many cases) or a wavelength shift (a few cases),17−19 which is ascribed to a change in the electronic push−pull effect of the substituted groups. For example, resorufin with the −OH group is a typical excellent fluorophore for designing fluorescent off−on probes; cresyl violet, modified via its free amino group, can produce two different fluorescence responses (off−on or ratiometric), depending on the excitation wavelength used.19 Therefore, such superior fluorophores with possible signal-tuning behavior by the recognition moiety are our first choice in preparing off−on and ratiometric probes for enzymes, as illustrated below in detail.

2. DESIGN STRATEGY FOR FLUORESCENT PROBES There are three basic kinds of response modes for fluorescent probes, namely, on−off (also called fluorescence quenching), off−on (also called fluorescence enhancement), and ratiometric, which are caused by disturbing various photophysical processes.9−16 Fluorescence detection of trace levels of enzymes is still difficult. Because of this, a fluorescence on− off probe shows no obvious advantage because of its high background signal. On the contrary, fluorescence off−on probes exhibit low background signal, which benefits high detection sensitivity. Sometimes, to accomplish an accurate quantitative analysis of intracellular species, ratiometric probes are preferred. As shown in Figure 1A, fluorescent off−on probes can be prepared by directly incorporating a recognition moiety into fluorophores, where the fluorophore’s fluorescence is quenched by the recognition moiety. This may be achieved

3. RECOGNITION MOIETIES OF FLUORESCENT PROBES FOR TYROSINASE Tyrosinase, a copper-containing oxidase, can catalyze the tranformation of a monophenol into the o-diphenol and subsequently convert the o-diphenol into the o-quinone.4,15,20 This function determines that the structure of the recognition moiety for tyrosinase should contain a phenol unit, and the ortho position of the phenolic hydroxyl group should be vacant to facilitate the formation of o-diphenol. At present, phenols substituted at the 4-position or 3-position are two main types of tyrosinase recognition moieties; the former (e.g., 4hydroxyphenyl) sometimes suffers from interference by ROS, but the latter (e.g., 3-hydroxyphenyl), proposed by our group (vide infra), can overcome the problem by virtue of the different reaction mechanisms of tyrosinase and ROS. 3.1. 4-Hydroxyphenyl

4-Hydroxyphenyl, whose representative derivative is 4-aminophenol, is a traditional tyrosinase recognition moiety and can be linked to fluorophores via an amino group to form fluorescent probes. The recognition moiety in the probes can be hydroxylated by tyrosinase at the position ortho to the −OH group and further oxidized to an unstable o-quinone, which undergoes an intramolecular rearrangement−elimination, thereby causing a fluorescence change. By the introduction of 4-aminophenol to 1,8-naphthalimide bearing

Figure 1. Design strategies for fluorescent probes of enzymes. 1893

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Figure 2. (A) Structure of probe 1 and its reaction with tyrosinase. (B) Colocalization of probe 1 and DND-99 (commercial lysosome-targeting dye) in B16 cells pretreated with inulavosin for a different period of time. Differential interference contrast (DIC) images are given in the right column. Reproduced from ref 21. Copyright 2016 American Chemical Society.

Figure 3. (A) Traditional recognition moieties of tyrosinase. (B) Reaction mechanism of probe 2. (C, D) Fluorescence responses of probe 2 to (C) ROS and tyrosinase and (D) varied tyrosinase. (E) Real-time imaging of B16 and HeLa cells by probe 2 and (F) relative pixel intensities of the images. Reproduced with permission from ref 22. Copyright 2016 Wiley-VCH.

response to tyrosinase over ROS.21 In the presence of tyrosinase and oxygen, the 4-hydroxyphenyl moiety was

morpholine as a lysosome-targeting unit, probe 1 (Figure 2) was prepared, and it displayed a selective fluorescence off−on 1894

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Figure 4. (A) Structure of probe 3 and (B) its fluorescence response to varied tyrosinase. (C) Imaging of different cells using probe 3. (D) Relative pixel intensities of the images in (C). (E) Western blot analyses. Reproduced with permission from ref 23. Copyright 2017 Royal Society of Chemistry.

oxidized into o-quinone, followed by hydrolysis to release the fluorophore and fluorescence recovery. Probe 1, having a dual function of tyrosinase detection and subcellular localization, was applied to imaging of the tyrosinase distribution between melanosomes and lysosomes under inulavosin stimulation. As depicted in Figure 2B, fluorescence colocalization studies revealed that the lysosomal tyrosinase gradually increases, which is possibly from the melanosomes.21

To improve detection sensitivity, an effective measure is to decrease the background fluorescence. In this respect, as mentioned above, the fluorophore of resorufin is favorable, because its 7-hydroxy substitution results in an almost complete fluorescence quenching, which is beneficial for achieving an extremely low background signal and thus a sensitive assay. As shown in Figure 4, combining the 3hydroxyphenyl moiety with resorufin yields probe 3, which can also overcome the ROS interference with a lower detection limit of 0.04 unit/mL. This probe can be employed to discriminate the tyrosinase levels in different cells, as supported by Western blot analysis.23 It is noteworthy that the new 3hydroxyphenyl recognition moiety proposed by us has been rapidly adopted by other scientists,24,25 who have also demonstrated its effectiveness in eliminating the influence of ROS. The above probes 1−3 for imaging tyrosinase show different performance. Probe 1 has melanosome-targeting ability, and both probes 2 and 3 with the new 3-hydroxyphenyl recognition moiety circumvent the influence of ROS. Moreover, probe 2 with the NIR feature is suited for in vivo imaging of tyrosinase, and sensitive probe 3 can be used to detect trace amounts of tyrosinase.

3.2. 3-Hydroxyphenyl

Tyrosinase recognition moieties that are based on phenols substituted at the 4-position have the structural features a−d depicted in Figure 3A. Fluorescent probes with these traditional moieties often produce similar responses to tyrosinase and ROS and thus suffer from the ROS interference; even worse, some ROS like HOCl and H2O2 usually have higher concentrations than tyrosinase in cells. Therefore, the cross-response may lead to wrong results. To avoid the interference, we first proposed the phenol substituted at the 3position (i.e., 3-hydroxyphenyl) as a new recognition moiety for tyrosinase.22,23 Figure 3B shows such a typical example (3hydroxybenzyloxy), in which the presence of the unique 3hydroxyl (instead of 4-hydroxy) group facilitates the hydroxylation at the 4-position vacancy by tyrosinase but not by ROS, and the resulting hydroxylated unit can be spontaneously removed via the 1,6-rearrangement−elimination. By incorporating 3-hydroxybenzyloxy into a near-infrared (NIR) fluorophore of hemicyanine, we constructed 2, a specific NIR fluorescent off−on probe for imaging of tyrosinase. As shown in Figure 3C, tyrosinase displays a strong fluorescence off−on response, but ROS even at much higher levels than their physiological concentrations do not, demonstrating that our probe can indeed eliminate the influence of ROS. The detailed reason for this may be that ROS usually show good oxidizing but poor hydroxylating ability, which leads to the possible formation of an oxidized mquinone (a difficult leaving group). The limit of detection (LOD) of the probe is 2.76 units/mL tyrosinase (Figure 3D). Application of probe 2 to cell imaging demonstrated that the tyrosinase activity in B16 cells was nearly 2-fold higher than that in HeLa cells (Figure 3E,F), which was further validated by enzyme-linked immunosorbent assay (ELISA). Moreover, probe 2 was used to image tyrosinase in zebrafish.22

4. RECOGNITION MOIETIES OF FLUORESCENT PROBES FOR MAO-A MAO has two isoforms, MAO-A and MAO-B, both of which catalyze the oxidation of an amine substrate to the corresponding imine, which subsequently hydrolyzes to give an aldehyde.26 However, MAO-A is associated with depressive disorders, which can be greatly inhibited by clorgyline (a specific inhibitor); MAO-B is thought to be a main cause of several neurodegenerative diseases and can be effectively inhibited by pargyline.27 Thus, selective detection of each isoform is important to understand its biofunction. At present, the most commonly used recognition moiety for MAO is propylamine, but sometimes the resulting fluorescent probe with this moiety reacts with both MAO-A and MAO-B. This indicates that propylamine itself is not very specific for MAO-A or MAO-B. Moreover, current propylamine-based fluorescent probes are mainly suited for detecting either MAOB or the total MAO content, whereas probes specific for MAO1895

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Figure 5. (A) Reaction of probe 4 or 5 with MAO. (B) Fluorescence ratiometric responses of the probes to MAO. (C) Ratiometric images of different cells. (D) Relative ratio values from the images in (C). (E) Activity of MAO-A in the cells as determined by ELISA. Reproduced from ref 28. Copyright 2015 American Chemical Society.

A are still rare. Obviously, developing fluorescent probes and especially specific recognition moieties for MAO-A is rather necessary but challenging. Our effort toward this issue led to the appearance of a new recognition moiety specific for MAOA instead of MAO-B, namely, propylamine with the targeting unit of the inhibitor, which excludes the probe’s cross-response to MAO-B.

new recognition moiety into resorufin, we developed six MAOA probes (6−11) together with a control probe (CP) without the targeting unit. Reaction of probes 6−11 with MAO-A induced oxidative deamination, subsequently accompanied by β-elimination and 1,6-rearrangement−elimination to release resorufin. Probes 6−11 all displayed much larger specificity coefficients (2.4−42) for MAO-A over MAO-B than the CP (0.96); however, the CP showed nearly the same fluorescence response to both MAO-A and MAO-B (Figure 6F). This clearly demonstrates that the combination of propylamine with clorgyline’s characteristic structure can be used as a new more specific recognition moiety for MAO-A. There exist specificity differences among the probes. The reasons for this might be complex, but a possible explanation is that steric hindrance or electronic effects of the different substituents may also play a role in interacting with the active center of the enzyme. Among these probes, probe 8 exhibited the highest selectivity and sensitivity (probably because it has the shortest distance between its reactive site and the enzyme’s active site)29 and may be employed to image intracellular MAO-A without the influence of MAO-B. This applicability was confirmed by imaging of MAO-A in different cells such as SH-SY5Y (high level of MAO-A) and HepG2 (high level of MAO-B). As can be seen from Figure 6G−I, probe 8 produces almost no fluorescence in HepG2 cells but remarkable fluorescence in SH-SY5Y cells, indicating the high specificity of the probe for MAO-A over MAO-B in living cells. Various MAO-A plasmidtransfected experiments, together with a vector control, further verified the specificity of probe 8 (Figure 6J−L). We expect that the use of the characteristic structure of an inhibitor may serve as a general strategy to design specific recognition moieties of fluorescent probes for other enzymes.29

4.1. Propylamine

As noted above, propylamine often serves as a recognition moiety for both MAO-A and MAO-B, but in some cases the specificity of the resulting probe also depends on the whole structure of the substrate. As shown in Figure 5, for instance, probes 4 and 5, constructed by incorporating propylamine into 1,8-naphthalimide, surprisingly exhibit a selective ratiometric response to MAO-A rather than MAO-B, and probe 4 displays higher sensitivity. The response mechanism is based on oxidation of the amine and β-elimination to release the fluorophore. The reason for the higher selectivity for MAO-A over MAO-B may be that each enzyme has its own specific substrate, and the whole 1,8-naphthalimide skeleton is fit for the reaction site of MAO-A. This was supported by docking studies, which revealed that MAO-A had stronger binding ability with the probe than MAO-B and that a potential hydrogen bond existed between the probe and MAO-A. The use of probe 4 in cell imaging uncovered that the MAO-A concentration in HeLa cells was about 2-fold higher than that in NIH-3T3 cells, which was proved by ELISA (Figure 5C−E). This was the first semiquantitative information about the MAO-A contents in HeLa and NIH-3T3 cells.28 4.2. Propylamine with the Targeting Unit of the Inhibitor

Presented below is the design of propylamine with the targeting unit of the inhibitor as a new recognition moiety specific for MAO-A. In such a design (Figure 6), we notice that pargyline is a specific inhibitor of MAO-B but clorgyline with a similar structure is a specific inhibitor of MAO-A; moreover, it is known that a specific inhibitor prefers to bind to its target enzyme. Considering these facts, we envisioned that the characteristic structure of the MAO-A inhibitor (i.e., the substituted phenol of clorgyline) might serve as a targeting unit that can specifically bind with MAO-A rather than MAO-B. In other words, we may design a new recognition moiety for MAO-A by combining propylamine with the targeting unit to further increase the selectivity. As a result, by introducing the

5. RECOGNITION MOIETIES OF FLUORESCENT PROBES FOR NTR NTR can reduce nitro-containing compounds into the corresponding amines or hydroxylamines in the presence of reduced nicotinamide adenine dinucleotide (NADH),30−32 and therefore, its recognition moiety often contains the nitro group as an essential part. To date, 5-nitrofuran-2-yl alcohol, 5nitrothiophen-2-yl alcohol, 4-nitrobenzyl alcohol, and 2nitroimidazole alcohol have been identified as recognition moieties for NTR. With these recognition moieties in mind, 1896

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Figure 6. (A−E) Designs of fluorescent probes specific for MAO-A. (F) Ratios of fluorescence intensity of probes reacting with MAO-A and MAOB. (G, H) Imaging of (G) HepG2 and (H) SHSY5Y cells: (a, c) cells only; (b, d) cells incubated with probe 8; (e) cells incubated successively with clorgyline and probe 8. (I) Relative pixel intensities of the images in (G) and (H). (J) Imaging by probe 8 of HepG2 cells pretransfected under different conditions: (a) un-pretreated; (b) vector control; (c) MAO-A plasmid; (d) MAO-A plasmid followed by pretreatment with clorgyline. (K) Relative pixel intensities of the images in (J). (L) Western blot analyses of the cells in (J). Reproduced with permission from ref 29. Copyright 2017 Wiley-VCH.

fluorescent probes for NTR can be designed (1) by directly oxidizing the amino group of a fluorophore into a nitroaromatic or nitroheterocylic compound or (2) by incorporating the recognition moiety into a hydroxyl or amino group of the fluorophore.

ufin. This fluorescent off−on probe, with an LOD of 0.27 ng/ mL NTR, has been used to monitor the hypoxic status of tumor cells (HeLa and A549) with time via the imaging of NTR, revealing a nonlinear increase in the intracellular NTR concentration with decreasing O2 level.33

5.1. 5-Nitrofuran-2-yl Alcohol

5.2. 5-Nitrothiophen-2-yl Alcohol

Figure 7 shows the construction of probe 12 by introducing 5nitrofuran-2-yl alcohol as a recognition moiety for NTR into resorufin. Upon reaction with NTR in the presence of NADH, the probe is reduced to 5-amino- or 5-hydroxylaminofuran, followed by 1,6-rearrangement−elimination to release resor-

As is known, the S atom has a relatively strong electrondonating ability. We envisioned that this property may enable the S atom to serve as a strong fluorescence quencher for achieving low background signal and thus high detection sensitivity. Thus, we used the S atom to replace the O atom of 1897

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Figure 7. (A) Structures of probes 12 and 13. (B) Fluorescence response of probe 12 to varied NTR. (C) Imaging of HeLa cells under different hypoxia conditions for (a) 4 h and (c) 8 h using probe 12. (D) Fluorescence response of probe 13 to varied NTR. (E) (a) Fluorescence change of probe 13 with Escherichia coli and (b) absorbance change of E. coli. Reproduced from ref 33 and with permission from ref 34. Copyright 2013 American Chemical Society and Royal Society of Chemistry, respectively.

5-nitrofuran-2-yl alcohol, obtaining 5-nitrothiophen-2-yl alcohol, which might be a better fluorescence quenching and recognition moiety for NTR than 5-nitrofuran-2-yl alcohol. To test this idea, we prepared probe 13 by linking 5-nitrothiophen-2-yl alcohol to the same resorufin fluorophore (Figure 7).34 The resulting probe indeed showed extremely low background fluorescence (Φ ≈ 0.01), which was about 4fold lower than that of its analogue with 5-nitrofuran-2-yl alcohol (Φ = 0.04).33 Obviously, this lower background signal of probe 13 is due to the stronger quenching action of the S atom. Upon reaction with NTR, the probe exhibited a sharp fluorescence off−on response (LOD = 0.1 ng/mL), which led to the establishment of a highly sensitive and selective assay for NTR. With the probe, we monitored NTR produced by Escherichia coli in real time, and the results were consistent with the bacterial growth curve obtained by absorbance analysis (Figure 7E), implying that probe 13 may be utilized as a microbial growth indicator.34

Figure 8. (A) Structure of probe 14 and (B) its fluorescence response to varied NTR. (C) Imaging of zebrafish by probe 14. Reproduced with permission from ref 35. Copyright 2014 Elsevier.

5.3. 4-Nitrobenzyl Alcohol

4-Nitrobenzyl alcohol is another commonly used recognition moiety for NTR. By incorporating this moiety into a hydroxyl hemicyanine, we developed 14, an NIR-fluorescent probe for imaging of NTR (Figure 8). Similar to resorufin, hemicyanine

with a hydroxyl or amino group exhibits not only higher stability but also tunable spectroscopic on−off properties via substitution. Notably, the hydroxyl or amino hemicyanines possess NIR absorption and emission features, thereby serving 1898

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acids, dipeptides, or water.38,39 Therefore, γ-glutamyl or glutathione is the most frequently used recognition moiety for GGT. As shown in Figure 9, probe 15 was prepared by combining γ-glutamic acid with cresyl violet through an amide bond. Upon reaction with GGT, the γ-glutamyl unit is cleaved, accompanied by strong fluorescence recovery. The γ-glutamyl unit not only plays the role of recognition moiety but also has the function of fluorescence signal modulation. The probe (LOD = 5.6 milliunits/L) was applied to imaging of the GGT change in HepG2 cells regulated by sodium butyrate, which uncovered that this drug may induce GGT upregulation in a dose- and time-dependent manner.40 Glutathione bearing the γ-glutamyl unit can also function as a recognition moiety for GGT. Figure 10 shows such an example, where glutathione is incorporated into a hydroxyl hemicyanine via an acrylyl linker, forming an NIR-fluorescent off−on probe (16). Reaction of the probe with GGT caused cleavage of the γ-glutamyl bond of glutathione, followed by spontaneous intramolecular cyclization and release of the fluorophore. The probe (LOD = 0.5 unit/L) can be applied to imaging of GGT in zebrafish.41

as important platforms for the development of various NIR probes. Upon reaction with NTR, probe 14 displayed a strong NIR fluorescence off−on response at 705 nm (LOD = 14 ng/ mL). The probe was used to image the distribution of NTR in zebrafish in vivo, disclosing that NTR mainly appeared in the yolk sac.35

6. RECOGNITION MOIETIES OF FLUORESCENT PROBES FOR SEVERAL AMNIOPEPTIDASES Aminopeptidases specifically hydrolyze peptide bonds of some proteins,36,37 and therefore, their recognition moieties are

6.2. Leucyl for LAP

LAP can hydrolyze N-terminal leucyl groups of proteins.42,43 Thus, the leucyl group is often used as the recognition moiety for LAP, as exemplified by probe 17 (Figure 11A), in which leucine is incorporated into cresyl violet. After reaction with LAP, the leucyl group was removed, concomitant with fluorescence recovery (LOD = 0.42 ng/mL; Figure 11B). Using probe 17, we analyzed the change in LAP in cisplatinpretreated HepG2 cells and found that cisplatin can induce the upregulation of intracellular LAP (Figure 11C).44 Further, an NIR probe was developed by coupling of the leucyl group with an amino hemicyanine and then used to monitor the LAP change in drug-induced mice.45

Figure 9. (A) Structure of probe 15 and (B) its fluorescence response to varied GGT. Reproduced from ref 40. Copyright 2015 American Chemical Society.

usually constructed with the corresponding amino acids, which can be engineered onto fluorophores via an amide bond.17 In this way, we have developed different fluorescent probes for several aminopeptidases, including γ-glutamyl transpeptidase (GGT), leucine aminopeptidase (LAP), aminopeptidase N (APN), pyroglutamate aminopeptidase 1 (PGP-1), pantetheinase, dipeptide peptidase IV (DPPIV), and fibroblast activation protein (FAP).3,37 These studies also demonstrate that besides the specific interaction with the target enzyme, another function of the recognition moiety, when linked to the free amino or hydroxyl group of some fluorophores, is to tune the fluorescence signal. As noted above, this usually results in the formation of fluorescent off−on or ratiometric probes,17 which provides the basis for sensitive or accurate detection of the enzymes.

6.3. Alanyl for APN

Similarly, APN can remove the N-terminal alanyl group of a protein,46 and thus, its recognition moiety is the alanyl group. Figure 12A shows the structure of 18, an NIR APN probe engineered by linking of L-alanine to an amino hemicyanine. Upon reaction with APN, the alanyl unit is cleaved, resulting in large fluorescence enhancement (LOD = 0.8 ng/mL). Using the probe, we studied the role of APN in cell migration. As shown in Figure 12B, small interfering RNA (siRNA)transfected HepG2 cells display downregulation of APN and a dose-dependent decrease in the migration ability, implying that the cell migration is positively correlated with the APN activity. Moreover, this probe was applied to imaging of APN in mice (Figure 12C,D).47 Furthermore, a ratiometric fluorescence probe for APN, 19, was designed by connecting L-alanine to cresyl violet (Figure

6.1. γ-Glutamyl and Glutathione for GGT

GGT can transfer the γ-glutamyl unit from glutathione or other γ-glutamyl derivatives to suitable acceptors such as amino

Figure 10. Structure of probe 16 and its fluorescence response to GGT. Reproduced with permission from ref 41. Copyright 2016 Elsevier. 1899

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Figure 11. (A) Structure of probe 17 and (B) its fluorescence response to varied LAP. (C) Imaging of LAP in HepG2 cells using probe 17: (a) cells only; (b−e) cells pretreated with increasing cisplatin; (f) relative pixel intensities of the images in (a−e); (g) LAP concentrations in the cells in (b−e) as determined by ELISA. Reproduced from ref 44. Published by the Royal Society of Chemistry.

Figure 12. (A) Structure of probe 18 and its reaction with APN. (B) Imaging of siRNA-pretreated HepG2 cells and scratch assay. (C, D) Imaging of tumor-bearing mice using probe 18. Reproduced with permission from ref 47. Copyright 2017 Royal Society of Chemistry.

13).48 The probe itself showed fluorescence emission at 575 nm when excited at 525 nm, and its reaction with APN caused a red shift of the emission to 626 nm. Probe 19 (LOD = 33 pg/mL) was applied to distinguishing the APN levels in different cells on the basis of the fluorescence ratio.48

pyroglutamic acid into cresyl violet. The probe displays fluorescence off−on response to PGP-1 via pyroglutamyl removal. Probe 20 (LOD = 5.6 ng/mL) was employed to monitor the PGP-1 alteration in LO2 cells treated with the immunopotentiators Freund’s incomplete adjuvant (FIA) and lipopolysaccharide (LPS).51

6.4. Pyroglutamyl for PGP-1

6.5. Pantothenic Acid for Pantetheinase

Since PGP-1 can cleave the peptide bond of pyroglutamic acid linked to the N-terminus of proteins,49,50 the pyroglutamyl unit is an important recognition moiety for this enzyme. Figure 14 shows the formation of probe 20 by incorporation of

Pantetheinase hydrolyzes one of the two amide bonds of pantetheine to generate cysteamine and pantothenic acid (vitamin B5),52,53 and therefore, pantothenic acid is charac1900

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Figure 13. (A, B) Structure of probe 19 and its ratiometric response to varied APN. (C) Imaging of a mixture of LO2 and HepG2 cells with probe 19. White arrows indicate HepG2 cells prestained with Hoechst-33342 (nuclear dye; blue), and yellow arrows indicate LO2 cells. Reproduced from ref 48. Copyright 2017 American Chemical Society.

Figure 15. (A) Structure of probe 21 and its reaction with pantetheinase. (B) Ratiometric imaging by probe 21 of pantetheinase in LO2 cells pretreated with increasing RR6 (a pantetheinase inhibitor). (C) Relative pixel intensities of the images in (B). Reproduced from ref 54. Copyright 2017 American Chemical Society.

terized as an important part of the enzyme’s recognition moiety. As shown in Figure 15, incorporating pantothenic acid into cresyl violet yielded probe 21, which produces a ratiometric fluorescence response to pantetheinase when excited at 525 nm. The probe (LOD = 4.7 ng/mL) has been utilized to image pantetheinase in living cells.54

of the probes to cell imaging revealed an adverse action of DPPIV and FAP during proliferation of cancer cells.57

7. CONCLUSION In this Account, we have described the recognition moieties of several enzymes, especially the new recognition moieties for tyrosinase and MAO-A that effectively eliminate the interference from ROS and MAO-B, respectively. On the other hand, it can be seen from the above discussion that a recognition moiety is responsible for not only selectivity (the specific interaction with the target) but also sensitivity (modulation of the spectroscopic signal) in most cases. This dual function becomes particularly pronounced when the recognition moiety is combined with some fluorophores containing a free amino or hydroxyl group, among which 1,8-naphthalimide (many cases) and cresyl violet (excited at its isosbestic-point wavelength of about 525 nm) usually form

6.6. Gly-L-Pro for DPPIV and Boc-Protected Gly-L-Pro for FAP

DPPIV and FAP are isoenzymes.55,56 Glycyl-L-proline (Gly-LPro) and Boc-protected Gly-L-Pro have been identified as recognition moieties for DPPIV and FAP, respectively. Figure 16 shows the use of the two recognition moieties in developing fluorescent probes 22 and 23 for DPPIV and FAP, respectively. Upon reaction with DPPIV and FAP, the Gly-L-Pro and NBoc-Gly-L-Pro moieties in probes 22 and 23 were cleaved, causing large fluorescence enhancements with LODs of 0.35 and 2.7 ng/mL for DPPIV and FAP, respectively. Application

Figure 14. (A) Structure of probe 20 and (B) its fluorescence response to varied PGP-1 at λex = 585 nm. (C) Imaging by probe 20 of PGP-1 in LO2 cells pretreated with increasing (a1−e1) FIA and (a2−e2) LPS. (D) (f1, f2) Relative pixel intensities of the images in (C) and (g1, g2) Western blot analysis. Reproduced with permission from ref 51. Published by the Royal Society of Chemistry. 1901

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Accounts of Chemical Research

Figure 16. (A) Structures of probes 22 and 23 and (B) their fluorescence responses to DPPIV and FAP. (C) Fluorescence images and Western blot analysis of DPPIV and FAP in MGC803 cells pretreated with genistein. Reproduced from ref 57. Copyright 2016 American Chemical Society.

ratiometric probes, whereas resorufin, cresyl violet (excited at its normal excitation wavelength of about 585 nm), and hydroxy/amino hemicyanines result in off−on probes. It is also noteworthy that resorufin with more complete quenching and higher signal/background ratio often shows higher sensitivity than a hemicyanine, although the latter is more suitable for preparing NIR probes. Each kind of fluorophore modified with a recognition moiety has its own merits, and for a given purpose the right choice by a researcher is the key point. Using the above strategy, we have developed different fluorescence off−on or ratiometric probes for imaging of enzymes in living biosystems. We expect that this Account may be useful to promote the development of more specific recognition moieties and thus excellent fluorescent probes for other proteinases and even non-proteinases.

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Wen Shi is an associate professor at ICCAS. He earned his B.Sc. in 2005 from Wuhan University and his Ph.D. from ICCAS in 2010 and was a visiting researcher at Arizona State University in 2018. His research interests include molecular spectroscopy, biochemical analysis, and fluorescence imaging. Xiaohua Li is an associate professor at ICCAS. She received her Ph.D. in 2004 from ICCAS and was a postdoctoral researcher at Emory University and Regensburg University from 2004 to 2008. Her research interests include fluorescent probes and nanomaterials for bioapplications. Huimin Ma is a Professor of Chemistry at ICCAS. He obtained his Ph.D. in 1990 from ICCAS and was a Humboldt Research Fellow at Bremen University in Germany in 1996−1997. His research interests include spectroscopic probes, bioimaging, and bioanalysis.

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AUTHOR INFORMATION

REFERENCES

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Corresponding Author

*E-mail: [email protected]. ORCID

Wen Shi: 0000-0002-2368-3997 Xiaohua Li: 0000-0001-5656-4444 Huimin Ma: 0000-0001-6155-9076 Funding

We are grateful for financial support from the National Natural Science Foundation of China (21820102007, 21535009, 21675159, 91732104, 21775152, 21435007, and 21621062), the Chinese Academy of Sciences (XDB14030102), and the CAS Youth Innovation Promotion Association (2016027). Notes

The authors declare no competing financial interest. Biographies Xiaofeng Wu obtained his B.Sc. in chemistry from Anhui Normal University in 2013 and his Ph.D. from the Institute of Chemistry, Chinese Academy of Sciences (ICCAS) in 2018 under the supervision of Prof. Huimin Ma, working on fluorescent probes for proteinases. 1902

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