A Ratiometric Fluorescent Probe for Monitoring Leucine

Subscriber access provided by University of Virginia Libraries & VIVA (Virtual Library of Virginia)

Article

A ratiometric fluorescent probe for monitoring Leucine aminopeptidase in living cells and zebrafish model Zhe Zhou, Feiyi Wang, Guichun Yang, CuiFen Lu, Junqi Nie, Zuxing Chen, Jun Ren, Qi Sun, Chunchang Zhao, and Wei-Hong Zhu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A ratiometric fluorescent probe for monitoring Leucine aminopeptidase in living cells and zebrafish model Zhe Zhou,† Feiyi Wang,*,† Guichun Yang,† Cuifen Lu,† Junqi Nie,† Zuxing Chen,† Jun Ren,† Qi Sun,*,‡ Chunchang Zhao,*,§ and Wei-Hong Zhu § †

Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & Hubei Collaborative Innovation Center for Advanced Organic chemical Materials, Hubei University, Wuhan 430062, P. R. China. ‡ Key Laboratory for Green Chemical Process of Ministry of Education and School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, P. R. China. § Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science & Technology, Shanghai 200237, P. R. China. ABSTRACT: Leucine aminopeptidase (LAP) is an important cancer-related biomarker, which shows significant overexpression in malignant tumor cells like liver cancer, Developing an effective method to monitor LAP in tumor cells holds great potential for cancer diagnosis, treatment and management. In this work, we report a novel BODIPY-based fluorescent probe (BODIPY-C-Leu) capable of monitoring LAP in vitro and in vivo in both ratiometric and turn-on model. BODIPY-C-Leu contains an asymmetrical BODIPY dye for fluorescent signaling, and a dipeptide (Cys-Leu) as the triggered moiety. Activation occurs by cleavage of the amide bond in dipeptides and subsequent an intramolecular S→N conversion to convert sulfur-substituted BODIPY to aminosubstituted BODIPY, resulting in a dramatic fluorescence variation to realize the detection of LAP. Furthermore, we have successfully employed BODIPY-C-Leu to monitor LAP activity in different cancer cells, indicating that HeLa cells have a higher level of LAP activity than A549 cells. Importantly, we demonstrated the capability of the probe for real-time monitoring the drug-induced LAP level changes in zebrafish.

Proteases play an essential role in various biological and physiological events, including protein digestion, cell proliferation and apoptosis.1 It is of great potential to accurately and realtime monitor the protease activities in living samples. Leucine aminopeptidase (LAP) is one of the most important exopeptidase which selectively catalyzes the hydrolysis of the Nterminal in leucine residues of proteins or peptides. It widely exists in mammals, plants, microbes and living cancer cells,2,3 and plays crucial roles in many physiological and pathological processes.4,5 Abnormal expression of LAP could lead to several human diseases, such as ovarian epithelial malignancy and breast cancer etc.,6-9 enabling LAP to be an important cancerrelated biomarker.10-14 Therefore, developing an effective method to monitor LAP activity in living systems will provide an important reference for cancer diagnosis, treatment and management.15-19 L-Leucine-p-nitroanilide-based colorimetric assay is conventionally used to monitor LAP activity. However, it shows poor anti-interference ability and low stability, which is not suitable for real-time imaging in living samples. Fluorescent probes are powerful tools which have attracted extensive attention for its excellent spatiotemporal resolution, ultra sensitivity and high selectivity.20-35 To date, several fluorescent probes have been developed for the detection of LAP.36-39 However, these probes simply linked leucine with a fluorophore, and adopt a fluorescence turn-on mode for the recognition of LAP, which was susceptible to complex experimental conditions and not convenient in quantitative analysis. Thus, it is urgently necessary to develop novel fluorescent probes to overcome aforementioned disadvantages.

In this contribution, we present the synthesis and biological evaluations of a novel fluorescent probe (BODIPY-C-Leu) which could monitor LAP activity in both ratiometric and turn-on model. Notably, ratiometric responsiveness provides an important self-calibration effect and eliminates interference in analysis experiment. The new probe contains an asymmertrical BODIPY dye as the fluorescence reporter, and a dipeptide (Cys-Leu) with free thiol function covalently attached to the BODIPY scaffold as the triggered moiety. Our design fully explored the well-known features of BODIPYs that show distinct photophysical properties by varying the substitutions on the dipyrromethene core. For instance, different optical properties can be observed for sulfenyl- and amino-substituted BODIPYs. Such target-triggered the aromatic hydrocarbon transfer between the sulfur and nitrogen atoms in BODIPYs have previously used for devising fluorescent probes.40-43 It can be reasoned that LAP could also introduce these cascade reactions in BODIPY-C-Leu and thus enable the sensitive detection of LAP. As demonstrated (Scheme 1), the amide bond between cystein and leucine could be cleavaged by LAP, and the released amino group in cystein then undergoes intermolecular displacement of thiolate to yield amino-substituted BODIPY-Cys adduct through five-membered cyclic transition state, which exhibits dramatically different optical features compared with the sulfur-substituted BODIPY. In this way, BODIPY-C-Leu can provide an alternative method to monitor LAP activity in vitro and in vivo with a high signal-to-noise ratio.

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Proposed mechanism of LAP triggering fluorescence change of BODIPY-C-Leu.

Page 2 of 8

Leu was determined using rhodmine 6G as a reference. Specially, using rhodmine 6G (Φ=0.94, ethanol) as a reference, BODIPY-C-Leu was prepared in PBS (0.02 M, pH=7.4) buffer solution and diluted to a suitable concentration with the absorption less than 0.05. Then, the absorbance and integral area were measured at this concentration. Finally, the fluorescence quantum yield of BODIPY-C-Leu was calculated via the following equation by Abbe’s refractometer.

EXPERIMENTAL SECTION Materials and Instruments. All chemicals were purchased from commercial suppliers and were used without further purification. LAP and other enzyme were purchased from Sigma. Solvents were purified before use. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer at room temperature. Mass spectra were measured on a HP 1100 LC-MS spectrometer. UV-vis absorption spectra were obtained on a SHIMADZU UV-1800 spectrophotometer. Fluorescence spectra were measured by Agilent Cary Eclipse Fluorescence spectrophotometer. Synthesis of BODIPY-C-Leu. BODIPY-Cl (50 mg, 0.1262 mmol) and dipeptide (35.5 mg, 0.1514 mmol) was dissolved in 100 ml acetonitrile-HEPES buffer (1:1, v/v, 20 mM, pH 7.4) and the mixture was stirred at room temperature for 2 h under argon. The solvent was removed under reduced pressure after the reaction. The residual was purified by silica gel chromatography with CH2Cl2/MeOH to get the target BODIPY-C-Leu (22 mg, 0.0371 mmol), yield was 30%. 1H NMR (CD3OD, 600 MHz): δ 7.65-7.53 (m, 3H), δ 7.47-7.36 (m, 3H), δ 7.01 (s, 1H), δ 6.86-6.85 (d, 1H), δ 6.74 (d, 1H), δ 6.66-6.65 (d, 1H), δ 4.77-4.73 (m, 1H), δ 3.98-3.93 (m, 1H), δ 3.92-3.87 (s, 3H), δ 3.86-3.81 (m, 1H), δ 3.59-3.51 (m, 1H), δ 1.82-1.77 (m, 2H), δ 1.76-1.72 (s, 3H), δ 1.72-1.69 (m, 1H), δ 1.03-0.99 (m, 6H);13C NMR (CD3OD, 150 MHz): δ 171.05, 164.68, 160.25, 150.32, 146.44, 142.36, 135.37, 134.94, 132.61, 128.53, 124.17, 119.98, 115.83, 111.23, 95.87, 56.11, 52.98, 47.85, 41.65, 25.35, 22.02, 12.25, 9.26; HRMS (ESI) calcd for C30H33BF2N4O4SNa: 617.2181, Found: 617.2183 [M + Na]+. (Figure S1) General optical measurements. For absorption and fluorescence measurements, all samples were firstly dissolved in DMSO to obtain stock solutions (5.0 mM), and then diluted to desired concentrations in PBS buffer for measurement. The excitation wavelength was provided at 480 nm and slit widths were both at 5 nm. All spectroscopic measurements were performed under physiological conditions (PBS buffer, pH 7.4 at 37 °C). Determination of quantum yield. According to the reported method, 44,45 the fluorescence quantum yield for BODIPY-C-

• •

Determination of IC50 values. To measure the IC50 values, different concentration of inobestin (0, 5, 10, 20, 50, 100, 200 µM) were load into the reaction system (BODIPY-C-Leu (10 -1 µM) + LAP (0.15 U mL )) to perform the enzyme inhibition assay. The increased fluorescence intensity of the incubated solution were measured at 578 nm after 5 min. The IC50 values were calculated by nonlinear regression analysis using OriginPro 8.1 SR3. To comparison, a commercial available LAP detector, L-Leucine-p-nitroanilide, was selected as the standard substrate, the same enzyme inhibitor assay was also performed in quartz cuvettes (1mL volume). Data, absorption values were measured at 380 nm after the reaction system incubated for 3 min. (Figure S10) Selectivity evaluation. To study the interference, BODIPY-CLeu was incubated with various analytes for 30 min, such as ions (Ca2+, Mg2+, Zn2+ and HS-), biothiols (Cys and GSH) and several other enzymes (glucose, lipase, aprotinin, trypsin, cellulase, α-amylase, sulfatase, GGT, ELA and α-Chy), respectively. Cell Cultures and Imaging. HeLa cells and A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin−streptomycin (0.5 U mL-1 of penicillin and 0.5 g mL-1 streptomycin) on a cell culture flask at 37 °C in an atmosphere of air with 5% CO2 and constant humidity. Each cell line was seeded in a 6-well plate for 24 h. The cells were initially incubated with BODIPY-C-Leu (10 µM) in culture medium for different time periods at 37 °C. After washing three times with phosphate-buffered saline (PBS), the imaging was carried out using inverted fluorescence microscopy (Olympus IX71, Japan). Flow Cytometry Analysis. The endogenous LAP of HeLa cells and A549 cells were determined using flow cytometry ( C6, BD biosciences) with BODIPY-C-Leu. Each cells were seed in 6-well plates and incubated with DMEM culture medium for a day. After removing the medium and washing with PBS three times, the cells were digested with trypsin (0.25%). This treated cells were centrifugated and re-suspended in PBS buffer at a density of 3×105 cells per mL. Then treatment with BODIPY-C-Leu (10 µM) for different times (10, 20, 30, 40 and 50 min ) at room temperature, the fluorescence signal intensity was collected in FL1-A detector channel by flow cytometry. Imaging of Zebrafish. The 3 to 7 days post-fertilization zebrafish were purchased from Eze-Rinka Company (Nanjing, China). The zebrafish were cultured in 5 mL embryo medium supplemented with 1-phenyl-2-thiourea (PTU) in 6-well plates for 24 h at 30 °C. The zebrafish were first pretreated with cisplatin (0.1 mg mL-1), which could induce LAP overexpression. After 12 h incubation, the zebrafish were washed three times to remove cisplatin, and further incubated with BODIPY-C-


Page 3 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Leu (10 µM) for 1 h at 30 °C. In the inhibitor experiment, the pretreated zebrafish were incubated in inobestin (100 µM) for 1 h before incubated with BODIPY-C-Leu (10 µM) . After removing the medium and washing zebrafish with PBS for three times, the fluorescence images were acquired with stereo microscopy (Olympus SZX16, Japan). In order to detect endogenous LAP contents, the zebrafish were directly treated with embryo medium containing BODIPY-C-Leu (10 µM) for 1 h, and then imaged by stereo microscopy after washing.

RESULTS AND DISCUSSION Boron dipyrromethene (BODIPY) derivatives are commonly used fluorophore which is sensitive to electronic distribution. For instance, sulfenyl- and amino-substituted BODIPYs displays distinct optical properties in absorption and emission spectra. In the light of these facts, our strategy for monitoring LAP relies on the cleavage of amide bond, along with a further intramolecular S→N conversion. In these processes, the dramatical photophysical change enables the design of an enzyme-activated fluorescent probe. BODIPY-C-Leu was synthesized by nucleophilic aromatic substitution (SNAr) of monochlorinated BODIPY derivative with free thiol group on dipeptides (Cys-Leu, Scheme 2). Its chemical structure was characterized by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry (HRMS) in supporting information (Figure S1).

signal change was triggered by LAP activity (Figure S4). These remarkable optical responses were ascribed to the cleavage of amide bond in dipeptide and the further released free amino group, which was followed by an intramolecular displacement of thiolate to yield an amino-substituted BODIPY-Cys adduct by a five-membered cyclic transition state (Scheme 1). The mechanism of this process was reported in our and Yang’s group previouly.40-43 Moreover, in order to further confirm the proposed mechanism of this reaction, the sample solution was analyzed by HPLC and HRMS. As illustrated in Figure S5, BODIPY-C-Leu showed a retention time at about 2.427 min. After treatment with LAP for 30 min, a new product with a retention time at 4.367 min was observed, whose mass peak at 480.1343 was identical to [BODIPY-Cys-H]-. In addition, the photophysical properties of the reaction system accord well with monochlorinated BODIPY +Cysteine (Figure S6). These results conformed that the fluorescence response was attributed to the action of LAP.

Scheme 2. The synthesis of BODIPY-C-Leu.

O + N

N

B F F

Cl

HS

OH O N H

N

O

Cys-Leu

OH

S

O

O BODIPY-Cl

N B F F

NH2

O HN

BODIPY-C-Leu

H2N

Sensing properties and proposed mechanism of BODIPY-C-Leu towards LAP. Spectroscopic evaluation of BODIPY-C-Leu toward LAP was performed in phosphatebuffered saline (PBS) aqueous solution. Under the optimized conditions (pH 7.4 , 37 °C, Figure S2), as shown in Figure 1, free BODIPY-C-Leu (10 µM) shows an intense absorption band at 578 nm and a relatively weak emission at 601 nm with a fluorescence quantum yield around 0.029. After incubation with LAP (0.15 U mL-1) under the physiological condition, the original absorption band at 578 nm became gradually decreased with simultaneous appearance of the blue-shifted new band at 505 nm. A well defined isosbestic point at 526 nm was observed. The fluorescence profiles of BODIPY-C-Leu treated with LAP was also systematically recorded. The emission maximum of BODIPY-C-Leu at 601 nm underwent a hypochromic shift to 578 nm, accompanied with a remarkable fluorescence enhancement. The fluorescence intensity at 578 nm and the intensity ratio (I578/I601, Figure S3) increased significantly and reached the plateau point in 25 min. These results allowed BODIPY-C-Leu monitoring LAP in both ratiometric and fluorescence turn-on model. However, this dramatic fluorescence change could be suppressed by inobestin, a well known inhibitor of LAP,46 demonstrating that the fluorescence

Figure 1. Time-dependent optical changes in absorption (a) and emission (b) of BODIPY-C-Leu (10 µM) in the presence of LAP (0.15 U mL-1) in aqueous solution (PBS buffer, pH = 7.4 ) at 37 °C (λex = 480 nm). Inset is the ratiometric fluorescence intensity (I578/I601) changes of BODIPY-C-Leu upon enzymatic reaction with LAP.

Titration experiments and enzyme kinetics parameters of BODIPY-C-Leu. To further evaluate the ability of LAP catalysis of BODIPY-C-Leu to release BODIPY-Cys, the titration experiments of the enzyme catalyzed reaction between LAP and BODIPY-C-Leu was investigated in the assay. With the increasing concentration of LAP (0-0.20 U mL-1) incubated with BODIPY-C-Leu (10 µM) under physiological conditions (Figure 2), the fluorescence intensity increased gradually with a blue-shift, while further adding of LAP (＞0.15 U mL1 ), no more change was noted. It reveals that 0.15 U mL-1 LAP can catalyze 10 µM BODIPY-C-Leu. Impressively, when 0.15 U mL-1 LAP was treated with different amounts of BODIPYC-Leu, the fluorescence intensity gave a dose-dependent variety, and 10 µM BODIPY-C-Leu was already sufficient to consume 0.15 U mL-1 LAP (as shown in Figure S7). These results demonstrated that 0.15 U mL-1 LAP was the optimal amount for 10 µM BODIPY-C-Leu. Having these data, further analysis was carried out, and the detection limit was determined to be 41.9 ng mL-1 for LAP following the previous method (Figure S8),38,39 which demonstrated that BODIPY-C-Leu was an ultra-sensitive probe capable of detecting trace amounts of intracellular LAP. Moreover, the apparent kinetic parameters of BODIPY-C-Leu against LAP was further determined according to Michaelis-Menten equation, and the Michaelis constant (Km) and maximum velocity (Vmax) values were 11.5306



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 8

µM and 2.3981 µM S-1. The Km was ~ 4-fold larger and the Vmax was ~10-fold higher than previous work.38,39 This analysis indicates that BODIPY-C-Leu can act as a fluorescence substrate and display higher affinity to LAP compared to other probes. (Figure S9)

Figure 2. The emission spectra of BODIPY-C-Leu (10 µM) in the presence of different amount of LAP (0, 0.01, 0.03, 0.05, 0.07, 0.10, 0.15, 0.20 U mL-1, respectively); Data were recorded 30 min after the addition of analytes.

IC50 value for BODIPY-C-Leu. To establish that our designed BODIPY-C-Leu was a promising fluorescent probe to detect LAP activity and suitable for inhibitor screening, a commercial LAP detector, L-Leucine-p-nitroanilide, was utilized as the standard substance. After using BODIPY-C-Leu (10 µM) and L-Leucine-p-nitroanilide (10 µM) as the substrate and incubated with a range of inobestin which contains 0.15 U mL-1 LAP, we measured the IC50 values for the two detectors, which were 5.14 µM (for BODIPY-C-Leu) and 5.91 µM (for L-Leucine-p-nitroanilide) respectively (Figure S10). The comparison of these results demonstrated that BODIPY-C-Leu could be used as a reliable detector for monitoring LAP activity in vitro condition. Selectivity of BODIPY-C-Leu. Considering that the intracellular environment was a complex biological system, the performance of BODIPY-C-Leu with some other bioanalytes were also explored, including some important metal ions, enzyme species and biomolecules. As shown in Figure 3, under the same PBS buffer solution, the fluorescence intensity at 578 nm showed a negligible change in the presence of other bioanalytes. While incubated with LAP, the fluorescence gave a remarkable enhancement. These results revealed that BODIPY-C-Leu was a highly specific bioprobe towards LAP over other competitive analytes. Based on this excellent selectivity, We further employed BODIPY-C-Leu to detect LAP in the urine and plasma of normal subjects. The results verified the ability of our probe for fluorescence sensing of LAP in complex biological system. (Figure S11)

Figure 3. Emission intensity of BODIPY-C-Leu (10 µM) at 578 nm in the presence of various species. All determinations were performed in PBS buffer solution (pH 7.4) at 37 °C. Bars represent relative fluorescence responses at 0, 15, 30 min after addition of bioanalytes (λex = 480 nm). Labels: a: free; b: Ca2+; c: Mg2+; d: Zn2+; e: Cys; f: GSH; g: NaHS; h: glucose; i: lipase; j: aprotinin; k: trypsin; l: cellulase; m: α-amylase; n: sulfatase; o: GGT; p: ELA; q: α-Chy; r: LAP.

Living cell imaging. Inspired by the excellent photophysical properties of BODIPY-C-Leu in monitoring the LAP activity in vitro, we further examined the capability of the probe to evaluate endogenous LAP activity in living samples. The cytotoxicity of free BODIPY-C-Leu was initially evaluated using typical MTT assays, and the results revealed that BODIPY-C-Leu coexisted well with living cancer cells. Since the probe shows good biocompatibility, we then investigate its performance in different cancer cells. Under the identical experimental conditions, HeLa cells and A549 cells were first incubated with BODIPY-C-Leu (10 µM) for 60 min. These two cells afforded a bright fluorescence signal in red channel, while in the green channel, a stronger fluorescence signal was observed in HeLa cells than A549 cells. The ratio of the fluorescence intensity from green channel to red channel was about 1.21 for HeLa cells and 0.74 for A549 cells (Figure S12). These results indicated that HeLa cells may have a higher level of intracellular LAP activity than A549 cells. In order to further confirm the above results, the endogenous LAP activity was detected in single cells by flow cytometry. The response of BODIPY-C-Leu (10 µM) to the endogenous LAP in HeLa cells and A549 cells was studied at different time points (10, 20, 30, 40 and 50 min). As shown in Figure 4, the results depicted that the fluorescence intensity in HeLa cells significantly increased over time (Figure 4A), while the emission intensity in A549 cells increased slowly (Figure 4B). These results indicate that BODIPY-C-Leu can accurately detect intracellular LAP in individual cells by flowcytometry, verifying that the LAP activity in HeLa cells was noticeably higher than that in A549 cells.

Figure 4. Flow cytometric analysis of HeLa Cells (A) and A549 cells (B) exposed to BODIPY-C-Leu (10 µM) at different incuba-


Page 5 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tion time (10, 20, 30, 40 and 50 min). (C) Plot of the relative fluorescence intensity versus the incubation time against HeLa cells (red line) and A549 cells (blue line) in flow cytometry. Data represent average values of three independent experiments.

Subsequently, more detailed analysis of intracellular LAP activity was carried out in HeLa cells model. After incubation with BODIPY-C-Leu (10 µM) for 20 min, the samples gave a significant fluorescence in the red channel, while the green channel was almost nonfluorescent. The ratio of the two emissions from green channel to red channel was about 0.01 (Figure 5 (A-D)). However, with the variation of incubation time, a remarkable fluorescence enhancement in green channel was observed, and after extending the incubation time to 60 min, the fluorescence intensity in green channel reached the plateau point, the ratio from green channel to red channel unambiguously exhibited a time-dependent change and improved to be around 1.23. Furthermore, as shown in Figure 5 (U-X), the fluorescence in green channel could be blocked significantly in the presence of inobestin, a typical LAP inhibitor, and the ratio decreased to 0.04. Overall, these imaging experiments reveal that the fluorescence signal change was selectively triggered by intracellular LAP, and BODIPY-C-Leu was suitable for monitoring LAP activity in living cancer cells.

Figure 5. Fluorescent imaging of HeLa cells incubated with BODIPY-C-Leu (10 µM) for 20 min (A-D); 30 min (E-H); 40 min (I-L); 50 min (M-P); 60 min (Q-T); and (U-X) pretreated HeLa

cells with inobestin (50 µM) for 30 min, and then further incubated with BODIPY-C-Leu (10 µM) for 60 min. (A,E,I,M,Q,U) bright field image; (B,F,J,N,R,V) green channel (510 nm-550 nm); (C,G,K,O,S,W) red channel (590 nm-650 nm ); (D,H,L,P,T,X) ratio/merge image generated from green channel to red channel; (Y) average intensity ratios from green channel to red channel in fluorescence image. Data represent mean standard error (n=3), the scale bar is 20 µm.

Zebrafish imaging. Using excessive amount of certain drugs usually causes damage to biological tissue, we next sought to investigate the drug-induced LAP level changes. In this endogenous experiment, zebrafish were chosen as the animal model, and divided into two groups: the control group (no cisplatin was added), and experimental group (0.1 mg L-1 cisplatin was added for inducing LAP expression). After culturing both group for 12 h, BODIPY-C-Leu (10 µM) was added, and followed by incubation for another 1 h. As compared to the strong fluorescence of two groups caused in the red channel (Figure 6C and G), the pretreated zebrafish samples exhibited significant fluorescence in the green channel (Figure 6F), while the control group had minimal fluorescence enhancement (Figure 6B). The ratio of the two emission intensity from green channel to red channel was about 0.18 for control group and 0.87 for experimental group. However, this dramatic phenomenon in experimental group could be significantly suppressed by inobestin (Figure 6I-L). These results demonstrated that BODIPY-C-Leu could perform LAP imaging in living organisms with high endogenous sensitivity.

Figure 6. Fluorescent images for zebrafish larvae: (A-D) the control group: zebrafish samples incubated with BODIPY-C-Leu (10 µM) for 1 h at 30 °C; The experimental group: zebrafish samples pretreated with cisplatin (0.1 mg L-1) for 12 h and (E-H) loaded with BODIPY-C-Leu (10 µM) for another 1 h at 30 °C; (I-L) the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

pretreated zebrafish samples were incubated with inobestin (100 µM) for 1 h before incubated with BODIPY-C-Leu (10 µM) (M) ratio from green channel (510 nm-550 nm) to red channel (590 nm-650 nm ); Values are the mean ± s.d. for n = 3 zebrafish larvae.

7 8 9 10

11

CONCLUSIONS In summary, we have designed a novel fluorescent probe (BODIPY-C-Leu) capable of selectively monitoring LAP activity in both ratiometric and turn-on model. Upon exposure to LAP, the amide bond in dipeptides is cleavaged to release a free amino group. Then the residue undergoes an intramolecular rearrangement to yield an amino-substituted BODIPY via the transfer between sulfur and nitrogen atoms through a fivemembered cyclic transition state, occurring coincidently with a dramatic photophysical change to realize the LAP detection. More importantly, BODIPY-C-Leu is cell-membranepermeable with great potential in cell imaging. As demonstrated, HeLa cells have a higher level of LAP activity than that in A549 cells. Impressively, BODIPY-C-Leu can achieve the endogenous detection of LAP activity in living zebrafish model for the first time. This effort makes an important contribution to better understanding of endogenous LAP activity in living samples, providing a novel alternative to trace LAP in complicated biological environments.

12 13 14 15 16

17 18 19 20 21

ASSOCIATED CONTENT

22

Supporting Information Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website.

23 24 25

AUTHOR INFORMATION

26

Corresponding Author * Email: [email protected] * Email: [email protected] * Email: [email protected]

28

Notes

29

27

The authors declare no competing financial interest.

30

ACKNOWLEDGMENT We gratefully acknowledge the financial support by the National Science Foundation of China (21676075).

31 32

REFERENCES 1

2

3

4 5 6

Harbut, M. B.; Velmourougane, G.; Dalal, S.; Reiss, G.; Whisstock, J. C.; Onder, O.; Brisson, D.; McGowan, S.; Klemba, M.; Greenbaum, D. C. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 526-534. Kondo, C.; Shibata, K.; Terauchi, M.; Kajiyama, H.; Ino, K.; Nomura, S.; Nawa, A.; Mizutani, S.; Kikkawa, F. Int. J. Cancer 2006, 118, 1390-1394. Sakabe, M.; Asanuma, D.; Kamiya, M.; Iwatate, R. J.; Hanaoka, K.; Terai, T.; Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2013, 135, 409-414. Matsui, M.; Fowler, J. H.; Walling, L. L. Biol. Chem. 2006, 387, 1535-1544. Huang, H.; Tanaka, H.; Hammock, B. D.; Morisseau, C. Anal. Biochem. 2009, 391, 11-16. Yamazaki, T.; Akada, T.; Niizeki, O.; Suzuki, T.; Miyashita, H. Blood 2004, 104, 2345-2352.

33 34

35

36 37 38

39

Page 6 of 8

Pearse, A. G. E.; Tremblay, G. Nature 1958, 181, 1532-1533. Bardawill, C.; Chang, C. Can. Med. Assoc. J. 1963, 89, 755-761. Reichling, J. J.; Kaplan, M. M. Dig. Dis. Sci. 1988, 33, 16011614. Mizutani, S.; Shibata, K.; Kikkawa, F.; Hattori, A.; Tsujimoto, M.; Ishii, M.; Kobayashi, H. Expert Opin. Ther. Targets 2007, 11, 453-461. Zhang, H.; Fan, J. L.; Wang, K.; Li, J.; Wang, C. X.; Nie, Y. M.; Jiang, T.; Mu, H. Y.; Peng, X. J. and Jiang, K. Anal. Chem. 2014, 86, 9131-9138 Lee, M. H.; Kim J. S. and Sessler, J. L. Chem. Soc. Rev. 2015, 44, 4185-4191. Mei, J. G.; Kim, D. H.; Ayzner, A. L.; Toney, M. F. and Bao, Z. N. J. Am. Chem. Soc. 2011, 133, 20130-20133 Bittner, G. C. V.; Bertozzi, C. R. and Chang, C. J. J. Am. Chem. Soc. 2013, 135, 1783-1795. Zhang, X.; Waibel, M.; Hasserodt, J. Chem. Eur. J. 2010, 16, 792-795. Kushida, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.; Ueno, T.; Yoshida, K.; Uchiyama, M.; Nagano, T. Bioorg. Med. Chem. Lett. 2012, 22, 3908-3911. Jiang, W.; Fu, Q.; Fan, H.; Ho J. and Wang, W. Angew. Chem. Int. Ed. 2007, 44, 8597-8600. Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J. H. and Yoon, J. J. Am. Chem. Soc. 2014, 136, 5351-5358. Wang, D.; Chen, J.; Ren, L.; Li, Q. L.; Li, D. D. and Yu, J. H. Inorg. Chem. Front. 2017, 4, 468-472. Wu, X. F.; Li, X. H.; Li, H. Y.; Shi, W.; Ma, H. M. Chem. Commun. 2017, 53, 2443-2446. Sun, Q.; Li, J.; Liu, W. N.; Dong, Q. J.; Yang, W. C. and Yang, G. F. Anal. Chem. 2013, 85, 11304–11311 Thorn-Seshold, O.; Vargas-Sanchez, M.; McKeon, S.; Hasserodt, J. Chem. Commun. 2012, 48, 6253-6255. He, X. Y.; Wu, X. F.; Shi, W.; Ma, H. M. Chem. Commun. 2016, 52, 9410-9413. Asanuma, H.; Akahane, M.; Niwa, R.; Kashida, H.; Kamiya, Y. Angew. Chem. Int. Ed. 2015, 54, 4315-4319. Lou, Z. R.; Li, P. and Han, K. L. Acc. Chem. Res. 2015, 48, 1358-1368. Yu, F. B.; Li, P.; Wang, B. S. and Han, K. L. J. Am. Chem. Soc. 2013, 135, 7674-7680. He, X. Y.; Xu, Y. H.; Shi, W. and Ma, H. M. Anal. Chem. 2017, 89, 3217-3221. Yu, F. B.; Li, P.; Li, G. Y.; Zhao, G. J.; Chu, T. S. and Han, K. L. J. Am. Chem. Soc. 2011, 133, 11030-11033. Yang, J.; Li, K.; Hou, J. T.; Lu, C. Y.; Li, L. L.; Yu, K. K.; Yu, X. Q. Sci. China Chem. 2017, 60, 793-798. Gu, K. Z.; Xu, Y. S.; Li, H.; Guo, Z. Q.; Zhu, S. J.; Zhu, S.; Shi, P.; James, T. D.; Tian, H.; Zhu, W. H. J. Am. Chem. Soc. 2016, 138, 5334-5340. Zhao, J. Z.; Xu, K. J.; Yang, W. B.; Wang Z. J. and Zhong, F. F. Chem. Soc. Rev. 2015, 44, 8904-8939. Song, J. X.; Tang, X. Y.; Zhou, D. M.; Zhang, W. Q.; James, T. D.; He, X. P. and Tian, H. Mater. Horiz. 2017, 4, 431-436. Pak, Y. L.; Li, J.; Ko, K. C.; Kim, G.; Lee, J. Y.; and Yoon, J. Anal. Chem. 2016, 88, 5476-5481. Tong, H. J.; Zheng, Y. J.; Zhou, L.; Li, X. M.; Qian, R.; Wang, R.; Zhao, J. H.; Lou, K. Y. and Wang, W. Anal. Chem. 2016, 88, 10816-10820. Surender, E. M.; Bradberry, S. J.; Bright, S. A.; McCoy, C. P.; Williams, D. C. and Gunnlaugsson, T. J. Am. Chem. Soc. 2017, 139, 381-388. Yoon, H. Y.; Shim, S. H.; Baek, L. J.; Hong, J. Bioorg. Med. Chem. Lett. 2011, 21, 2403-2405. Ho, N.; Weissleder, R.; Tung, C. Tetrahedron 2006, 62, 578585. Gu, K. Z.; Liu, Y. J.; Guo, Z. Q.; Lian, C.; Yan, C. X.; Shi, P.; Tian, H.; Zhu, W. H. ACS Appl. Mater. Interfaces 2016, 8, 26622-26629. Gong, Q. Y.; Shi, W.; Li, L. H.; Ma, H. M. Chem. Sci. 2016, 7, 788-792.


Page 7 of 8 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

41

42

43 44 45 46 47

Analytical Chemistry Niu, L. Y.; Guan, Y. S.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z. J. Am. Chem. Soc. 2012, 134, 18928-18931. Wang, F. Y.; Zhu, Y.; Zhou, L.; Pan, L.; Cui, Z. F.; Fei, Q.; Luo, S. H.; Pan, D.; Huang, Q.; Wang, R.; Zhao, C. C.; Tian, H.; Fan, C. H. Angew. Chem. Int. Ed. 2015, 54, 7349-7353. Wang, F. Y.; Zhou, L.; Zhao, C. C.; Wang, R.; Fei, Q.; Luo, S. H.; Guo, Z. Q.; Tian, H. and Zhu, W. H. Chem. Sci. 2015, 6, 2584-2589. Wang, F. Y.; Guo, Z. Q.; Li, X.; Li, X. A.; Zhao, C. C. Chem. Eur. J. 2014, 20, 11471-11478. Sun, Q.; Yang, S. H.; Wu, L.; Dong, Q. J.; Yang, W. C.; and Yang, G. F. Anal. Chem. 2016, 88, 6084-6091. Li, J.; Zhang, C. F.; Yang, S. H.; Yang, W. C.; and Yang, G. F. Anal. Chem. 2014, 86, 3037-3042. Burley, S. K.; David, P. R.; Lipscomb, W. N. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 6916-6920. Goldbarg, J. A.; Rutenburg A. M. Cancer 1958, 11, 283-291.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only


Page 8 of 8

A Ratiometric Fluorescent Probe for Monitoring Leucine

Recommend Documents