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Developing Activity Localization Fluorescence Peptide Probe Using ThiolEne Click Reaction for Spatially Resolved Imaging of Caspase-8 in Live Cells Wei Liu, Si-Jia Liu, Yong-Qing Kuang, Feng-Yan Luo, and Jian-hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02174 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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Analytical Chemistry

Developing Activity Localization Fluorescence Peptide Probe Using Thiol-Ene Click Reaction for Spatially Resolved Imaging of Caspase-8 in Live Cells Wei Liu‡,a, Si-Jia Liu‡,b, Yong-Qing Kuang*,a, Feng-Yan Luoa and Jian-Hui Jiang*,a a

Institute of Chemical Biology & Nanomedicine, State Key Laboratory of Chemo/Biosensing & Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, China

b

Guangxi Collaborative Innovation Center for Biomedicine & School of Preclinical Medicine, Guangxi Medical University, Nanning 530021, China

*

Corresponding Author: Fax: +86-731-88821916; E-mail: [email protected], [email protected]

ABSTRACT: Small molecule probes suitable for high-resolution fluorescence imaging of enzyme activity pose a challenge in chemical biology. We developed a novel design of activity localization fluorescence (ALF) peptide probe, which enables spatially resolved, highly sensitive imaging of peptidase in live cells. The ALF probe was synthesized by a facile thiol-ene click reaction of a cysteine-appended peptide with an acryloylated fluorophore. Upon cleavage by peptidase, the probe undergoes a seven-memberd intramolecular cyclization and releases the fluorophore with excited-state intramolecular photon transfer (ESIPT) effect. A highly fluorescent, insoluble aggregate was formed around the enzyme, which facilitates high-sensitivity and high-resolution imaging. This design is demonstrated for detection of caspase-8 activation. The results show that our design allows easy, high-yield synthesis of the probe, and the probe affords high sensitivity for caspase-8 detection. Live cell imaging reveals that the probe is able to render highly localized and high-contrast fluorescence signal for caspase-8. Our design holds the potential as a generally applicable strategy for developing high-sensitivity and high-resolution imaging peptide probes in cell biology and diagnostics.

INTRODUCTION Small molecule-based probes are useful tools for monitoring and imaging of biological events in live cells. Because of their structural tailorability, small molecule probes provide excellent cell permeability and wide adaptability to a wide range of biological targets.1-4 Among them, enzymatic probes, which allow real-time and in situ visualization of enzymatic regulation within cells, are of considerable significance.5-6 Most enzymatic probes utilize substrate reporters, which, upon catalysis by enzyme, activate fluorescence signals for deciphering the enzymatic activity.7-9 These probes, if not subcellularly localized in specific organelles, typically fail to provide spatially resolved information about enzymatic activity, because of rapid diffusion of the activated fluorescent molecules away from the reaction sites. Development of small molecule probes suitable for high-resolution fluorescence imaging of enzyme activity has posed a challenge in chemical biology. Fluorescently quenched activity-based probes (qABPs) represent a smart approach to address this challenge.10-11 Such probes are capable of forming covalent bonds with active sites of enzymes with concomitant activation of fluorescence signals,10-11 thereby providing the possibility

for high-resolution imaging of active enzymes. The qABPs, however, may suffer from limited sensitivity, since their reaction-mediated conjugation mechanisms do not offer signal amplification for fluorescence detection.12 An improved qABPs approach with signal amplification has been proposed based on the formation of reactive intermediates through multiple enzyme turnovers.12 The intermediates are capable of covalently conjugating to nucleophiles in proximity to enzyme, rendering a localized fluorescence signal for enzymatic activity.12 However, the signal amplification qABPs are in large molecular sizes, raising concerns in their solubility and cell permeability. An alternative strategy for spatially resolved imaging of enzymatic activity is the signal-localized probes which are water-soluble before enzymatic reactions and form insoluble precipitate comprising a number of fluorophores during enzyme turnovers. With suitable design, these precipitate may display very limited diffusion, rendering high-resolution and high-contrast signals for enzymatic activity and localization. There are two prominent examples for fluorophores of this type, such as aggregation induced emission (AIE) fluorophores13-14 and some dyes with excited-state intramolecular photon transfer (ESIPT) effect15-16. Despite the success of AIE fluorophores,13-14 ESIPT probes for spatially resolved imag-

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ing of enzymatic activity have been largely unexplored. A commercialized signal-localized ESIPT probe ELF-97 phosphate, which is applicable for detection of phosphatase activity, has been developed.17 This ESIPT probe has also been adapted using a structurally slightly different version for protease assays.18 However, this protease probe is challenged by the difficulty and low yield in synthesis. To our knowledge, current signal-localized ESIPT probes have not been realized for live cell imaging applications. Herein, we report a novel design of activity localization fluorescence (ALF) peptide probe based on the ESIPT effect, which, for the first time, enables spatially resolved, highly sensitive imaging of peptidase activities in live cells, as illustrated in Scheme 1. The ALF peptide probe can be synthesized based on a facile thiol-Michael addition or thiol-ene click reaction19-20 of a cysteine-appended peptide substrate with an acryloylated ESIPT fluorophore, such as 2-(5’-chloro-2-hydroxyl-phenyl)-6-chloro-4-(3H)-quinazolinone (CHCQ) (Scheme S1 in Supporting Information).17 The peptide-linked fluorophore is supposed to be weakly fluorescent, because the ESIPT process is blocked by acylation of the hydroxyl group. Fluorescence activation of this peptide probe relies on an intramolecular cyclization reaction known for the synthesis of substituted 1, 4thiazepines.21 Upon reacting with intracellular peptidase, the probe is cleaved at the peptide bond between cysteine and the substrate peptide followed by intramolecular cyclization,22 yielding a seven-membered ring thiazepine byproduct while releasing the fluorophore CHCQ. As a result of the intramolecular hydrogen bonds which substantially decrease water solubility, the fluorophore forms localized aggregate in proximity to the enzyme and delivers bright green fluorescence with a large Stokes shift. This localized fluorescence probe thus enables highsensitivity and high-resolution imaging of the peptidase activity. Scheme 1. Illustration of the ALF peptide probe for highresolution peptidase imaging.

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To our knowledge, this is the first time that the thiolMichael addition click reaction has been utilized for synthesis of peptide probes for bioimaging. This design provides a generally applicable and facile strategy, which enables the development of a variety of peptide probes for peptidases. Moreover, it allows the use of peptide substrates with natural peptide bonds between the enzymerecognizing sequence and cysteine, potentially affording maximal enzymatic activities and improved sensitivity. In particular, with the choice of ESIPT a fluorophore, cleavage of the probes by the target peptidase produces localized precipitate comprising a large number of fluorescent molecules with unusually large Stokes shifts. This characteristic not only realizes spatially resolved imaging, but also substantially enhances the contrast and alleviates the autofluorescence background. Therefore, the ALF probe design may provide a new promising paradigm in developing smart, responsive probes for high-sensitivity and high-resolution imaging applications. As the proof of principle, we choose an apoptotic signal, caspase-8 activation,23-24 as the case of study. Caspase-8 is an initiator enzyme accepting signals from death receptors and activating downstream effectors caspases, acting as a key mediator in the extrinsic apoptosis pathway.23-24 Despite of the importance of caspase-8 in this process, there has been no probes for spatially resolved imaging of caspase-8 activity. Herein we demonstrate the development of a new probe for selectively visualizing caspase-8 activity in living cells with high sensitivity and high spatial resolution.

EXPERIMENT SECTION Reagents and Materials. Recombinant human caspase-8 (active form) was purchased from IBL International GmbH. Cbz-Ile-Glu-Thr-Asp-FMK(Z-IETDFMK) was obtained from Biovision Inc. Mitomycin C (MMC) was purchased from Merck (Darmstadt, Germany). Mitotracker®Red (MitoTracker®Red CMH2XRos) was obtained from Invitrogen (Gaithersburg, MD, USA). Cbz-Ile-Glu-Thr-Asp-Cys (Z-IETDC) peptide was synthesized by Sangon Biotech Co., Ltd. HeLa cells were obtained from the cell bank of Central Laboratory at Xiangya Hospital (Changsha, China). Thin-layer chromatography (TLC) was performed on precoated silical gel 60 F254 plates and column chromategraphy was conducted over silica gel (mesh 200-300), both of which were obtained from Qingdao Ocean Chemicals (Qingdao, China). All solvents used were purified by standard methods prior to use. All other reagents were commercially available from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and used as received without further purification. Ultrapure water used was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) and had an electric resistance >18.25 MΩ. In Vitro Assays. In an assay for caspase-8, 10 μL sample with caspase-8 of a given concentration was added in the reaction mixture containing 10 μL negative HeLa


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cell lysate, 170 μL of 10 mM Tris-HCl buffer (pH 7.4) and 10 μL of 200 μM ALF probe followed by incubation at 37 °C. After the reaction, the mixture was subjected to fluorescence measurements. The excitation wavelength was 360 nm, and the emission wavelengths were in the range from 450 to 630 nm with both excitation and emission slits of 5 nm. Fluorescence Imaging of Living Cells. HeLa cells were grown in RPMI-1640 medium (Thermo Scientific HyClone) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in a humidified atmosphere incubator containing 5% wt/vol CO2. The cell density was determined using a TC20™ automated cell counter (BIORAD, USA). Fluorescence imaging of living cells was performed as follows: HeLa cells were plated on a 35 mm Petri dish with 10 mm bottom well in RPMI-1640 medium for 24 h. The cells were then treated with 3 µM ALF probe (or together with 10 µM Z-IETD-FMK) in 1 mL RPMI-1640 medium for 30 min. After washed three times with cold PBS, the cells were incubated with a fresh RPMI-1640 medium containing MMC of a given concentration for additional 1 h at 37 °C. The cells were washed twice with cold PBS before imaging. Fluorescence imaging of caspase-8 localization was performed using the same protocol except for an additional incubation step after MMC induction with 1 ng/mL Mitochondria tracer (Mitotracker®Red) for 5 min before imaging. The endpoint fluorescence images were collected at a sampling speed of 31 μs/pixel. Fluorescence emission was collected at 510-590 nm under excitation at 403 nm for ALF probe and 570-620 nm under excitation at 560 nm for Mitotracker®Red.

by oxidation with DDQ to obtain the acryloylated CHCQ (82% yield). It is noteworthy that the CHCQ could not be directly acryloylated, in which the tautomer of CHCQ also reacted with acryloyl chloride to yield a regioisomeric product difficult to separate from the desired acryloylated CHCQ.27 Subsequently, the ALF probe for caspase-8 was readily synthesized by the thiol-Michael addition click reaction (93% yield). Such a high-yield and facile synthesis indicated that our design created an efficient paradigm for developing the ALF peptide probes. The synthetic intermediates and ALF probe were characterized by 1H-NMR, 13C-NMR and Mass spectrometry (MS) (Figures S1-S8 in Supporting Information).

RESULTS AND DISCUSSION Synthesis and Characterization of ALF probe. The ALF probe for caspase-8 was synthesized from a cysteineappended, caspase-8-specific substrate peptide and the acryloylated CHCQ (Scheme S2 in Supporting Information). The substrate peptide, Z-IETDC,25 was obtained using standard solid phase synthesis. In the peptide the carboxylic acid side chains of Glu and Asp were masked as methyl esters to improve its cell membrane permeability.26 Once inside the cell, intracellular esterases can cleave the ester to regenerate the active substrates.26 The appended D-cysteine at Cterminal, which was resistant to degradation from nonspecific peptidases, was used for click chemistry conjugation to the acryloylated CHCQ. The acryloylated CHCQ was synthesized by a two-step procedure: First, 5chloro-salicylaldehyde reacted with acryloyl chloride in dry CH2Cl2 in the presence of a small amount of dry Et3N to synthesize 4-chloro-2-formyl-phenyl acrylate (90% yield); Second, 4-chloro-2-formylphenyl acrylate was refluxed with 2-amino-5-chloro-benzamide in EtOH in the presence of a catalytic amount of TsOH•H2O followed

Figure 1. (a) Fluorescence spectral response of ALF probe. I, probe plus positive mixture; II, probe plus positive lysate; III, probe plus negative lysate; IV, probe plus positive mixture and Z-IETD-FMK; V, ALF probe. (b) Fluorescence spectral responses to caspase-8 of varying concentrations.

Response of ALF probe to caspase-8. The fluorescence response of the as-synthesized ALF probe was investigated for caspase-8 assay (Figure 1a). The probe (10 μM) merely displayed a very weak fluorescence signal, evidencing efficient fluorescence quenching of the CHCQ fluorophore owing to the inhibition of ESIPT process via masking the hydroxyl group. Incubation of the probe with a lysate of HeLa cells with no induced apoptosis (negative lysate) also showed little change in the signal, implying that complex biological matrices without caspase-8 had no interference with the probe in caspase-8 assay. In con-



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trast, the probe showed an intense fluorescence peak after incubated with a positive mixture of the negative cell lysate with 3 nM caspase-8. This result suggested that caspase-8 activated the fluorescence of the probe in the cell lysate matrix, indicating the potential of the ALF probe for caspase-8 detection. Incubation of the probe with a positive lysate of cells treated with MMC, a known inducer of cell apoptosis with caspase-8 activation,28-29 also gave a strong fluorescence signal, revealing that caspase-8 activation in apoptotic cells gave rise to a turnon fluorescense signal for the ALF probe. Moreover, addition of a caspase-8 inhibitor (10 μM), Z-IETD-FMK,30 in the positive mixture followed by incubation with the probe showed almost complete attenuation of the fluorescence, confirming the specificity for the probe in response to caspase-8. Besides its specificity, the ALF probe also provided high sensitivity for caspase-8 assay. With increasing concentrations of caspase-8 in the range of 30 pM to 3 nM, the fluorescence signals displayed dynamic increase (Figure 1b) with a saturated response achieved at 3 nM. This saturated signal seemed attributed to complete depletion of the probe by 3 nM enzyme, indicating thousands of turnovers of an enzyme in catalyzing the cleavage of the probe. Such a large turnover number gave direct evidence for high sensitivity of the ALF probe for caspase-8 detection. The fluorescence intensities at 520 nm were found to be linearly correlated to caspase-8 concentrations with an estimated detection limit of 10 pM (Figure S9 in Supporting Information). Such sensitivity was much higher than those for existing methods, 25, 31-33, 34 suggesting that the ALF probe afforded a highly sensitive strategy for caspase8 detection. The sensitivity improvement might be ascribed to the large Stokes shift of the dye and the use of peptide substrates with a natural peptide bond at the cleavage site. As a matter of fact, a close investigation revealed that the Michaelis constant Km was determined to be ~20 μM for caspase-8 (Figure S10 in Supporting Information), which was much smaller than the literature reported value (~66 μM).34 Such a smaller Michaelis constant indicated higher affinity of the probe to capspase-8, confirming that the natural peptide bonds between the enzyme-recognized sequence and cysteine in our probe rendered an improved enzymatic activity. Further experiments were performed to interrogate the mechanism and performance for the ALF probe. MS analysis revealed that enzymatic reaction yielded two new peaks corresponding to the CHCQ (calcd for C14H8Cl2N2O2 [M-H]- m/z 304.9890, found 305.1171) (Figue 2a) and the seven-membered ring thiazepine by-product (calcd for C6H9NO3S [M+H]+ m/z 176.0376, found 176.0113) (Figure 2b), giving clear evidence for its response mechanism of the probe. Analyses using UV-vis absorption spectroscopy and TLC showed that the probe (10 μM), after reacting with 3 nM caspase-8, was almost quantitatively converted into CHCQ, confirming the large turnover number (~3333) of this probe in caspase-8 assay (Figures

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S11-S12 in Supporting Information). Investigation of the effects of pH and temperature manifested that the probe was chemically stable under conditions of varying pH and temperature, and the optimal activity was achieved at physiological conditions of pH 7.4 and 37 oC, suggesting the utility of this probe for live cell imaging applications (Figure S13-S14 in Supporting Information). Cytotoxicity assay using HeLa cells revealed that the probe only generated marginal toxicity (cell viability decreased by ~7% after 6 h incubation) at a concentration up to 20 µM (Figure S15 in Supporting Information), supporting the ability of this probe for live cell imaging at a working concentration below 20 µM.

Figure 2. MS analysis of the proposed response mechanism. (a) negative and (b) positive MS of the reaction product from the reaction of ALF probe with caspase-8.

Fluorescence Imaging of caspase-8 in Live Cells. We investigated the applicability of this probe to imaging caspase-8 activity and localization in living cells (Figure 3). As anticipated, no appreciable fluorescence signal was obtained when incubating 3 µM probe with HeLa cells for 30 min in a RPMI-1640 medium. In contrast, after the cells were incubated with 3 µM probe followed by treatment with 40 µg/mL MMC for 60 min, the bright green fluorescence was observed in the cells. A control experiment with cells treated with 40 µg/mL MMC and 10 µM ZIETD-FMK also did not give bright green fluorescence image, verifying the specificity of the probe for fluorescence imaging of caspase-8. Previous studies in histochemistry had found that the commercialized probe, ELF97, produced CHCQ precipitates localized in close proximity to phosphatase.17 This finding suggested that


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CHCQ precipitates might be adsorbed on some hydrophobic protein sites or pockets around the active enzyme. A closer observation of the fluorescence image revealed that the turn-on signals also appeared as spatially isolated and highly localized spots rather than continuous areas. Further imaging experiments at different sections through z-axis also gave densely distributed but spatially separate fluorescent spots inside the cytoplasma (Figure S16 in Supporting Information). Based on the proved localization property of CHCQ, these results demonstrated the ability of the ALF probe for spatially resolved imaging of caspase-8 activity. Considering the formation of green fluorophores into insoluble aggregates only occurred at the site where the probe reacted with caspase-8, these fluorescent spots could give indicators for the localization of caspase-8. Moreover, since the localized signals typically comprised a large number of fluorophores, the ALF probe was able to afford higher contrast for imaging, providing high sensitivity for caspase-8 detection.

fragmented bubble-like morphology of mitochondria with slight decreased fluorescence contrast for Mitotracker®red appeared since 60 min, indicators of slight loss of mitochondrial membrane potential and progression of cell apoptosis.35 These results suggested that in MMC induced apoptosis, caspase-8 activation occurred much earlier than MOMP, a typical temporal order for extrinsic apoptosis pathway.36-37 This result also indicated a dominant role of extrinsic apoptosis pathway in MMC treated HeLa cells, which was consistent with previous findings for other cell lines.36-37

Figure 3. Fluorescence (1) and differential interference contrast (DIC) merged (2) images for HeLa cells. (a) cells treated with 3 µM probe for 30 min, (b) cells treated with probe followed by 40 µg/mL MMC for 60 min, (c) cells treated with probe and 10 µM Z-IETD-FMK followed by MMC.

To further expand the utility of our ALF probe for caspase-8 imaging, real-time monitoring of caspase-8 activation in cells was performed (Figure 4). For the cells treated with 20 µg/mL MMC, no appreciable activation of caspase-8 was observed in 30 min, but substantial caspase-8 activity was found after 45 min. At this time, fluorescence staining with Mitotracker®red showed that mitochondria remained a branched filamentous structure, a typical morphology of mitochondria without outer membrane permeabilization.35 This finding implied that caspase-8 activation was an event occurring earlier than mitochondrial outer membrane permeabilization (MOMP) in MMC induced apoptosis in HeLa cells. As time increased up to 120 min, enhanced fluorescence signals were achieved for caspase-8 activation with a concomitant shrinkage of the cells. Interestingly, a small portion of

Figure 4. Real-time fluorescence images for HeLa cells treated with 3 µM probe followed by 20 µg/mL MMC for 30 (a), 45 (b), 60 (c) and 120 (d) min, respectively. Staining of mitochondrials was obtained by treating cells with 1.0 ng/mL Mitotracker®red for 5 min followed immediate collections of images.

A further experiment was performed to investigate cells treated for 1 h with MMC of increasing concentrations (Figure 5). Clear dose-dependent fluorescence activation was observed. Very few bright spots appeared for cells treated with 10 µg/mL MMC, and much more densely distributed fluorescence spots were obtained with increasing MMC concentrations. A close investigation with images collected at lower resolution for more cells gave further evidence for the dose-dependent fluorescence



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signals arising from caspase-8 activities in the cells (Figure S17 in Supporting Information). It was reported that increased dose of MMC induced higher caspase-8 enzymatic activity.28-29 Therefore, these results actually implied the potential of the ALF probe for quantitative imaging of caspase-8 activity in live cells.

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‡ These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (21527810, 21190041, 21521063) and National Key Basic Research Program (2011CB911000).

REFERENCES

Figure 5. Fluorescence (1) and DIC merged (2) images for HeLa cells treated for 1 h with MMC of different concentrations: (a) 0 µg/mL; (b) 10 µg/mL; (c) 20 µg/mL; (d) 40 µg/mL; (e) 60 µg/mL.

CONLUSIONS We develop a novel design of activity localization fluorescence (ALF) peptide probes based on excited-state intramolecular photon transfer (ESIPT) effect for spatially resolved, highly sensitive imaging of peptidase activity in live cells. This is the first time that the thiol-Michael addition click reaction has been utilized for constructing peptide probes for bioimaging. This design provides a generally applicable and facile strategy for synthesis of a variety of peptide probes. With the use of ESIPT fluorophores, the probe is able to produce insoluble fluorescent aggregate comprising many fluorophores localized around the enzyme, affording high-sensitivity and high-resolution imaging of peptidase activity. Results show that our design creates a peptide probe with high affinity to capase-8, and enables high-selectivity, high-sensitivity, highresolution imaging for capase-8 while sheds light on the mechanism of apoptosis. Therefore, the ALF probe may provide a new promising paradigm for developing smart, responsive peptide probes in high-sensitivity and highresolution imaging applications.

ASSOCIATED CONTENT Supporting Information Instrumentation, synthesis and characterization of the CHCQ and ALF probe, extraction of HeLa cell lysate, and additional figures. These materials are available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected].

Author Contributions

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Developing Activity Localization Fluorescence ... - ACS Publications

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