A General Strategy Toward Highly Fluorogenic Bioprobes Emitting

Article pubs.acs.org/JACS

A General Strategy Toward Highly Fluorogenic Bioprobes Emitting across the Visible Spectrum Haoliang Chen, Xiujing He, Meihui Su, Wenhao Zhai, Hao Zhang, and Changhua Li* State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Pharmacy, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China S Supporting Information *

ABSTRACT: A general approach toward highly fluorogenic probes across the visible spectrum for various analytes offers significant potential for engineering a wide range of bioprobes with diverse sensing and imaging functions. Here we show a facile and general strategy that involves introducing a new fluorogenic mechanism in boron dipyrromethene (BODIPY) dyes, based on the principle of stimuli-triggered dramatic reduction in the electron-withdrawing capabilities of the meso-substituents of BODIPYs. The fluorogenic mechanism has been demonstrated to be applicable in various BODIPYs with emission maxima ranging from green to far red (509, 585, and 660 nm), and the synthetic strategy allows access to a panel of highly fluorogenic bioprobes for various biomolecules and enzymes (H2O2, H2S, and protease) via introducing specific triggering motifs. The potency of the general design strategy is exemplified by its application to develop a mitochondria-targeting far-red probe capable of imaging of endogenous H2O2 in living cells.

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INTRODUCTION Fluorescence imaging techniques have emerged as powerful tools for noninvasive monitoring of biological analytes and processes in real time with high spatiotemporal resolution.1 Despite the diverse palette of available fluorescence probes that are essential for fluorescence imaging, there is still a demand for more specific, sensitive, and versatile probes, especially fluorogenic bioprobes.2 A unified design strategy toward highly fluorogenic bioprobes sensitive and selective for variable analytes of interest with tunable emission maxima throughout the visible spectral region provides significant potential to engineer a broad range of bioprobes with desired sensing and imaging capabilities for biological research and clinical needs. As one class of highly fluorescent dyes, members of the boron dipyrromethene (BODIPY) family have been widely employed as scaffolds for functional fluorogenic bioprobes due to their outstanding spectroscopic and photophysical properties, including excellent environmental stability, large extinction coefficients, and sharp fluorescence spectrum with high quantum yield.3 Most importantly, the wide versatility of synthetic routes to BODIPYs allows the facile synthesis of fluorophores with desired emission maxima ranging from cyan to far-red region. The excellent features of BODIPY make it a viable candidate for a universal scaffold to access diverse fluorogenic probes with various photophysical properties. The key to establishing such a platform was the identification of a fluorogenic mechanism that could be applied in BODIPYs with emission maxima across the visible region. However, at present, most BODIPY-based fluorogenic © 2017 American Chemical Society

probe designs are based on the principle of photo-induced electron transfer (PeT)4 or Förster resonant energy transfer (FRET),5 which requires electron or energy transfer between donors and acceptors, rendering them difficult to apply in a set of BODIPYs with different emission wavelengths. Recently, the fluorescence properties of BODIPYs were found to be highly dependent on the electronic properties of the meso-substituents of the dye.6 For example, enzymatic hydrolysis of highly electron-withdrawing meso-ester groups to electrondonating meso-carboxylate anions induces dramatic enhancement of fluorescence at 550 nm in aqueous solution.6a Recognizing that the fluorescence turn on is merely due to the dramatic reduction in the electron-withdrawing capabilities of the meso-substituents, we hypothesized that this “ester-to-carboxylate” conversion-based fluorogenic mechanism could be potentially applied in various BODIPYs across the entire visible spectrum. On the other hand, it is known that carboxyl groups (−COOH) could be modularly caged by a self-immolating linker via formation of an ester bond and selectively decaged in response to specific stimuli of interest.7 We envisioned that by incorporating of self-immolative chemistry, meso-COOH-substituted BODIPYs can be modularly derivatized to afford a palette of fluorogenic probes for variable analytes of interest throughout the visible spectrum. As depicted in Figure 1, in the presence of analyte of interest, the nonfluorescent meso-ester-substituted Received: June 8, 2017 Published: June 27, 2017 10157

DOI: 10.1021/jacs.7b05920 J. Am. Chem. Soc. 2017, 139, 10157−10163

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Table 1. Photophysical Properties of All BODIPY-COOHs and Bioprobes compound a

B-COOH-G B-COOH-Oa B-COOH-FRa P-HP-Gb P-HP-Oc P-HP-FRa P-HP-FR-tppc P-HS-FRa P-PGA-FRa

λabs (nm)

ε (M−1 cm−1)

λem (nm)

Φfl (%)

495 565 640 513 595 688 688 688 688

132 800 123 200 86 000 109 700 103 000 57 100 57 200 58 000 58 600

509 585 660 509 585 660 660 660 660

52 43 11 0.12 0.24 0.053 0.057 0.062 0.042

a Data were collected in 10 mM PBS (pH 7.4) at 37 °C. bData were collected in 10 mM PBS (pH 7.4; 8% v/v DMSO) at 37 °C. cData were collected in 10 mM PBS (pH 7.4; 1% v/v DMSO) at 37 °C.

Figure 1. Detection mechanism of the highly fluorogenic BODIPYbased bioprobes with tunability in both emitting wavelength and sensing analytes.

The fluorescence quantum yields of the meso-carboxylatesubstituted BODIPY-COOHs and corresponding meso-estersubstituted H2O2 probes were measured in PBS as shown in Table 1. Gratifyingly, we found that the fluorescence quantum yields of all three BODIPY-COOHs are significantly higher than the corresponding meso-ester-substituted H2O2 probes, indicating that the “ester-to-carboxylate” conversion should turn on the fluorescence of the BODIPYs possessing emission maxima that range from green to far-red wavelengths. Thus, the successful conversion of meso-ester groups to meso-carboxylate groups via self-immolation upon treatment with special analytes becomes pivotal to the design strategy shown in Figure 1. Most attention has been paid to the triggered release of phenol or amine groups via self-immolative chemistry, while relatively few reports on the triggered release of carboxyl groups.7,9,10 To assess the feasibility of the fluorogenic probes, P-HP-G, P-HP-O, and P-HP-FR (0.5 μM) were treated with H2O2 in PBS at 37 °C. All the three P-HP probes underwent significant fluorescence enhancement (P-HP-G, 320-fold; P-HP-O, 220-fold; P-HP-FR, 133-fold) upon addition of 100 μM H2O2 within 60 min (Figure 3a−c). Concomitantly, the color of the three probe solutions changes obliviously as shown in Figure 3d. The H2O2induced fluorescence turn-on can also be visualized by naked eye, as evidenced by the transition from non-fluorescence to the intense emission (Figure 3d). The absorption spectra showed obvious blue-shifts during the incubation due to the H2O2induced “ester-to-carboxylate” conversion (Figure 4a−c). HPLC analysis of the assay solution further confirmed the complete conversion to meso-COOH-substituted products in the presence of H2O2 as shown in Figure 4d−f. All the three P-HP probes are highly stable in the absence of H2O2 (Figures S5−S7). As shown in Figures 5a and S8, the fluorescence intensities have an excellent linear relationship with the concentrations of H2O2 in 0−50 μM. The detection limit was calculated to be as low as 0.15, 0.20, and 0.25 μM for P-HP-G, P-HP-O, and P-HP-FR, respectively. Collectively, the above experiments clearly demonstrated that the proposed fluorogenic mechanism (Figure 1) can be applied in several BODIPYs, to produce highly sensitive fluorogenic H2O2 probes across the visible spectrum. It was also demonstrated that the three P-HP probes are highly selective for H2O2 over other ROSs and some typical physiological nucleophiles (Figure 5b). Mitochondria-Targeting Far-Red Probe for Live Cell Imaging of H2O2. Since the overproduction of H2O2 is concerned with several diseases and mitochondria are the primary sources of H2O2,9a,11 our next goal was to design a far-red probe

bioprobes would be converted to highly labile intermediates, followed by the linker self-immolation to form meso-carboxylatesubstituted BODIPYs with strong fluorescence emission.

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RESULTS AND DISCUSSION Applying the Proposed Fluorogenic Mechanism in BODIPYs Emitting throughout the Visible Spectrum. To test our hypothesis that the proposed fluorogenic mechanism could be applied in BODIPY fluorophores with tunable emission maxima, we synthesized three typical meso-carboxylsubstituted BODIPY-COOH fluorophores (Figure 2a and

Figure 2. Chemical structures of (a) three typical meso-carboxylsubstituted BODIPYs, B-COOH-G, B-COOH-O, and B-COOH-FR, and (b) the corresponding three H2O2 probes P-HP-G, P-HP-O, and P-HP-FR.

Supporting Information), which exhibit emission maxima at 509 nm (green, B-COOH-G), 585 nm (orange, B-COOH-O), and 660 nm (far-red B-COOH-FR) in phosphate buffer solution (PBS; 10 mM, pH 7.4) (Figure S1). Notably, the emission intensities of the three synthesized BODIPY-COOHs are almost constant irrespective of solution pH in the physiologically relevant pH range 4−9 (Figures S2−S4).6a,8 Following a facile synthetic route shown in Scheme S1, the BODIPY-COOHs were reacted with H2O2-responsive arylboronate triggers,9 trigger-HP, to afford the corresponding three H2O2 probes, i.e., P-HP-G, P-HP-O, and P-HP-FR (Figure 2b). 10158

DOI: 10.1021/jacs.7b05920 J. Am. Chem. Soc. 2017, 139, 10157−10163

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Figure 3. Fluorescence emission spectra recorded for 0.5 μM solution of (a) P-HP-G, (b) P-HP-O, and (c) P-HP-FR upon incubating with H2O2 (100 μM) for varying time intervals (0−60 min) at 37 °C; inset in each figure shows the time-dependent increases in normalized fluorescence intensities at 509, 585, and 660 nm, respectively. (d) Optical photographs recorded under ambient light (top panel) and UV (365 nm) light (bottom panel) for P-HP-G, P-HP-O, and P-HP-FR in the absence (left) or presence (right) of H2O2 after 60 min.

Figure 4. UV−vis absorption spectra recorded for 0.5 μM solution of (a) P-HP-G, (b) P-HP-O, and (c) P-HP-FR upon incubating with H2O2 (100 μM) for varying time intervals at 37 °C. HPLC chromatograms of (d) P-HP-G, (e) P-HP-O, and (f) P-HP-FR without treatment of H2O2 (top); with H2O2 (200 equiv) treatment for different time as indicated at 37 °C (middle); and the corresponding meso-carboxyl-substituted BODIPY-COOH only (bottom). The samples were analyzed by HPLC using an isocratic elution program with a solvent system of methanol/deionized water (95/5, V/V) at a flow rate of 1.0 mL/min with detection wavelength at 500, 570, and 656 nm for P-HP-G, P-HP-O, and P-HP-FR, respectively.

moieties can specifically localize in mitochondria.4b,12 We then synthesized a tpp functionalized far-red probe, P-HP-FR-tpp (Figure 6 and Supporting Information). As shown in Figures 7a

that can be specifically localized in mitochondria and evaluate its performance in cell imaging of endogenous H2O2. It is well-known that lipophilic triphenylphosphonium cation (tpp) 10159

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Figure 5. (a) H2O2 concentration dependence of normalized fluorescence intensities after incubating 0.5 μM of (●) P-HP-G, (○) P-HP-O, or (□) P-HP-FR with H2O2 (0−50 μM) for 30 min at 37 °C. (b) Fluorescence response of P-HP-G (green), P-HP-O (orange), or P-HP-FR (red) toward various ROSs and some physiological nucleophiles.

and S9a, this P-HP-FR analogue maintained the enhancement in fluorescence of P-HP-FR while possessing a mitochondriatargeting moiety, making it well-suited for selective monitoring endogenous H2O2 in mitochondria of living cells. The stability of P-HP-FR-tpp was assessed by incubating it in PBS in the absence of H2O2. Only small fluorescence enhancement (2.2-fold) was observed after incubation for 1.5 h at 37 °C (Figures 7a and S9b). To examine the selectivity, the probe (0.5 μM) was treated with various potentially interfering species, including ROSs and physiological nucleophiles under standard assay conditions.

Figure 6. Chemical structure of lipophilic triphenylphosphonium cation (tpp)-functionalized H2O2 probe, P-HP-FR-tpp.

Figure 7. (a) Time evolution of normalized emission intensities at 660 nm recorded for P-HP-FR-tpp (0.5 μM) in the absence (◇) or presence (○) of H2O2 (100 μM). (b) Fluorescence response of P-HP-FR-tpp toward various ROSs and some physiological nucleophiles. (c) Cell viability of HeLa cells in the presence of P-HP-FR-tpp with indicated concentrations determined by MTT. Error bars represent mean ± SD (n = 5). (d−f) Confocal fluorescence imaging of endogenous H2O2 with B-TEG4-Me (as control) or P-HP-FR-tpp in HeLa cells. Cells were treated with 0.2 μM Mito-Tracker Green (MTG) and 0.5 μM B-TEG4-Me (d) or P-HP-FR-tpp (e,f) for 15 min, then incubating for 30 min at 37 °C; note that cells were pretreated (d,e) without or (f) with PMA (1 μg/mL) for 1 h. Scale bar: 20 μm. (g) Normalized probe (red) channel emission intensities quantified from CLSM images. (h) Scatter plot of red and green pixel intensities of HeLa cells shown in the right panel of (f). 10160

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Figure 8. Chemical structures of (a) hydrogen sulfide probe, P-HS-FR, and (b) PGA probe, P-PGA-FR.

Figure 9. (a) Fluorescence emission spectra recorded for 0.5 μM solution of P-HS-FR upon incubating with NaSH (100 μM) for varying time intervals (0−180 min) at 37 °C; inset shows the time-dependent increases in normalized fluorescence intensities at 660 nm. (b) HPLC chromatograms of P-HSFR without treatment of NaSH (top), with NaSH (200 equiv) treatment for different time as indicated at 37 °C (middle), and B-COOH-FR only (bottom). The samples were analyzed by HPLC using an isocratic elution program with a solvent system of methanol/deionized water (95/5, V/V) at a flow rate of 1.0 mL/min with detection wavelength at 656 nm. (c) Fluorescence response of P-HS-FR toward various biologically relevant species. (d) NaSH concentration dependence of normalized emission intensities at 660 nm after incubating P-HS-FR (0.5 μM) with NaSH (0−50 μM) for 60 min at 37 °C.

As shown in Figure 7b, various species up to 100 μM, including • OH, ClO−, tBuO•, 1O2, Cys, GSH, and H2S, induced no obvious changes in fluorescence intensities. In addition, the low cytotoxicity of P-HP-FR-tpp was also demonstrated by methyl thiazolyltetrazolium (MTT) assay (Figure 7c). We next tested P-HP-FR-tpp for visualization of endogenous H2O2 in living cells. A H2O2-insensitive meso-ester-substituted BODIPY dye, B-TEG4-Me, was synthesized as a control compound (see Supporting Information). It is quite stable in the absence or presence of H2O2 (Figure S10). HeLa cells were then incubated with B-TEG4-Me and P-HP-FR-tpp. After incubation for 30 min, control cells treated with B-TEG4-Me showed no detectable fluorescence (Figure 7d), whereas the fluorescence of P-HP-FR-tpp treated cells was visible but weak (Figure 7e). This should be ascribed to the endogenous H2O2-induced partial conversion to B-COOH-FR. HeLa cells were then pretreated with phorbol-12-myristate-13-acetate (PMA; an intracellular H2O2 stimulator13) for 1 h before incubation with P-HP-FR-tpp to promote the intracellular H2O2 production. Compared to untreated cells, CLSM images revealed a significant increase

of red channel emission intensities for PMA-pretreated cells (Figure 7f). Quantitative analysis revealed that red emission intensities increased 4-fold (untreated) and 13-fold (PMApretreated) for P-HP-FR-tpp incubated HeLa cells, respectively, as compared to B-TEG4-Me incubated HeLa cells (Figure 7g). In addition, the red-channel fluorescence showed excellent overlay with green-channel fluorescence of Mito-Tracker Green (MTG), with Pearson’s correlation coefficient at 0.86 ± 0.04 (evaluated by ImageJ software), indicating the probes specifically localized in mitochondria of the HeLa cells (Figure 7h). Assessment of the Generality of the Proposed Strategy. Having established that the fluorogenic mechanism can work in a set of BODIPYs, and demonstrated the feasibility of P-HP-FR-tpp as mitochondria-targeting fluorogenic bioprobe for endogenous H2O2 in living cells, we then evaluated the generality of the proposed strategy (Figure 1). The flexibility of the synthetic strategy enabled us to prepare a H2S probe (P-HS-FR) containing H2S-sensitive benzyl azide as triggerable motif14 (Figure 8a and Supporting Information), which is very stable in PBS (Figure S11). Upon addition of 100 μM NaHS (a standard 10161

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Figure 10. (a) Fluorescence emission spectra recorded for 0.5 μM P-PGA-FR in PBS (10 mM, pH 7.4) after the addition of 5 U/mL PGA for varying time intervals (0−120 min) at 37 °C. (b) Time-dependent increases in normalized emission intensity at 660 nm recorded for P-PGA-FR (0.5 μM) under varying concentrations of PGA (from bottom to top: 0, 1, 2, 5, and 10 U/mL).

source of H2S), the fluorescence intensity of P-HS-FR increased by up to nearly 227-fold before reaching a plateau after 150 min incubation (Figure 9a). HPLC analysis of the assay solution confirmed the full conversion to B-COOH-FR in the presence of NaSH (Figure 9b). The high selectivity of P-HS-FR for H2S over other biological species including glutathione (GSH) and cysteine was also demonstrated (Figures 9c and S12). Fluorescence titration experiments indicated an excellent linear relationship with the concentrations of NaSH in 0−50 μM with detection limit of 0.19 μM (Figures 9d and S13). Finally, we expanded the design strategy for highly fluorogenic probe sensing of protease that is known to be upregulated in several diseases such as cancer, rheumatoid arthritis, and neurodegenerative disorders.15 A water-soluble far-red probe, P-PGA-FR, for penicillin G amidase (PGA; selected as a model protease) with phenyl acetamide as triggerable motif16 was synthesized as a paradigm (Figure 8b and Supporting Information). Having demonstrated its stability in PBS (Figure S14a), we then investigated the capability of P-PGA-FR in detection of PGA activity. As shown in Figures 10 and S14b−d, upon gradually increasing PGA levels (0−10 U/mL), fluorescence intensities at 660 nm of the decaged B-COOH-FR increased more abruptly. Specifically, the enzyme-induced conversion was finished within 60 min in the presence of 10 U/mL PGA. Together, the above results indicated that meso-carboxylate-substituted BODIPYs could be modularly derivatized to generate variable highly sensitive fluorogenic probes for biomolecules and enzymes by facilely replacing the trigger moiety of interest (Figure 1 and Scheme S1).

mechanism and the versatility of the synthetic strategy, we envisage that the concept disclosed herein could be exploited for customization of diverse bioprobes in both emission wavelength and sensing analytes for biological research and clinical needs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b05920. Experimental details, imaging methods, supplementary UV/vis and fluorescence spectra, HPLC chromatograms, and NMR spectra, including Figures S1−S40 and Scheme S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Changhua Li: 0000-0003-4688-7551 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from Nankai University, National Natural Science Foundation of China (Grant No. 51673101 and 81601590), and Natural Science Foundation of Tianjin of China (Grant No. 15JCZDJC65800) is gratefully acknowledged.

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CONCLUSION With the aim of developing a general strategy toward highly fluorogenic bioprobes across the visible spectrum for diverse analytes, we designed and successfully synthesized a set of BODIPY-based bioprobes with the meso-carboxyl moieties caged by a stimuli-responsive, self-immolative linker via an ester bond. As proof-of-concept examples, we established that the meso-ester moieties can be converted to meso-carboxylates upon stimuli (H2O2, H2S, PGA), and the conversion induces a significant reduction in the electron-withdrawing capabilities of the meso-substituents of BODIPYs, leading to the dramatic enhancement of fluorescence emission. Moreover, the fluorogenic mechanism has been successfully applied in various BODIPYs emitting throughout the visible spectrum (509, 585, and 660 nm). Further application to the visualization of endogenous H2O2 in mitochondria was also validated for P-HP-FR-tpp in living cells. Given the generality of the highly fluorogenic

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