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Development of large Stokes shift, near-infrared fluorescence probe for rapid and bioorthogonal imaging of nitroxyl (HNO) in living cells
Author’s Accepted Manuscript Development of Large Stokes Shift, Near-infrared Fluorescence Probe for Rapid and Bioorthogonal Imaging of Nitroxyl (HNO) in Living Cells Chun-Xia Zhang, Mei-Hao Xiang, Xian-Jun Liu, Fenglin Wang, Ru-Qin Yu, Jian-Hui Jiang www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)30975-5 https://doi.org/10.1016/j.talanta.2018.09.062 TAL19076
To appear in: Talanta Received date: 8 August 2018 Revised date: 12 September 2018 Accepted date: 18 September 2018 Cite this article as: Chun-Xia Zhang, Mei-Hao Xiang, Xian-Jun Liu, Fenglin Wang, Ru-Qin Yu and Jian-Hui Jiang, Development of Large Stokes Shift, Nearinfrared Fluorescence Probe for Rapid and Bioorthogonal Imaging of Nitroxyl (HNO) in Living Cells, Talanta, https://doi.org/10.1016/j.talanta.2018.09.062 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development
of
Large
Stokes
Shift,
Near-infrared
Fluorescence Probe for Rapid and Bioorthogonal Imaging of Nitroxyl (HNO) in Living Cells Chun-Xia Zhang, Mei-Hao Xiang, Xian-Jun Liu, Fenglin Wang*, Ru-Qin Yu, Jian-Hui Jiang* Institute of Chemical Biology & Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing & Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, P. R. China
*
To whom correspondence should be addressed: E-mail: [email protected]; [email protected].
Abstract: Nitroxyl (HNO), as an electron reduced and protonated form of nitric oxide, is emerging as a potential diagnostic and therapeutic biomarker. It is still of great interest to develop probes of desirable properties to study its biological functions. Here we develop a near infrared fluorescence probe for detecting and visualizing exogenous and endogenous HNO in living cells. The probe is designed by coupling a HNO-responsive moiety, diphenylphosphinobenzoyl group, with a near infrared fluorophore with large of Stokes shift via an ester linker. The probe was initially nonfluorescent. HNO-catalysed oxidation reaction generates an aza-ylide, which
intramolecularly attacks the carbonyl carbon, liberating the initial fluorophore with activated fluorescence signals. The probe is proportional to the concentrations of HNO in the range of 2.0 μM -80 μM with a limit of detection of 0.05 μM. Furthermore, the probe also exhibits high selectivity and fast response (reaching plateau within 600 s) towards HNO in vitro. Moreover, imaging studies reveal that the probe is capable of detecting exogenous HNO with dose-dependent fluorescence signals. Its ability to image endogenous HNO without or with induction is also demonstrated in living cells. This turn-on fluorescence probe provides a useful tool for studying HNO in living cells.
Graphical abstract
A near Infrared fluorescent probe with large stokes shift, high sensitivity and fast response is designed for HNO.
Keywords: near-infrared fluorescence probe, large Stokes shift, exogenous HNO, endogenous HNO 1. Introduction Nitroxyl (HNO), a member of the reactive nitrogen species (RNS) obtained via one-electron reduction and protonation of nitric oxide (NO), is known as an important signaling molecule in many physiological processes [1，2 ]. Earlier studies revealed that HNO had emerged as a potential diagnostic and therapeutic biomarker for different diseases such as cardiovascular disorders [3, 4]. Despite of the advances about the biological roles of HNO, many physiological and pathological functions of HNO in living cell context still remain elusive. Development of fluorescence probes for rapid and sensitive detection of HNO in cellular studies has become a major interest in chemical biology. Most of reported fluorescent probes for HNO are designed based on the reduction of Cu (II) to Cu(I) [5-7], or nitroxide to hydroxylamine by HNO [8-10]. These probes are subjected to the risk of interferences by biological reductants such as ascorbic acid and glutathione (GSH) [11-13]. More recently, a thiol-based probe which could be oxidized by HNO, triggering the release of fluorophore was developed for HNO detection [14]. However, this design may have issues of stability and complicated synthetic procedures. Recent development has
demonstrated that HNO can react highly selectively with phosphines at a high reaction rate constant (up to 9×105 M-1 s-1) to generate an aza-ylide, which subsequently undergoes intramolecular cyclization reaction and release a hydroxyl- and amide-containing compound [15, 16]. This Staudinger ligation reaction is bioorthogonal, since phosphines are usually reactively inert toward other endogenous biological substances, which catalyzes the increasing utility of this mechanism for developing of fluorescence probes for HNO imaging. Until recent, a few activatable fluorescent probes were designed by deriving various fluorophores with this HNO-responsive bioorthogonal moiety [17-20]. To avoid autofluorescence background and minimize photodamage in biological systems, several near infrared fluorescence (NIR) probes were also developed, however, majority of them had short Stokes shifts and sluggish response rates, which may suffer from self-quenching and preclude fast detection and imaging of transient HNO in living cells [21-26]. Thus, the development of NIR probes with large Stokes shifts for detection and imaging of HNO concentrations in biological systems is still of great interest. Here we report a NIR fluorescent turn-on probe (DCX-TPP) for rapid and sensitive monitoring of exogenous and endogenous HNO level in living systems. DCX-OH, consisted of a dicyanomethylene-4H-chromene which is conjugated to a xanthene moiety, is chosen as the fluorophore because of its NIR infrared emission and large Stokes shift. DCX-TPP is obtained by masking the phenolic hydroxyl group of DCX-OH with a diphenylphosphinobenzoyl group, a bioorthogonal HNO-recognition moiety. The probe was initially nonfluorescent due to esterification of the hydroxyl group, leading to an
interrupted push-pull structure. Nonetheless, HNO could react with phosphine moiety, forming an aza-ylide, which intramolecularly attacked the carbonyl carbon, releasing the initial fluorophore with activated fluorescence signal, and 2-(diphenylphosphonyl) benzamide via intramolecular nucleophilic attack of the carbonyl carbon. DCX-TPP was responsive towards HNO with fast response, high selectivity and high sensitivity in vitro. The probe was also introduced to image exogenous and endogenous HNO activity in living cells with high sensitivity, high specificity and dose-dependent fluorescence signals. We believe our novel probe would provide a useful tool for detection and visualizing HNO in living cells, holding great potential in elucidating its biological functions. 2. Experimental section 2.1. Reagents and Apparatus All reagents were of analytical grade and were purchased from J&K Chemical (Beijing, China). Organic solvents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and used without further purification. Angeli’s salt (Na2N2O3, AS) was purchased from Cayman Chemical. 1H and
13
C NMR spectra were recorded on a Bruker DRX-400
spectrometer using CDCl3 and DMSO-d6 as solvent. The chemical shifts (δ) were reported in ppm (relative to TMS as internal standard) and coupling constants (J) were given in Hz. Signal multiplicities were represented by s (singlet), d (doublet), t (triplet) and m (multiplet). Ultraviolet spectrum was carried out on Shimadzu UV1800 (Japan). Fluorescence spectra were collected on spectrofluorometer FS5 (Edinburgh, UK). Electrospray ionization mass
spectrometry (ESI-MS) was determined with Finnigan LCQ Advantage MAX (Thermo
Finnigan).
Analytical
thin-layer
chromatography
(TLC)
was
performed on silica gel aluminum sheets with F-254 indicator. The column chromatography was conducted using 200-300 mesh SiO2 (Qingdao Ocean Chemical Products). 2.2. Synthesis of 2-bromocyclohex-1-ene-1-carbaldehyde (1) PBr3 (12.4 mL, 131 mmol) was slowly added to a solution of dimethylformamide (DMF, 11.2mL, 145 mmol) and CH2Cl2 (50 mL) at 0 °C and stirred for 45 min. Then cyclohexanone (10 mL, 96.8 mmol) was added and the mixture was stirred at room temperature for 12 h. The resulting red solution was then poured onto ice and NaHCO3 powder was slowly added until neutral. The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (500 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to provide a yellow oil (8.0 g) which was directly used in the next step. 2.3. Synthesis of 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde (2) To a solution of Compound 1 (0.41 g, 2.16 mmol) dissolved in DMF (8 mL), 2-hydroxy-4-methoxybenzaldehyde (0.27 g, 1.80 mmol) and Cs2CO3 (1.8 g, 5.4 mmol) were added at room temperature. The solution was stirred for 12 h at room temperature and an intense yellow spot appeared on the TLC plate. The precipitates were then filtered and the filtrate was concentrated. The residue was dissolved in CH2Cl2 (60 mL) and washed with water, brine and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using petroleum ether/ethyl acetate 4:1 (v/v) as the
eluent to yield Compound 2 as a yellow solid (0.35 g, 77%). 1H NMR (400 MHz, CDCl3) δ (ppm): 10.26 (s, 1H, Ar-H), 7.05(d, J = 8.4 Hz, Ar-H), 6.61-6.64 (3H, m, Ar-H), 3.8 (3H, s, CH2), 2.42-2.54 (2H, m, CH), 2.39-2.42 (2H, m, CH2), 1.65-1.71 (2H, m, CH2).
13
C NMR (100 MHz, CDCl3) δ (ppm):
187.49, 161.32, 160.79, 153.27, 127.46, 126.93, 126.42, 114.60, 112.43, 110.79, 100.43, 55.62, 29.86, 21.48, 20.35. MS m/z: 243.2 [M+H]+. 2.4. Synthesis of 2-bromocyclohex-1-ene-1-carbaldehyde (3) NaH (1.06 g, 44.1 mmol) was dissolved in anhydrous THF (10 ml) and it was slowly added to a solution of 2-hydroxyacetophenone (1.33 ml, 11.0 mmol) and ethyl acetate (2.48 g, 27.6 mmol) in anhydrous THF (10 ml) under stirring conditions. After stirring at 70 °C for 1 h, ice (100 g) was slowly add to the reaction solution, followed by the adjustment of pH to neutral. The aqueous solution was extract with CH2Cl2 and the organic layers were dried over Na2SO4, and concentrated in vacuum to afford compound 3 as a brown solid (0.9 g,46%), which was directly used in the following reactions. 2.5. Synthesis of 1-(2-hydroxyphenyl)butane-1,3-dione(4) Compound 3 (0.31 g, 1.7 mmol) and 98% H2SO4 (0.2 mL) were added to AcOH (4 mL), then the solution was refluxed for 0.5 h. Then it was poured into ice water and the aqueous solution was extract with CH2Cl2. The organic layer was dried with anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by column chromatography using ethyl acetate/petroleum ether 1:6 (v/v) as the eluent to yield compound 4 as white solid (0.19 g, 69.8 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.18-8.20 (1H, m, Ar-H), 7.62-7.67 (1H, m, Ar-H), 7.36-7.44 (2H, m, Ar-H), 6.19 (1H, s, CH=C), 2.40 (3H, s, CH3).
13
C
NMR (100 MHz, CDCl3) δ (ppm): 178.28, 166.21, 156.47, 133.45, 125.64,124.92, 123.55, 117.79, 110.57, 20.63. MS m/z: 161.16 [M+H]+. 2.6. Synthesis of 2-(2-methyl-4H-chromen-4-ylidene)malononitrile (5) Compound 4 (0.8 g, 5.0 mmol) and malononitrile (0.66 g, 2.5 mmol) were dissolved in acetic anhydride (10 mL), and the mixture was refluxed for 12 h. Then, the solvent was evaporated under reduced pressure and water (40 mL) was added to the residue, which was refluxed for another 0.5 h, followed by extraction with CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated in vacuum. The residue was purified by column chromatography using ethyl acetate/petroleum ether 1:4 (v/v) as the eluent to yield compound 5 as an orange solid (0.5 g, 46 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.88-8.90 (1H, m, Ar-H), 7.72-7.76 (1H, m, Ar-H), 7.43-7.50 (2H, m, Ar-H), 6.71 (1H, s, Ar-H), 2.46 (3H, s, CH3). 13C NMR (100 MHz, CDCl3) δ (ppm): 161.85, 153.26, 152.88, 134.66, 126.04, 125.70, 118.71, 117.49, 116.62, 115.49, 105.45, 20.53. MS m/z: 207.02 [M+H]+. 2.7. Synthesis of (E)-2-(3-(2-(6-methoxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)4H-chromen-4-ylidene)malononitrile (6) To a solution of Compound 5 (1 mmol) in dry CH3CN (20 mL), Compound 2 (0.29 g, 1.2 mmol) and piperidine (0.1 mL) were added. Then, the resulting solution was heated to reflux under nitrogen atmosphere for 24 h. After removal of solvent, the residues were purified by alumina column chromatography using petroleum ether/dichloromethane (1:1, v/v) as eluent to afford Compound 6 as a deep blue solid (0.41 g,77%).1H NMR (400 MHz, CDCl3) δ (ppm): 8.93 (1H, d, J = 8.3 Hz), 8.16 (1H, d, J = 17.4 Hz), 7.71-7.75
(1H, m, Ar-H), 7.54-7.59 (1H, m, Ar-H), 7.42-7.45 (1H, m, Ar-H), 7.05 (1H, d, J = 8.4 Hz) 6.79 (1H, s, Ar-H), 6.71 (1H, s, Ar-H), 6.65 (1H, d, J = 8.48 Hz), 6.52 (1H, s, CH=H), 6.10 (1H, d, J = 15.4 Hz), 3.90 (3H, s, CH2). 2.57-2.60 (2H, m, CH2), 2.49-2.52 (2H, m, CH2), 1.85-1.88 (2H, m, CH2). 13C NMR (100 MHz, CDCl3) δ (ppm): 161.11, 159.51, 154.04, 152.90, 152.66, 152.47, 133.88, 127.61, 127.11, 125.93, 124.56, 118.69, 116.69, 115.46, 113.34, 110.59, 109.91, 105.48, 100.87, 55.68, 29.71, 24.59, 20.77. MS m/z: 432.0 [M+H]+. 2.8. Synthesis of DCX-OH BBr3 (0.94 ml, 10 mmol) was added to a solution of Compound 6 (0.5 mmol) in dry CH2Cl2 (10 mL) at 0 °C and the solution was stirred at room temperature for 16 h. Then saturated NaHCO3 solution (30 mL) was added to quench the reaction, followed by extraction of the aqueous layer with CH 2Cl2. The organic layer was dried over Na2SO4 and concentrated in vacuum. The crude product was directly used in the next reaction without further purification. MS m/z: 417.0 [M-H]-. 2.9. Synthesis of DCX-TPP To a solution of 2-(diphenylphosphino) benzoic acid (0.10 g, 0.32 mmol) in dry CH2Cl2, dicyclohexylcarbodiimide（DCC）(0.08 g, 0.39 mmol), DCX-OH (0.15 g, 0.35 mmol) and 4-dimethylaminopyridine（DMAP) (0.02 g, 0.16 mmol) were added under N2 atmosphere. Then the solution was stirred at room temperature for 6 h and concentrated under vacuum，the crude product was purified by silica gel column chromatography using dichloromethane/petroleum ether 1:2 (v/v) as the eluent to yield Compound 8 as solid (0.5 g, 46 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.94 (1H, d, J = 8.3 Hz), 8.31 (1H, d, Ar-H),
8.10 (1H, m, J = 16.1 Hz), 7.69-7.73(1H, m, Ar-H), 7.64-7.66 (2H, m, Ar-H), 7.49-7.55(1H, m, Ar-H) 7.33-7.39 (9H, m, Ar-H), 7.06 (1H, s, J = 8.3 Hz), 6.86 (1H, s, Ar-H), 6.79 (1H, s, Ar-H), 6.65 (1H, d, J = 8.1 Hz), 6.49 (1H, s, Ar-H), 6.16 (1H, s, Ar-H), 5.33 (1H, s, Ar-H) 2.54 (4H, m, CH2), 1.88 (2H, m, CH2). MS m/z: 705.2 [M-H]2.10. General procedures for spectra measurement All UV–vis and fluorescence measurements were carried out in PBS buffer (10 mM, pH 7.4) solution, containing 50 % DMF. In a 1 mL tube, 40 μL DCX-TPP (100 μM) and 160 μL DMF were mixed, and then an appropriate volume of HNO (10 mM) was added. The final solution volume was adjusted to 0.4 mL with PBS buffer and incubated at 37 °C for 30 min unless otherwise indicated. Fluorescence spectra were recorded in the range from 600 to 850 nm unless otherwise indicated, with an excitation wavelength of 580 nm. The excitation and emission slit widths were both 5 nm. For studying the real time response of DCX-TPP towards HNO. DCX-TPP and HNO were rapidly mixed. Then, the fluorescence intensity at 740 nm was recorded in real time for 1400 s at 37 °C with an excitation wavelength of 580 nm on an F-7000 fluorescence spectrophotometer (Hitachi, Japan), using slit widths of 5 nm for both excitation and emission. 2.11. HPLC analysis The HPLC chromatograms of DCX-TPP, DCX-OH, and the reaction products of DCX-TPP and HNO were performed on a system with a C18 column (150 nm × 4.6 mm, 5 μm) and the conditions were as follows:
methanol/H2O = 95/5 (v/v); flow rate: 0.8 mL/min; detection wavelength: 580 nm. 2.12. Cytotoxicity assays The cytotoxicity of DCX-TPP against HeLa cells and Raw264.7 was studied using a standard methyl thiazolyltetrazolium (MTT) assay. The cells were seeded into a 96-well plate at 5×104 cells /well in complete medium, containing DMEM and 10% fetal bovine serum (FBS). Then, the cells were incubated for 24 h at 37 °C under 5 % CO2. Afterwards, the medium was removed and washed with PBS for three times. Cells were then incubated with fresh medium (serum-free DMEM) containing various concentrations of DCX-TPP (0-20 μM) for 24 h. 20 μL of MTT solution (5.0 mg/mL) was then added to each well. After 4 h, the supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. Cell viability was determined by measuring absorbance at 450 nm with a microplate reader. 2.13. Fluorescence imaging in living cells HeLa cells and RAW 264.7 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in an incubator, containing 5% wt/vol CO2. After 24 h, RAW 264.7 cells were washed with PBS and treated with DCX -TPP (10 μM) for 30 min. RAW 264.7 Cells were subsequently washed twice with PBS and further treated with different concentrations of AS (0, 10, 50 and 100 μM) for 30 min. As for HeLa cells, the cells were treated with sodium nitroprusside (SNP, 2 mM) for 8 h, followed by addition of NaASc (2 mM) and DCX-TPP (10 μM) and 30-min incubation. Cell imaging was performed after the cells
were washed twice with PBS. Fluorescence signals collected at 663-738 nm with an excitation wavelength of 560 nm. 3. Results and discussion 3.1. Design rationale and probe synthesis We aimed to design a NIR fluorescence probe (DCX-TPP) with large Stokes shift to detect HNO in vitro and vivo. DCX-OH, comprised of dicyanomethylene-4H-chromene as the electron-withdrawing group and a hydroxyl xanthene moiety as the electron-donating group, was chosen as the fluorophore owing to its large Stokes shift and near infrared emission, which could potentially provide high sensitivity and minimal autofluorescence background in biological matrix [27]. Then, DCX-TPP was obtained by deriving DCX-OH with a bioorthogonal diphenylphosphinobenzoyl moiety as the recognition group for HNO. We envision that the esterification of the hydroxyl group by the HNO-responsive would make DCX-TPP non-emissive and the HNO-induced cascade reactions would regenerate the hydroxyl groups of the fluorophores with dramatic enhancement in fluorescence. Motivated by this rationale, DCX-TPP was prepared in several steps with good yields. First, DCX-OH was prepared by conjugating a dicyanomethylene-4H-chromene derivative with a xanthene derivative. Then, DCX-OH was derived with the diphenylphosphinobenzoyl moiety via standard Steglich esterification reactions. The detailed synthetic procedures of the probe were outlined in Scheme 2. 3.2. Photophysical properties characterization The photophysical properties of the probe were first investigated with both
absorption
and
fluorescence
spectroscopy.
DCX-TPP
was
initially
non-emissive with an absorption maximum at ~540 nm. Upon reaction with HNO, using Angeli’s salt (Na2N2O3, AS) as an HNO donor, there was a remarkable fluorescence enhancement at 740 nm (21-fold) and the absorption maximum was bathochromically shifted to 580 nm (Figure 1). The red-shift in absorption and dramatic fluorescence enhancement were attributed to HNO-triggered cascade reactions, releasing the initial fluorophore, DCX-OH, with distinct intramolecular charge transfer (ICT). These results suggested that our probe was responsive towards HNO. Having demonstrated that DCX-TPP was responsive towards HNO, its sensitivity was then explored by measuring the fluorescence spectra in the presence of various concentrations of AS as shown in Figure 2a. The fluorescence responses of DCX-TPP exhibited a dynamic correlation to the concentrations of HNO (0-100 μM). Good linearity was obtained between the fluorescence intensity at 740 nm and the HNO concentrations in the range of 2.0 μM -80 μM (Figure 2b). The limit of detection was calculated to be 0.05 μM, which is comparable to the existing near infrared fluorescence probes for HNO (Table S1). Then, the response rate of DCX-TPP towards HNO was investigated by recording the fluorescence intensity increments of DCX-TPP (10 μM) in different concentrations of HNO in real time. Figure 3a showed that there was a drastic enhancement in fluorescence intensities in the presence of either 20 μM HNO or 100 μM HNO, which reached the plateaux at 400 s and 600 s, respectively. The pseudo-first order rate constant was calculated to
be 0.00801 s-1 (Figure 3b). Moreover, similar response rates were obtained when 5 μM DCX-TPP was reacted with 10 and 50 μM HNO (Figure 3c). The pseudo-first order rate constant was calculated to be 0.0077 s-1 (Figure 3d). These results suggested that our probe could response towards HNO with rapid kinetics. Taken together, these results demonstrated that our probe could respond to HNO with high sensitivity and fast response in vitro. 3.3. Reaction mechanism The reaction products of DCX-TPP with HNO were analyzed with ESI-MS and HPLC to investigate the response mechanism. DCX-TPP and DCX-OH had retention times of 16.368 min and 7.402 min, respectively (Figure S1). Upon reaction with HNO, the peak at 16.346 min representing DCX-TPP decreased significantly and was accompanied by the appearance of a new peak at 7.389 min, indicating the release of DCX-OH. Moreover, ESI-MS analyses further verified the generation of DCX-OH as a reaction product. The ESI-MS spectrum from the reaction solution of DCX-TPP with AS showed a major peak at m/z=417.0 [M-H]-, corresponding to DCX-OH ([M-H]- 417.1) (Figure S2), which clearly verified that the reaction of DCX-TPP with HNO produced DCX-OH. These results demonstrated that the reaction of DCX-TPP with HNO was indeed attributed to the HNO-catalyzed oxidation reaction as depicted in Scheme 1. 3.4. Selectivity and pH dependence of DCX-TPP The selectivity of DCX-TPP to HNO and the pH dependence of DCX-TPP were then investigated to demonstrate its potential application in biological conditions.
First, the selectivity test was performed by incubating DCX-TPP (10 μM) with other biologically relevant species (100 μM of AS, ONOO-, .O2-, .NO-, H2O2, ClO-, and NO2-, 1 mM of Hcy, S2-, Fe3+, Na+ and K+, 10 mM of GSH, Cys). No obvious fluorescence intensity increase at 740 nm was observed in the presence of the tested biological relevant species. On the contrary, the addition of AS caused a remarkable enhancement of the fluorescence intensity at 740 nm, attributed to the biorthogonality of the HNO-responsive diphenylphosphinobenzoyl group (Figure 4a). Then, the effect of pH on the fluorescence intensity of DCX-TPP was also investigated. As can be seen from Figure 4b, there were no fluorescence changes of DCX-TPP as pH was varied from 5.0 to 10.0, indicating that the ester group was stable in this pH range. Upon addition of 100 μM HNO, the fluorescence intensity of DCX-TPP at 740 nm remarkably increased in the pH range of 6.0-8.0. The relative smaller increase in fluorescence intensity when the pH was smaller than 6.0 might be due to the decomposition of AS, resulting in decreased generation of HNO. Taken together, these results indicated DCX-TPP could be used for detecting HNO in physiological conditions with high selectivity. 3.5. Fluorescence imaging of HNO in living cells Encouraged by the superior advantages of DCX-TPP including excellent sensitivity, rapid response and high selectivity, we then investigated whether DCX-TPP could be applied in imaging of HNO in living cells. First, the potential cytotoxicity of DCX-TPP towards RAW 264.7 and HeLa cells was evaluated with a standard MTT assay. As is shown in Figure 5, the cells had over 90% viability upon
treatment with various concentrations of DCX-TPP (10, 20, 30, 40 and 50 μM) at 37 °C for 24 h, indicating good biocompatibility and low cytotoxicity of DCX-TPP. Then, the ability of DCX-TPP to detect exogenous HNO was tested by incubating RAW 264.7 cells with DCX-TPP for 30 min, followed by treating with different concentrations of HNO (10, 50 and 100 μM). Both bright field and confocal fluorescence images were then recorded. There were no fluorescence signals for cells incubated with DCX-TPP, indicating that RAW 264.7 cells barely generated HNO, consistent with previous reports [28-30]. In contrast, there was gradual increase in fluorescence signals when increasing concentrations of exogenous HNO were added. Therefore, these results demonstrated that DCX-TPP was cell membrane permeable and was capable of detecting exogenous HNO in living cells. The capability of DCX-TPP to detect and image endogenous HNO in normal culturing or induced conditions were further investigated. HeLa cells, with a low level of endogenous HNO, were used to study whether DCX-TPP possessed potential application for monitoring endogenous HNO. As shown in Figure 6, there were weak but evident fluorescence signals when HeLa cells were only treated with DCX-TPP (10 μM). Furthermore, as it was reported that the reaction of L-ascorbate with NO in biological media could form intracellular HNO, we then tried to test the ability of DCX-TPP towards HNO in HeLa cells under induced conditions [30-32]. HeLa cells were pretreated with SNP (2 mM), a commercial NO donor for 8 h, then NaASc (2 mM) and DCX-TPP (10 μM) were added. Fluorescence images were obtained after another incubation of 30 min. As shown in Figure 7, HeLa cells displayed bright fluorescence signals. These results demonstrated that our probe could not only detect
low level of endogenous HNO but also could visualize HNO in living cells under induced conditions. 4. Conclusions In summary, we have developed a novel NIR fluorescent turn-on probe for the detection and imaging of exogenous and endogenous HNO in living cells. The probe was composed of a NIR fluorophore with a large Stokes shift of 160 nm, which was derived with a diphenylphosphinobenzoyl moiety as the HNO-responsive unit. The probe was non-emissive in the absence of HNO. However, there was a rapid increase (within 600 s) in fluorescence upon addition of HNO. The probe was demonstrated to exhibit excellent sensitivity with a limit of detection of 0.05 μM, high selectivity towards HNO and great stability under physiological conditions. Furthermore, its ability to detect and visualize exogenous HNO and endogenous HNO without or with induction was demonstrated in different living cells. We believe the combined advantages of our probe would provide an invaluable tool for studying the biological functions of HNO in living cells. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants 21527810, 91753107, and 21205034).
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Mater. Chem. B 4 (2016) 1263−1269. [27] Y. Qi ,Y. Huang, B. Li，F. Zeng,S. Wu, Real-time monitoring of endogenous cysteine levels in vivo by near-Infrared turn-on fluorescent probe with large stokes shift, Anal. Chem. 90 (2018) 1014–1020 [28] C. Liu, Y. Wang, C. Tang, et al, A reductant-resistant ratiometric, colorimetric and far-red fluorescent probe for rapid and ultrasensitive detection of nitroxyl, J. Mater. Chem. B 5 (2017) 3557--3564. [29] F. Ali, S. Sreedharan, A. H. Ashoka, et al, A super resolution probe to monitor HNO levels in the endoplasmic reticulum of cells, Anal. Chem. 89 (2017) 12087– 12093 [30] S. A. Suarez, N. I. Neuman, M. Muñoz, et al. Nitric oxide is reduced to HNO by proton-coupled nucleophilic attack by ascorbate, tyrosine, and other alcohols. A new route to HNO in biological media, J. Am. Chem. Soc. 137 (2015) 4720–4727 [31] K. Hamilton and A. MacKenzie, Vasc, Aldehyde dehydrogenase 2 plays a role in the bioactivation of nitroglycerin in humans, Pharmacol. 71 (2015) 208–214 [32] H. Li, Q. Yao, F. Xu, et al. Recognition of exogenous and endogenous nitroxyl in living cells via a two-photon fluorescent probe, Anal. Chem. 90 (2018) 4641−4648
Scheme 1. Sensing mechanism of DCX-TPP for HNO detection
Scheme 2. Synthesis of DCX-TPP
Figure 1. (a) Fluorescence spectra of DCX-TPP (10 μM, black line), DCX-TPP (10 μM, red line) reacted with HNO (100 μM) at 37 oC for 30 min; (b) UV-vis absorption spectra of DCX-TPP before (20 μM, black line) and after (20 μM, red line) reaction with HNO (200 μM) at 37 oC for 30 min.
Figure 2. (a) The fluorescence emission spectra of DCX-TPP (10 μM) in the presence of different concentrations of HNO (0-100 μM); (b) Linear fitting of fluorescence intensity (F) toward the concentrations (C) of HNO from 0 to 150 μM. λex/em = 580/740 nm.
Figure 3. (a) Time dependent fluorescence emission intensity (λex/em = 580/740) of DCX-TPP (10 μM) upon addition of different concentrations of HNO, 100 μM (blue line), 20 μM (red line), and without HNO (black line); (b) Pseudo first-order kinetic plot of the reaction of DCX-TPP (10 μM) with HNO (100 μM), slope = 0.0080 s-1; (c) Time dependent fluorescence emission intensity (λex/em = 580/740) of DCX-TPP (5 μM) upon addition of different concentrations of HNO concentration, 50 μM (pink line), 10 μM (blue line), and without HNO (black line) ; (d) Pseudo first-order kinetic plot of the reaction of DCX-TPP (5 μM) with HNO (50 μM), slope = 0.0077 s-1.
Figure 4. (a) Fluorescence responses of DCX-TPP (10 μM) towards different species: (1) 100 μM ONOO-; (2) 100 μM .O2-; (3) 100 μM NO-; (4) 100 μM H2O2; (5) 100 μM ClO-; (6) 1 mM Hcy; (7) 1 mM S2-; (8) 1 mM Fe3+; (9) 1 mM Na+; (10) 1 mM K+; (11) 100 μM .NO2-; (12) 10 mM GSH; (13)10 mM Cys; (14) 100 μM HNO; (b) Effect of pH value on the fluorescence intensity of DCX-TPP (10 μM) and after being reacted with HNO (100 μM) at 37 oC for 60 min. λex/em = 580/740 nm
Figure 5. Effects of DCX-TPP (0-50 μM) on the viability of HeLa cells and Raw 264.7 cells. The viability of cells without DCX-TPP is defined as 100%. The results are the means ± SD of three experiments.
Figure 6. Images of RAW264.7 cells: (a-e) bright field; (f) Fluorescence image of cells without any treatments. (g-j) Fluorescence images of cells pretreated with 10 μΜ DCX-TPP, followed by addition of different concentrations of exogenous HNO (0, 10 μΜ, 50 μΜ, and 100 μΜ). (k-o) Overlay images of bright field and fluorescence field. λex = 560 nm, collection channel: 663-738 nm, scale bar = 50 μm.
Figure 7. Images of HeLa cells: (a-c) control group without any treatments; (d-f) cells only treated with10 μΜ DCX-TPP; (g−i) cells were pretreated with 2 mM SNP and then incubated with 2 mM NaASc and 10 μΜ DCX-TPP; λex = 560 nm, collection channel: 663-738 nm, scale bar = 50 μm.
Highlights (1) The probe DCX-TPP is designed by masking a fluorophore with large Stokes shift and near infrared emission with a bioorthogonal HNO-responsive moiety. (2) The probe DCX-TPP exhibits high sensitivity, fast response and excellent selectivity towards HNO in vitro. (3) The probe DCX-TPP has been successfully introduced to image both exogenous and endogenous HNO in living cells.
PII: DOI: Reference:
S0039-9140(18)30975-5 https://doi.org/10.1016/j.talanta.2018.09.062 TAL19076
To appear in: Talanta Received date: 8 August 2018 Revised date: 12 September 2018 Accepted date: 18 September 2018 Cite this article as: Chun-Xia Zhang, Mei-Hao Xiang, Xian-Jun Liu, Fenglin Wang, Ru-Qin Yu and Jian-Hui Jiang, Development of Large Stokes Shift, Nearinfrared Fluorescence Probe for Rapid and Bioorthogonal Imaging of Nitroxyl (HNO) in Living Cells, Talanta, https://doi.org/10.1016/j.talanta.2018.09.062 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development
of
Large
Stokes
Shift,
Near-infrared
Fluorescence Probe for Rapid and Bioorthogonal Imaging of Nitroxyl (HNO) in Living Cells Chun-Xia Zhang, Mei-Hao Xiang, Xian-Jun Liu, Fenglin Wang*, Ru-Qin Yu, Jian-Hui Jiang* Institute of Chemical Biology & Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing & Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, P. R. China
*
To whom correspondence should be addressed: E-mail: [email protected]; [email protected].
Abstract: Nitroxyl (HNO), as an electron reduced and protonated form of nitric oxide, is emerging as a potential diagnostic and therapeutic biomarker. It is still of great interest to develop probes of desirable properties to study its biological functions. Here we develop a near infrared fluorescence probe for detecting and visualizing exogenous and endogenous HNO in living cells. The probe is designed by coupling a HNO-responsive moiety, diphenylphosphinobenzoyl group, with a near infrared fluorophore with large of Stokes shift via an ester linker. The probe was initially nonfluorescent. HNO-catalysed oxidation reaction generates an aza-ylide, which
intramolecularly attacks the carbonyl carbon, liberating the initial fluorophore with activated fluorescence signals. The probe is proportional to the concentrations of HNO in the range of 2.0 μM -80 μM with a limit of detection of 0.05 μM. Furthermore, the probe also exhibits high selectivity and fast response (reaching plateau within 600 s) towards HNO in vitro. Moreover, imaging studies reveal that the probe is capable of detecting exogenous HNO with dose-dependent fluorescence signals. Its ability to image endogenous HNO without or with induction is also demonstrated in living cells. This turn-on fluorescence probe provides a useful tool for studying HNO in living cells.
Graphical abstract
A near Infrared fluorescent probe with large stokes shift, high sensitivity and fast response is designed for HNO.
Keywords: near-infrared fluorescence probe, large Stokes shift, exogenous HNO, endogenous HNO 1. Introduction Nitroxyl (HNO), a member of the reactive nitrogen species (RNS) obtained via one-electron reduction and protonation of nitric oxide (NO), is known as an important signaling molecule in many physiological processes [1，2 ]. Earlier studies revealed that HNO had emerged as a potential diagnostic and therapeutic biomarker for different diseases such as cardiovascular disorders [3, 4]. Despite of the advances about the biological roles of HNO, many physiological and pathological functions of HNO in living cell context still remain elusive. Development of fluorescence probes for rapid and sensitive detection of HNO in cellular studies has become a major interest in chemical biology. Most of reported fluorescent probes for HNO are designed based on the reduction of Cu (II) to Cu(I) [5-7], or nitroxide to hydroxylamine by HNO [8-10]. These probes are subjected to the risk of interferences by biological reductants such as ascorbic acid and glutathione (GSH) [11-13]. More recently, a thiol-based probe which could be oxidized by HNO, triggering the release of fluorophore was developed for HNO detection [14]. However, this design may have issues of stability and complicated synthetic procedures. Recent development has
demonstrated that HNO can react highly selectively with phosphines at a high reaction rate constant (up to 9×105 M-1 s-1) to generate an aza-ylide, which subsequently undergoes intramolecular cyclization reaction and release a hydroxyl- and amide-containing compound [15, 16]. This Staudinger ligation reaction is bioorthogonal, since phosphines are usually reactively inert toward other endogenous biological substances, which catalyzes the increasing utility of this mechanism for developing of fluorescence probes for HNO imaging. Until recent, a few activatable fluorescent probes were designed by deriving various fluorophores with this HNO-responsive bioorthogonal moiety [17-20]. To avoid autofluorescence background and minimize photodamage in biological systems, several near infrared fluorescence (NIR) probes were also developed, however, majority of them had short Stokes shifts and sluggish response rates, which may suffer from self-quenching and preclude fast detection and imaging of transient HNO in living cells [21-26]. Thus, the development of NIR probes with large Stokes shifts for detection and imaging of HNO concentrations in biological systems is still of great interest. Here we report a NIR fluorescent turn-on probe (DCX-TPP) for rapid and sensitive monitoring of exogenous and endogenous HNO level in living systems. DCX-OH, consisted of a dicyanomethylene-4H-chromene which is conjugated to a xanthene moiety, is chosen as the fluorophore because of its NIR infrared emission and large Stokes shift. DCX-TPP is obtained by masking the phenolic hydroxyl group of DCX-OH with a diphenylphosphinobenzoyl group, a bioorthogonal HNO-recognition moiety. The probe was initially nonfluorescent due to esterification of the hydroxyl group, leading to an
interrupted push-pull structure. Nonetheless, HNO could react with phosphine moiety, forming an aza-ylide, which intramolecularly attacked the carbonyl carbon, releasing the initial fluorophore with activated fluorescence signal, and 2-(diphenylphosphonyl) benzamide via intramolecular nucleophilic attack of the carbonyl carbon. DCX-TPP was responsive towards HNO with fast response, high selectivity and high sensitivity in vitro. The probe was also introduced to image exogenous and endogenous HNO activity in living cells with high sensitivity, high specificity and dose-dependent fluorescence signals. We believe our novel probe would provide a useful tool for detection and visualizing HNO in living cells, holding great potential in elucidating its biological functions. 2. Experimental section 2.1. Reagents and Apparatus All reagents were of analytical grade and were purchased from J&K Chemical (Beijing, China). Organic solvents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and used without further purification. Angeli’s salt (Na2N2O3, AS) was purchased from Cayman Chemical. 1H and
13
C NMR spectra were recorded on a Bruker DRX-400
spectrometer using CDCl3 and DMSO-d6 as solvent. The chemical shifts (δ) were reported in ppm (relative to TMS as internal standard) and coupling constants (J) were given in Hz. Signal multiplicities were represented by s (singlet), d (doublet), t (triplet) and m (multiplet). Ultraviolet spectrum was carried out on Shimadzu UV1800 (Japan). Fluorescence spectra were collected on spectrofluorometer FS5 (Edinburgh, UK). Electrospray ionization mass
spectrometry (ESI-MS) was determined with Finnigan LCQ Advantage MAX (Thermo
Finnigan).
Analytical
thin-layer
chromatography
(TLC)
was
performed on silica gel aluminum sheets with F-254 indicator. The column chromatography was conducted using 200-300 mesh SiO2 (Qingdao Ocean Chemical Products). 2.2. Synthesis of 2-bromocyclohex-1-ene-1-carbaldehyde (1) PBr3 (12.4 mL, 131 mmol) was slowly added to a solution of dimethylformamide (DMF, 11.2mL, 145 mmol) and CH2Cl2 (50 mL) at 0 °C and stirred for 45 min. Then cyclohexanone (10 mL, 96.8 mmol) was added and the mixture was stirred at room temperature for 12 h. The resulting red solution was then poured onto ice and NaHCO3 powder was slowly added until neutral. The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (500 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to provide a yellow oil (8.0 g) which was directly used in the next step. 2.3. Synthesis of 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde (2) To a solution of Compound 1 (0.41 g, 2.16 mmol) dissolved in DMF (8 mL), 2-hydroxy-4-methoxybenzaldehyde (0.27 g, 1.80 mmol) and Cs2CO3 (1.8 g, 5.4 mmol) were added at room temperature. The solution was stirred for 12 h at room temperature and an intense yellow spot appeared on the TLC plate. The precipitates were then filtered and the filtrate was concentrated. The residue was dissolved in CH2Cl2 (60 mL) and washed with water, brine and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using petroleum ether/ethyl acetate 4:1 (v/v) as the
eluent to yield Compound 2 as a yellow solid (0.35 g, 77%). 1H NMR (400 MHz, CDCl3) δ (ppm): 10.26 (s, 1H, Ar-H), 7.05(d, J = 8.4 Hz, Ar-H), 6.61-6.64 (3H, m, Ar-H), 3.8 (3H, s, CH2), 2.42-2.54 (2H, m, CH), 2.39-2.42 (2H, m, CH2), 1.65-1.71 (2H, m, CH2).
13
C NMR (100 MHz, CDCl3) δ (ppm):
187.49, 161.32, 160.79, 153.27, 127.46, 126.93, 126.42, 114.60, 112.43, 110.79, 100.43, 55.62, 29.86, 21.48, 20.35. MS m/z: 243.2 [M+H]+. 2.4. Synthesis of 2-bromocyclohex-1-ene-1-carbaldehyde (3) NaH (1.06 g, 44.1 mmol) was dissolved in anhydrous THF (10 ml) and it was slowly added to a solution of 2-hydroxyacetophenone (1.33 ml, 11.0 mmol) and ethyl acetate (2.48 g, 27.6 mmol) in anhydrous THF (10 ml) under stirring conditions. After stirring at 70 °C for 1 h, ice (100 g) was slowly add to the reaction solution, followed by the adjustment of pH to neutral. The aqueous solution was extract with CH2Cl2 and the organic layers were dried over Na2SO4, and concentrated in vacuum to afford compound 3 as a brown solid (0.9 g,46%), which was directly used in the following reactions. 2.5. Synthesis of 1-(2-hydroxyphenyl)butane-1,3-dione(4) Compound 3 (0.31 g, 1.7 mmol) and 98% H2SO4 (0.2 mL) were added to AcOH (4 mL), then the solution was refluxed for 0.5 h. Then it was poured into ice water and the aqueous solution was extract with CH2Cl2. The organic layer was dried with anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by column chromatography using ethyl acetate/petroleum ether 1:6 (v/v) as the eluent to yield compound 4 as white solid (0.19 g, 69.8 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.18-8.20 (1H, m, Ar-H), 7.62-7.67 (1H, m, Ar-H), 7.36-7.44 (2H, m, Ar-H), 6.19 (1H, s, CH=C), 2.40 (3H, s, CH3).
13
C
NMR (100 MHz, CDCl3) δ (ppm): 178.28, 166.21, 156.47, 133.45, 125.64,124.92, 123.55, 117.79, 110.57, 20.63. MS m/z: 161.16 [M+H]+. 2.6. Synthesis of 2-(2-methyl-4H-chromen-4-ylidene)malononitrile (5) Compound 4 (0.8 g, 5.0 mmol) and malononitrile (0.66 g, 2.5 mmol) were dissolved in acetic anhydride (10 mL), and the mixture was refluxed for 12 h. Then, the solvent was evaporated under reduced pressure and water (40 mL) was added to the residue, which was refluxed for another 0.5 h, followed by extraction with CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated in vacuum. The residue was purified by column chromatography using ethyl acetate/petroleum ether 1:4 (v/v) as the eluent to yield compound 5 as an orange solid (0.5 g, 46 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.88-8.90 (1H, m, Ar-H), 7.72-7.76 (1H, m, Ar-H), 7.43-7.50 (2H, m, Ar-H), 6.71 (1H, s, Ar-H), 2.46 (3H, s, CH3). 13C NMR (100 MHz, CDCl3) δ (ppm): 161.85, 153.26, 152.88, 134.66, 126.04, 125.70, 118.71, 117.49, 116.62, 115.49, 105.45, 20.53. MS m/z: 207.02 [M+H]+. 2.7. Synthesis of (E)-2-(3-(2-(6-methoxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)4H-chromen-4-ylidene)malononitrile (6) To a solution of Compound 5 (1 mmol) in dry CH3CN (20 mL), Compound 2 (0.29 g, 1.2 mmol) and piperidine (0.1 mL) were added. Then, the resulting solution was heated to reflux under nitrogen atmosphere for 24 h. After removal of solvent, the residues were purified by alumina column chromatography using petroleum ether/dichloromethane (1:1, v/v) as eluent to afford Compound 6 as a deep blue solid (0.41 g,77%).1H NMR (400 MHz, CDCl3) δ (ppm): 8.93 (1H, d, J = 8.3 Hz), 8.16 (1H, d, J = 17.4 Hz), 7.71-7.75
(1H, m, Ar-H), 7.54-7.59 (1H, m, Ar-H), 7.42-7.45 (1H, m, Ar-H), 7.05 (1H, d, J = 8.4 Hz) 6.79 (1H, s, Ar-H), 6.71 (1H, s, Ar-H), 6.65 (1H, d, J = 8.48 Hz), 6.52 (1H, s, CH=H), 6.10 (1H, d, J = 15.4 Hz), 3.90 (3H, s, CH2). 2.57-2.60 (2H, m, CH2), 2.49-2.52 (2H, m, CH2), 1.85-1.88 (2H, m, CH2). 13C NMR (100 MHz, CDCl3) δ (ppm): 161.11, 159.51, 154.04, 152.90, 152.66, 152.47, 133.88, 127.61, 127.11, 125.93, 124.56, 118.69, 116.69, 115.46, 113.34, 110.59, 109.91, 105.48, 100.87, 55.68, 29.71, 24.59, 20.77. MS m/z: 432.0 [M+H]+. 2.8. Synthesis of DCX-OH BBr3 (0.94 ml, 10 mmol) was added to a solution of Compound 6 (0.5 mmol) in dry CH2Cl2 (10 mL) at 0 °C and the solution was stirred at room temperature for 16 h. Then saturated NaHCO3 solution (30 mL) was added to quench the reaction, followed by extraction of the aqueous layer with CH 2Cl2. The organic layer was dried over Na2SO4 and concentrated in vacuum. The crude product was directly used in the next reaction without further purification. MS m/z: 417.0 [M-H]-. 2.9. Synthesis of DCX-TPP To a solution of 2-(diphenylphosphino) benzoic acid (0.10 g, 0.32 mmol) in dry CH2Cl2, dicyclohexylcarbodiimide（DCC）(0.08 g, 0.39 mmol), DCX-OH (0.15 g, 0.35 mmol) and 4-dimethylaminopyridine（DMAP) (0.02 g, 0.16 mmol) were added under N2 atmosphere. Then the solution was stirred at room temperature for 6 h and concentrated under vacuum，the crude product was purified by silica gel column chromatography using dichloromethane/petroleum ether 1:2 (v/v) as the eluent to yield Compound 8 as solid (0.5 g, 46 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.94 (1H, d, J = 8.3 Hz), 8.31 (1H, d, Ar-H),
8.10 (1H, m, J = 16.1 Hz), 7.69-7.73(1H, m, Ar-H), 7.64-7.66 (2H, m, Ar-H), 7.49-7.55(1H, m, Ar-H) 7.33-7.39 (9H, m, Ar-H), 7.06 (1H, s, J = 8.3 Hz), 6.86 (1H, s, Ar-H), 6.79 (1H, s, Ar-H), 6.65 (1H, d, J = 8.1 Hz), 6.49 (1H, s, Ar-H), 6.16 (1H, s, Ar-H), 5.33 (1H, s, Ar-H) 2.54 (4H, m, CH2), 1.88 (2H, m, CH2). MS m/z: 705.2 [M-H]2.10. General procedures for spectra measurement All UV–vis and fluorescence measurements were carried out in PBS buffer (10 mM, pH 7.4) solution, containing 50 % DMF. In a 1 mL tube, 40 μL DCX-TPP (100 μM) and 160 μL DMF were mixed, and then an appropriate volume of HNO (10 mM) was added. The final solution volume was adjusted to 0.4 mL with PBS buffer and incubated at 37 °C for 30 min unless otherwise indicated. Fluorescence spectra were recorded in the range from 600 to 850 nm unless otherwise indicated, with an excitation wavelength of 580 nm. The excitation and emission slit widths were both 5 nm. For studying the real time response of DCX-TPP towards HNO. DCX-TPP and HNO were rapidly mixed. Then, the fluorescence intensity at 740 nm was recorded in real time for 1400 s at 37 °C with an excitation wavelength of 580 nm on an F-7000 fluorescence spectrophotometer (Hitachi, Japan), using slit widths of 5 nm for both excitation and emission. 2.11. HPLC analysis The HPLC chromatograms of DCX-TPP, DCX-OH, and the reaction products of DCX-TPP and HNO were performed on a system with a C18 column (150 nm × 4.6 mm, 5 μm) and the conditions were as follows:
methanol/H2O = 95/5 (v/v); flow rate: 0.8 mL/min; detection wavelength: 580 nm. 2.12. Cytotoxicity assays The cytotoxicity of DCX-TPP against HeLa cells and Raw264.7 was studied using a standard methyl thiazolyltetrazolium (MTT) assay. The cells were seeded into a 96-well plate at 5×104 cells /well in complete medium, containing DMEM and 10% fetal bovine serum (FBS). Then, the cells were incubated for 24 h at 37 °C under 5 % CO2. Afterwards, the medium was removed and washed with PBS for three times. Cells were then incubated with fresh medium (serum-free DMEM) containing various concentrations of DCX-TPP (0-20 μM) for 24 h. 20 μL of MTT solution (5.0 mg/mL) was then added to each well. After 4 h, the supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. Cell viability was determined by measuring absorbance at 450 nm with a microplate reader. 2.13. Fluorescence imaging in living cells HeLa cells and RAW 264.7 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in an incubator, containing 5% wt/vol CO2. After 24 h, RAW 264.7 cells were washed with PBS and treated with DCX -TPP (10 μM) for 30 min. RAW 264.7 Cells were subsequently washed twice with PBS and further treated with different concentrations of AS (0, 10, 50 and 100 μM) for 30 min. As for HeLa cells, the cells were treated with sodium nitroprusside (SNP, 2 mM) for 8 h, followed by addition of NaASc (2 mM) and DCX-TPP (10 μM) and 30-min incubation. Cell imaging was performed after the cells
were washed twice with PBS. Fluorescence signals collected at 663-738 nm with an excitation wavelength of 560 nm. 3. Results and discussion 3.1. Design rationale and probe synthesis We aimed to design a NIR fluorescence probe (DCX-TPP) with large Stokes shift to detect HNO in vitro and vivo. DCX-OH, comprised of dicyanomethylene-4H-chromene as the electron-withdrawing group and a hydroxyl xanthene moiety as the electron-donating group, was chosen as the fluorophore owing to its large Stokes shift and near infrared emission, which could potentially provide high sensitivity and minimal autofluorescence background in biological matrix [27]. Then, DCX-TPP was obtained by deriving DCX-OH with a bioorthogonal diphenylphosphinobenzoyl moiety as the recognition group for HNO. We envision that the esterification of the hydroxyl group by the HNO-responsive would make DCX-TPP non-emissive and the HNO-induced cascade reactions would regenerate the hydroxyl groups of the fluorophores with dramatic enhancement in fluorescence. Motivated by this rationale, DCX-TPP was prepared in several steps with good yields. First, DCX-OH was prepared by conjugating a dicyanomethylene-4H-chromene derivative with a xanthene derivative. Then, DCX-OH was derived with the diphenylphosphinobenzoyl moiety via standard Steglich esterification reactions. The detailed synthetic procedures of the probe were outlined in Scheme 2. 3.2. Photophysical properties characterization The photophysical properties of the probe were first investigated with both
absorption
and
fluorescence
spectroscopy.
DCX-TPP
was
initially
non-emissive with an absorption maximum at ~540 nm. Upon reaction with HNO, using Angeli’s salt (Na2N2O3, AS) as an HNO donor, there was a remarkable fluorescence enhancement at 740 nm (21-fold) and the absorption maximum was bathochromically shifted to 580 nm (Figure 1). The red-shift in absorption and dramatic fluorescence enhancement were attributed to HNO-triggered cascade reactions, releasing the initial fluorophore, DCX-OH, with distinct intramolecular charge transfer (ICT). These results suggested that our probe was responsive towards HNO. Having demonstrated that DCX-TPP was responsive towards HNO, its sensitivity was then explored by measuring the fluorescence spectra in the presence of various concentrations of AS as shown in Figure 2a. The fluorescence responses of DCX-TPP exhibited a dynamic correlation to the concentrations of HNO (0-100 μM). Good linearity was obtained between the fluorescence intensity at 740 nm and the HNO concentrations in the range of 2.0 μM -80 μM (Figure 2b). The limit of detection was calculated to be 0.05 μM, which is comparable to the existing near infrared fluorescence probes for HNO (Table S1). Then, the response rate of DCX-TPP towards HNO was investigated by recording the fluorescence intensity increments of DCX-TPP (10 μM) in different concentrations of HNO in real time. Figure 3a showed that there was a drastic enhancement in fluorescence intensities in the presence of either 20 μM HNO or 100 μM HNO, which reached the plateaux at 400 s and 600 s, respectively. The pseudo-first order rate constant was calculated to
be 0.00801 s-1 (Figure 3b). Moreover, similar response rates were obtained when 5 μM DCX-TPP was reacted with 10 and 50 μM HNO (Figure 3c). The pseudo-first order rate constant was calculated to be 0.0077 s-1 (Figure 3d). These results suggested that our probe could response towards HNO with rapid kinetics. Taken together, these results demonstrated that our probe could respond to HNO with high sensitivity and fast response in vitro. 3.3. Reaction mechanism The reaction products of DCX-TPP with HNO were analyzed with ESI-MS and HPLC to investigate the response mechanism. DCX-TPP and DCX-OH had retention times of 16.368 min and 7.402 min, respectively (Figure S1). Upon reaction with HNO, the peak at 16.346 min representing DCX-TPP decreased significantly and was accompanied by the appearance of a new peak at 7.389 min, indicating the release of DCX-OH. Moreover, ESI-MS analyses further verified the generation of DCX-OH as a reaction product. The ESI-MS spectrum from the reaction solution of DCX-TPP with AS showed a major peak at m/z=417.0 [M-H]-, corresponding to DCX-OH ([M-H]- 417.1) (Figure S2), which clearly verified that the reaction of DCX-TPP with HNO produced DCX-OH. These results demonstrated that the reaction of DCX-TPP with HNO was indeed attributed to the HNO-catalyzed oxidation reaction as depicted in Scheme 1. 3.4. Selectivity and pH dependence of DCX-TPP The selectivity of DCX-TPP to HNO and the pH dependence of DCX-TPP were then investigated to demonstrate its potential application in biological conditions.
First, the selectivity test was performed by incubating DCX-TPP (10 μM) with other biologically relevant species (100 μM of AS, ONOO-, .O2-, .NO-, H2O2, ClO-, and NO2-, 1 mM of Hcy, S2-, Fe3+, Na+ and K+, 10 mM of GSH, Cys). No obvious fluorescence intensity increase at 740 nm was observed in the presence of the tested biological relevant species. On the contrary, the addition of AS caused a remarkable enhancement of the fluorescence intensity at 740 nm, attributed to the biorthogonality of the HNO-responsive diphenylphosphinobenzoyl group (Figure 4a). Then, the effect of pH on the fluorescence intensity of DCX-TPP was also investigated. As can be seen from Figure 4b, there were no fluorescence changes of DCX-TPP as pH was varied from 5.0 to 10.0, indicating that the ester group was stable in this pH range. Upon addition of 100 μM HNO, the fluorescence intensity of DCX-TPP at 740 nm remarkably increased in the pH range of 6.0-8.0. The relative smaller increase in fluorescence intensity when the pH was smaller than 6.0 might be due to the decomposition of AS, resulting in decreased generation of HNO. Taken together, these results indicated DCX-TPP could be used for detecting HNO in physiological conditions with high selectivity. 3.5. Fluorescence imaging of HNO in living cells Encouraged by the superior advantages of DCX-TPP including excellent sensitivity, rapid response and high selectivity, we then investigated whether DCX-TPP could be applied in imaging of HNO in living cells. First, the potential cytotoxicity of DCX-TPP towards RAW 264.7 and HeLa cells was evaluated with a standard MTT assay. As is shown in Figure 5, the cells had over 90% viability upon
treatment with various concentrations of DCX-TPP (10, 20, 30, 40 and 50 μM) at 37 °C for 24 h, indicating good biocompatibility and low cytotoxicity of DCX-TPP. Then, the ability of DCX-TPP to detect exogenous HNO was tested by incubating RAW 264.7 cells with DCX-TPP for 30 min, followed by treating with different concentrations of HNO (10, 50 and 100 μM). Both bright field and confocal fluorescence images were then recorded. There were no fluorescence signals for cells incubated with DCX-TPP, indicating that RAW 264.7 cells barely generated HNO, consistent with previous reports [28-30]. In contrast, there was gradual increase in fluorescence signals when increasing concentrations of exogenous HNO were added. Therefore, these results demonstrated that DCX-TPP was cell membrane permeable and was capable of detecting exogenous HNO in living cells. The capability of DCX-TPP to detect and image endogenous HNO in normal culturing or induced conditions were further investigated. HeLa cells, with a low level of endogenous HNO, were used to study whether DCX-TPP possessed potential application for monitoring endogenous HNO. As shown in Figure 6, there were weak but evident fluorescence signals when HeLa cells were only treated with DCX-TPP (10 μM). Furthermore, as it was reported that the reaction of L-ascorbate with NO in biological media could form intracellular HNO, we then tried to test the ability of DCX-TPP towards HNO in HeLa cells under induced conditions [30-32]. HeLa cells were pretreated with SNP (2 mM), a commercial NO donor for 8 h, then NaASc (2 mM) and DCX-TPP (10 μM) were added. Fluorescence images were obtained after another incubation of 30 min. As shown in Figure 7, HeLa cells displayed bright fluorescence signals. These results demonstrated that our probe could not only detect
low level of endogenous HNO but also could visualize HNO in living cells under induced conditions. 4. Conclusions In summary, we have developed a novel NIR fluorescent turn-on probe for the detection and imaging of exogenous and endogenous HNO in living cells. The probe was composed of a NIR fluorophore with a large Stokes shift of 160 nm, which was derived with a diphenylphosphinobenzoyl moiety as the HNO-responsive unit. The probe was non-emissive in the absence of HNO. However, there was a rapid increase (within 600 s) in fluorescence upon addition of HNO. The probe was demonstrated to exhibit excellent sensitivity with a limit of detection of 0.05 μM, high selectivity towards HNO and great stability under physiological conditions. Furthermore, its ability to detect and visualize exogenous HNO and endogenous HNO without or with induction was demonstrated in different living cells. We believe the combined advantages of our probe would provide an invaluable tool for studying the biological functions of HNO in living cells. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants 21527810, 91753107, and 21205034).
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Scheme 1. Sensing mechanism of DCX-TPP for HNO detection
Scheme 2. Synthesis of DCX-TPP
Figure 1. (a) Fluorescence spectra of DCX-TPP (10 μM, black line), DCX-TPP (10 μM, red line) reacted with HNO (100 μM) at 37 oC for 30 min; (b) UV-vis absorption spectra of DCX-TPP before (20 μM, black line) and after (20 μM, red line) reaction with HNO (200 μM) at 37 oC for 30 min.
Figure 2. (a) The fluorescence emission spectra of DCX-TPP (10 μM) in the presence of different concentrations of HNO (0-100 μM); (b) Linear fitting of fluorescence intensity (F) toward the concentrations (C) of HNO from 0 to 150 μM. λex/em = 580/740 nm.
Figure 3. (a) Time dependent fluorescence emission intensity (λex/em = 580/740) of DCX-TPP (10 μM) upon addition of different concentrations of HNO, 100 μM (blue line), 20 μM (red line), and without HNO (black line); (b) Pseudo first-order kinetic plot of the reaction of DCX-TPP (10 μM) with HNO (100 μM), slope = 0.0080 s-1; (c) Time dependent fluorescence emission intensity (λex/em = 580/740) of DCX-TPP (5 μM) upon addition of different concentrations of HNO concentration, 50 μM (pink line), 10 μM (blue line), and without HNO (black line) ; (d) Pseudo first-order kinetic plot of the reaction of DCX-TPP (5 μM) with HNO (50 μM), slope = 0.0077 s-1.
Figure 4. (a) Fluorescence responses of DCX-TPP (10 μM) towards different species: (1) 100 μM ONOO-; (2) 100 μM .O2-; (3) 100 μM NO-; (4) 100 μM H2O2; (5) 100 μM ClO-; (6) 1 mM Hcy; (7) 1 mM S2-; (8) 1 mM Fe3+; (9) 1 mM Na+; (10) 1 mM K+; (11) 100 μM .NO2-; (12) 10 mM GSH; (13)10 mM Cys; (14) 100 μM HNO; (b) Effect of pH value on the fluorescence intensity of DCX-TPP (10 μM) and after being reacted with HNO (100 μM) at 37 oC for 60 min. λex/em = 580/740 nm
Figure 5. Effects of DCX-TPP (0-50 μM) on the viability of HeLa cells and Raw 264.7 cells. The viability of cells without DCX-TPP is defined as 100%. The results are the means ± SD of three experiments.
Figure 6. Images of RAW264.7 cells: (a-e) bright field; (f) Fluorescence image of cells without any treatments. (g-j) Fluorescence images of cells pretreated with 10 μΜ DCX-TPP, followed by addition of different concentrations of exogenous HNO (0, 10 μΜ, 50 μΜ, and 100 μΜ). (k-o) Overlay images of bright field and fluorescence field. λex = 560 nm, collection channel: 663-738 nm, scale bar = 50 μm.
Figure 7. Images of HeLa cells: (a-c) control group without any treatments; (d-f) cells only treated with10 μΜ DCX-TPP; (g−i) cells were pretreated with 2 mM SNP and then incubated with 2 mM NaASc and 10 μΜ DCX-TPP; λex = 560 nm, collection channel: 663-738 nm, scale bar = 50 μm.
Highlights (1) The probe DCX-TPP is designed by masking a fluorophore with large Stokes shift and near infrared emission with a bioorthogonal HNO-responsive moiety. (2) The probe DCX-TPP exhibits high sensitivity, fast response and excellent selectivity towards HNO in vitro. (3) The probe DCX-TPP has been successfully introduced to image both exogenous and endogenous HNO in living cells.