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A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes
Journal Pre-proof A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes Lei Shi, Sheng Yang, Hao-Jia Hong, Yong Li, Hui-Juan Yu, Guang Shao, Kai Zhang, Sheng-Zhao Gong PII:
S0003-2670(19)31200-0
DOI:
https://doi.org/10.1016/j.aca.2019.10.004
Reference:
ACA 237138
To appear in:
Analytica Chimica Acta
Received Date: 10 May 2019 Revised Date:
26 September 2019
Accepted Date: 7 October 2019
Please cite this article as: L. Shi, S. Yang, H.-J. Hong, Y. Li, H.-J. Yu, G. Shao, K. Zhang, S.-Z. Gong, A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
Graphical Abstract Figure
Graphical Abstract A novel dual-activatable fluorogenic probe CS has been rationally designed. This probe exhibited good sensitivity, high specificity and fast response towards HClO under the lysosomal acidic conditions, and it has been successfully applied for visualizing exogenous or endogenous HClO in lysosomes of living cells.
A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes Lei Shi a, Sheng Yang b*, Hao-Jia Hong a, Yong Li c, Hui-Juan Yu c,d, Guang Shao c,d*, Kai Zhang e, Sheng-Zhao Gong a* a. Guangdong Engineering Technical Research Center for Green Household Chemicals, Guangdong Industry Polytechnic, Guangzhou, Guangdong, 510300, P. R. China. Email: 1996103022@ gdip.edu.cn. b. Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources, School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, Hunan 410114, P. R. China. Email: [email protected]. c. School of Chemistry, Sun Yat-sen University, Guangzhou, Guangdong, 510275, P. R. China. Email: [email protected]. d. Shenzhen Research Institute, Sun Yat-sen University, Shenzhen, 518057, P. R. China. e. College of Preclinical Medicine, Southwest medical university, Luzhou, Sichuan, 646000, P. R. China. *To whom correspondence should be addressed.
ABSTRACT: Hypochlorite (HClO) is involved in various physiological and pathological processes as well as regulation of lysosomal functions. Thus, it is appreciated to develop efficient molecule tools for precisely detecting HClO in lysosomes. Although several lysosomal-targetable fluorogenic probes for HClO have been developed to date, they still suffered from the discounted sensing performance under lysosomal acidic condition. Herein, on the basis of the “AND” logic gate strategy, a novel dual-activatable fluorogenic probe CS has been rationally designed by simultaneously incorporating HClO recognition site and pH-sensitive group with lysosomal-targetable characteristic into a coumarin fluorophore. Different from the single-activated ones previously reported, CS exhibited good sensitivity, high specificity and fast response towards HClO under the acidic conditions but was out of operation in the neutral or alkaline environment. Importantly, it had been successfully applied for spatial-resolution imaging of exogenous or endogenous HClO in lysosomes.
1. Introduction As one of the most important reactive oxygen species (ROS), hypochlorous acid (HClO) has essential roles in biological systems. The endogenous hypochlorite (HClO/ClO-) is generated from the biological oxidation reaction between hydrogen peroxide (H2O2) and chloride ion (Cl-) by the catalysis of myeloperoxidase (MPO)[1,2]. The HClO in vivo could act as a formidable oxidant and the pathogens-killer in the innate immune systems, and play a key part in many biologically vital processes[3-7]. However, misplaced or excessive HClO would result in serious damage to tissues and lead to various diseases, such as cardiovascular diseases, neuron degeneration, arthritis, and even cancers[8-10]. The lysosome as a kind of membrane-bound cytoplasmic organelles is generally recognized as the cellular “incinerators” for the degradation and recycling of cellular wastes. Recently, there is already compelling evidence that lysosome is also involved in many other fundamental processes, such as secretion,
plasma
membrane
repair,
signaling,
energy
metabolism,
and
lipid
degradation[11,12]. Furthermore, a tight relationship between the HClO level and the redox balance in lysosomes has been found, and aberrant endogenous or exogenous HClO level could lead to the dysfunction of lysosomes and might even cause apoptosis by the rupture of lysosomes[13,14]. Hence, it is very necessary to develop a facile and accurate analytical method for detecting HClO in lysosomes. Comparing with other analytical techniques, fluorimetry based on the organic fluorescent probes is regarded as an ideal tool for real-time monitoring biomolecules in living biosystem due to its high sensitivity, excellent selectivity, non-invasiveness and high spatiotemporal resolution[15-20]. In recent years, a large number of HClO fluorescence probes have been developed[21-24], but many of them were not very appropriate for HClO detection at subcellular organelles because of the deficiency in lysosome-specificity. To address this issue, some lysosome-targeting groups such as morpholine were incorporated into the skeleton of probes with the purpose of guiding the probes into lysosomes[25-31]. However, all of them were only able to show similar fluorescent responses in different pH conditions. Considering the imperfect targeting abilities of the guiding groups and the widespread existence of HClO in cells, lots of interfering signals would be caused in the neutral cytosol and other organelles. In order to solve this problem, we have previously proposed a new strategy for specifically visualizing the targeted analytes in a specific organelle by a fluorescent probe with the target and location dual-controlled molecular switches[32]. As a continuation of our research and considering the acidic environment of lysosomes (pH 4.0-6.0), we rationally designed a novel HClO and pH dual-activatable fluorescent probe CS. As depicted in Scheme
1 and Fig.1, N,N-dimethyl-thiocarbamate (DMTC) was separated due to the oxidation of HClO, resulting in the weak fluorescence (fluorescence quantum yield Φ = 6%) in neutral aqueous condition. By contrast, this probe would display much stronger blue fluorescence (Φ = 22%) under the acidic environment due to the protonation of phenolic anion and morpholine group, indicating its specific detection performance at lysosomal acidic condition. To the best of our knowledge, such a dual-locked model system that activates the fluorescent signals by the acidic condition and HClO remains unreported.
Scheme 1. The fluorescence response mechanisms of probe CS for sensing H+ and HClO alone or both of them.
(B)
d
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0.4
0.3
a,c 0.2
0.1
b 0.0 250
300
350
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b
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Fluorescence Intensity
UV absorbance
(A)
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400
450
500
d
6.0x105 4.0x105 2.0x10
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6
5
0.0 375
a,c 400
425
450
475
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550
575
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Fig. 1. The UV absorption and fluorescence spectra of probe CS (10 µmol/L) under different conditions (λex = 345 nm). (a) In buffer solution (10% DMF, pH 5.0); (b) with addition of NaClO (8 eq) in the same buffer solution; (c) in PBS buffer solution (10% DMF, pH 7.4); (d) with addition of NaClO (8 eq) in the same buffer solution.
2. Experimental section 2.1. Apparatus and chemicals 1
H NMR (400 MHz) and
13
C NMR (100 MHz) spectra were conducted by a Bruker
Avance 300 spectrometer. High-resolution mass spectrometry (HRMS) involved a Q-TOF6510 spectrograph (Agilent). UV–vis spectra were measured by a Hitachi U-4100 spectrophotometer. Quartz cuvettes used for all measurements were 1 cm path length and 3
mL volume. Perkin-Elmer LS-55 and Edinburgh F35 luminescence spectrophotometer were used to measure fluorescent spectra (slit widths: 0.5 nm/1.0 nm). A Mettler Toledo pH meter was used to test the pH values. The twice-distilled water was used throughout all experiments. All reagents were purchased from Energy Chemical and used without further purification unless otherwise stated. 2.2 General procedure for absorption and fluorescence analysis A stock solution (1.0 mmol/L) of probe CS was prepared in DMF, and its test solution (10 µmol/L) was prepared by the dilution of the stock solution with the buffer solution (containing 10% DMF) under an indicated pH value. Then various analytes were added separately, and the absorption and fluorescence measurements were carried out after being incubated for 10 min. For fluorescence measurements, the excitation wavelength was set at 345 nm with a slit width of 0.5×1.0 nm. Additionally, the absolute fluorescence quantum yield (Φ) values were determined by using an integrating sphere according to the previous report[33]. 2.3 Computational methods All the calculations were performed using density functional theory (DFT) and the Gaussian 09 program[34]. The ground state structures of the compounds were optimized by the density functional theory (DFT) using a B3LYP. The 6-31+G (d, p) basis sets were employed for CS and 2-O-, and 6-31G (d, p) basis sets were employed for CS+H+ and 2+H+. The excited-state calculations were carried out with the time-dependent density functional theory (TD-DFT) with the optimized structure of the ground state. The solvent effects were modeled with the polarizable continuum model (PCM). 2.4 Cell cytotoxic assays and imaging HeLa cells were seed in DMEM medium (Thermo Scientific HyClone) and cultured with a mixture of 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin and 100 U/mL gentamicin, Then the cells were grown at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The cytotoxic effects of the probe CS were tested by the MTT assay according to the previous report[27]. Before imaging, the HeLa cells were incubated with the probe CS (10.0 µmol/L) and washed three times with PBS buffer. Then the cells were treated by NaClO (0 ~ 250 µmol/L) and washed with PBS buffer. After that, the cells were sent to imaging analysis, and the fluorescent images (400-500 nm) were acquired by excitation with a multi Ar laser (345 nm). For monitoring endogenous HClO in the lysosomes, the cells were pretreated with probe CS (10 µmol/L) and lysosome tracker LysoTracker red (1.0 µmol/L) for 30 min, and then the
cells were incubated with phorbol 12-myristate 13-acetate (PMA) for 30 min. After rinsing cells with PBS buffer again, the imaging analysis was carried out by the confocal laser scanning microscopy.
3. Results and discussion 3.1. Design and synthesis of the probe CS In order to further understand the roles and functions of HClO in lysosomes, we hope to construct a novel fluorescent probe which could detect HClO precisely and specifically in lysosomes. With this goal in mind, we utilized the pH difference between the lysosome and other subcellular organelles, and developed a novel fluorescent probe dual-controlled by HClO and pH values. In this research, 7-hydroxy-coumarin was chosen as the fluorophore due to its high quantum yield, good stability, and biological compatibility. And the morpholine group was incorporated as a guiding group for the lysosome. More importantly, the phenolic hydroxyl group and the morpholine group were also acted as the binding sites for H+, leading to the changes
of
fluorescence
under
the
different
pH
conditions.
Additionally,
the
dimethylthiocarbamate (DMTC) was employed as the recognition group for HClO owing to its fast and selective oxidation reaction towards HClO[35-38]. Therefore, a novel lysosome-targetable fluorescent probe CS with the dual recognizing sites for HClO and H+ was designed and synthesized as shown in scheme S1. Besides, compound 3, 4 and 5 were also synthesized with the purpose of understanding the pH effects of this probe. All these compounds were characterized by NMR and HRMS spectra (Fig. S17-S27). (A) 3.0x10 (B) 1000 6
Probe Probe + 4 eq HClO Probe + 8 eq HClO Compound 2
2.6x106 6
Fluorescent Intensity
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900 800
Fluorescent Intensity
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700 600 500 400 300
Add ClO-
200
Add OH-
Add H+
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4
5
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7
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9
0
50
pH Value
100
150
200
250
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400
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500
Time (s)
Fig. 2. (A) The fluorescence intensity variation of compound 2 and probe CS under different pH conditions (10 µmol/L, λex = 345 nm). (B) The Time-dependent fluorescence changes of probe CS (10 µmol/L) upon addition of NaClO (8 eq), NaOH (adjust to pH 10) and hydrochloric acid (adjust to pH 5), and the test was carried out under the excitation at 345 nm.
3.2. The pH effects of probe CS In this study, the hydroxyl and morpholine group were employed as the pH-sensing sites
and combined into the coumarin fluorophore, and we studied the pH effects of the two moieties, respectively. According to previous reports[39,40], the UV-absorbance and excitation spectra of 7-hydroxycoumarin would be red-shifted in the basic aqueous solution by comparison with that in the neutral or acidic solutions. Besides, the maximum fluorescent emission bands were almost the same during the pH range of 3~10 due to the rapid excited state acid-base equilibrium which took place during the singlet excited state lifetime of 10-8 seconds[39]. Therefore,
the
fluorescent
intensity
at
the
maximum
emission
wavelength
of
7-hydroxycoumarin would become pH-dependent by giving an excitation light at a fixed wavelength. In order verify the above hypothesis, the compound 4 and 5 were synthesized and investigated. As shown in Fig. S3 and S4, the fluorescent intensity at 451 nm of compound 4 enhanced greatly with increasing pH values under the irradiation light of 405 nm, but it became decreased when excited at 345 nm as a result of the red-shift on its UV-absorbance and excitation spectra. In contrast, the compound 5 as a hydroxyl-ether derivative of 4, emitted much stable fluorescent performance during the pH range of 3.2~9.2, illustrating the great pH-sensitive ability of the hydroxyl moiety. Subsequently, we determined the pH-dependent property of compound 3 to investigate the pH effect of the morpholine unit. Unlike compound 4, the fluorescence of 3 greatly enhanced when the pH value decreased from 8 to 4, which was due to the protonated nitrogen and blocked PET process from the morpholine moiety to the fluorophore[41]. By combining the two pH sensing sites of hydroxyl and morpholine group, compound 2 was also an excellent pH-sensitive probe. As the pH increased from 3 to 10, the UV-absorption spectra of 2 displayed a large red-shift, and its fluorescent intensity at 451 nm also showed greatly pH-dependent(Fig. S8-S11). In addition, the pKa values of compound 2 was calculated to be 6.06 according to the Henderson-Hasselbalch equation. As expected, the probe CS as a derivative of 2, displayed much stronger fluorescent responses to HClO in acidic conditions(Fig. 2A). The extraordinary fluorescent responses under acidic conditions provided a feasible way for constructing an ideal lysosome-targeted probe for visualizing HClO. Furthermore, the responses of CS to HClO and the pH variations were quite fast, which could be completed within a few seconds (Fig. 2B). The results suggested that probe CS would be very favorable for the real-time monitoring of HClO in lysosomal acid conditions. 3.3 The DFT/TDDFT calculations In order to gain a thorough comprehension of the changes in the photophysical properties
of compound 2 and CS, the theoretical calculations based on DFT and TDDFT were carried out. The pictorial drawing of their optimized geometries and the frontier molecular orbitals are shown in Fig 3, and the calculated data are summarized in Table 1.
Fig. 3. The frontier molecular orbital plots of compound 2 and CS in water (PCM model). Table 1. Frontier molecular orbital profiles of 2 and CS based on DFT/TD-DFT calculations. Compoun d
UV-vis Absorption
Emission
energy gap (nm)
fa
major contribution
CS
463.72 (2.67 eV) 390.38 (3.18 eV) 366.05 (3.39 eV) 317.68 (3.90 eV)
0.01 0.04 0.02 0.54
HOMO → LUMO (99.80%) HOMO-1 → LUMO (98.72%) HOMO-2 → LUMO (99.13%) HOMO-3 → LUMO (90.97%)
820.16 0.04 (1.51 eV)
HOMO → LUMO (99.84%)
CS+H+
387.91 (3.20 eV) 359.43 (3.45 eV) 320.05 (3.87 eV)
0.05 0.01 0.62
HOMO → LUMO (97.71%) HOMO-1 → LUMO (98.74%) HOMO-2 → LUMO (93.56%)
540.83 0.03 (2.29 eV)
HOMO → LUMO (99.51%)
2+H+
325.88 (3.80 eV)
0.63
HOMO → LUMO (96.72%)
368.20 0.83 (3.37 eV)
HOMO → LUMO (98.70%)
2-O
381.58 (3.25 eV)
0.74
HOMO → LUMO (96.96%)
433.67 0.87 (2.86 eV)
HOMO → LUMO (98.58%)
energy gap (nm)
fb
major contribution
fa is the oscillator strength of the absorption coefficient. fb is the oscillator strength of the emission coefficient.
As shown in Fig. 3, the HOMO and LUMO of compound 2-O and 2+H+ were localized on the coumarin fluorophore, which would facilitate the internal charge transfer (ICT) process upon excitation. It should be pointed out that the allowed HOMO → LUMO transition with the oscillator strength (f = 0.74 and 0.63) were observed for both derivatives (2-O and 2+H+) of compound 2. By comparing with 2+H+ (4.12 eV), a lower energy gap between the HOMO and LUMO was found for 2-O (3.49 eV). This might be the result of the relatively higher electron-releasing ability of the phenoxide anion in comparison with the phenol group[42]. As
a result, the ICT process of 2-O would be enhanced, leading to the redshift of its absorption spectra. This prediction has been proved by the experimental results as shown in Fig. S6. When the pH conditions varied from acid to base, the maximum absorption wavelength and the maximum excitation wavelength of compound 2 were red-shifted into a lower-energy region (345 nm to 405 nm). Consequently, when environmental pH value increased, a decrease of fluorescent intensities was observed under the irradiation light at 345 nm, while an enhancement of its fluorescence would appear with the excitation light source of 405 nm (Fig. S8~S11). Unlike compound 2, the HOMO of probe CS was localized on the morpholine moiety, and the HOMO of CS+H+ was mainly dominated by the DMTC group (Fig. 4). The occupation of HOMO by this group would prevent electronic relaxation from LUMO to HOMO, resulting in their fluorescence quenching[43,44]. Furthermore, for two forms of the probe (CS and CS+H+), the electron transitions of HOMO → LUMO were forbidden due to the extremely low oscillator strength (f = 0.01 and 0.05). Even though HOMO-3 → LUMO and HOMO-2 → LUMO might be the allowed transition, the emissive state of organic fluorophores was usually occurred during the HOMO → LUMO transition. Furthermore, considering the very low the oscillator strength of emission coefficient (f = 0.04 and 0.03), the radiative LUMO →HOMO transition of the probe was extremely unlikely to happen, implying that probe CS and CS+H+ were non-fluorescent according to the principle of Kasha's rule[45,46]. However, when the DMTC part was oxidized off by HClO, the electronic transition occurred completely in the coumarin moiety, and the oscillator strength of emission coefficient rose sharply (2+H+: f = 0.83; 2-O: f = 0.87), indicating the recovery of their strong fluorescence. The above predictions were in full agreement with the experimental results. 3.4 Proposed sensing mechanism of the probe According to the experimental results and the published reports[34,47,48], we proposed the possible mechanism of probe CS for detecting HClO as depicted in Scheme S2. The strong oxidation capability of HClO to sulfide moiety would induce the formation of unstable imine moiety via the release of SO2, and then the next hydrolysis reaction resulted in the generation of the coumarin fluorophore which would turn into the strong fluorescent compound 2 under the acidic condition. To prove this proposed sensing mechanism, the reaction mixture of probe CS and HClO was characterized by MS spectrum. After addition of NaClO, the peaks for probe CS (m/z 406.48, [M+H]+) lowered obviously, while a new peak for the predicted product (m/z 319.45, [M+H]+) emerged (Fig. S16). The MS analysis revealed the fact that the fluorescence turn-on
process of probe CS involved the oxidation and separation of DMTC group by HClO.
(B)1.8x10
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Fluorescence Intensity
1.6x106
140 µM
1.4x106 1.2x106
0 µM
1.0x106 8.0x105 5
6.0x10
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Fluorescence Intensity
(A)1.8x10
pH = 5.0
6
1.6x106
y = 18216x+67995 R2 = 0.995
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y = 5584x+12017 R2 = 0.998
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pH = 7.4 0
Wavelength (nm)
10
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100
Concentration (µmol/L)
Fig. 4. (A) The fluorescence titration spectra of CS (10 µmol/L) toward varying concentrations of NaClO in acetate buffer (pH 5.0, containing 10% DMF) at 25 oC. Exited at 345 nm. (B) Standard
curve for relationship between the concentration of HClO and the fluorescence intensity at 451 nm of probe CS in the buffer solutions of pH 5.0 and pH 7.4.
Fig. 5. Fluorescence responses of probe CS (10 µmol/L) towards various analytes (200 µmol/L except for 100 µmol/L HClO) in acetate buffer (pH 5.0, containing 10% DMF). Data shown in turn: (1) blank, (2) NaClO, (3) K+, (4) Na+, (5) Mg2+, (6) Zn2+, (7) Ca2+, (8) Cu2+, (9) Pb2+, (10) Al3+, (11) Fe3+, (12) NO3-, (13) NO2-, (14) I−, (15) SO42-, (16) PO43-, (17) S2O82- (18) H2O2, (19) ⋅OH, (20) TBHP,·(21) O2-, (22) ONOO−.
3.5 The spectroscopic properties of probe CS to HClO Encouraged by the above result, the fluorescent properties of probe CS were systematically analyzed. In the acidic condition, the probe displayed a fast and dramatic enhancement of its fluorescence around 451 nm upon addition of HClO, and its fluorescent intensity at 451 nm has a great linear relationship (R2 = 0.995) in the concentration range of 0~90 µmol/L (Fig. 4 and S13). However, a much milder enhancement of its fluorescence could be observed by replacement of the neutral buffer solution, suggesting that the fluorescent response of CS was really pH-dependent. Moreover, the quantum yield of the reaction
mixture of probe CS and HClO was up to 22% in comparison with probe CS and CS+H+ (Φ = 0.1% and 0.2%), indicating the huge enhancement of its fluorescence and extremely low background. Moreover, the limit of detection of probe CS was evaluated to be 24.3 nmol/L according to the formula of LOD = 3σ/Slope, implying the excellent sensitivity of probe CS for detecting HClO. 3.6 Selectivity of probe The selectivity is a very important parameter for evaluating the application performance of the fluorescent probe in the biological systems. Thus, we investigated the influence of representative metal cations (K+, Na+, Mg2+, et al), anions (NO3-, NO2-, et al), RNS (ONOO-), and other ROS (H2O2, ⋅OH, TBHP, et al) to probe CS. As shown in Fig. 5, no significant fluorescent response could be observed in the presence of the above analytes except for HClO, suggesting the excellent selectivity of probe CS towards HClO over other bioactive substances. Furthermore, the competition experiments revealed that the response of probe CS toward HClO would not be disrupted in the presence of these species, which is helpful for monitoring HClO in cells or actual samples.
(M) Piexl Intensity
8
CS+PMA
6
4
2 CS only
CS+ABAH CS+BMA/BFA
0
Fig. 6. The Bright field and fluorescent images of HeLa cells. (A) Cells only(control); (B) by incubation with PMA (25 mg/L) for 2 h in the presence of CS (10 µmol/L); (C) by incubation with ABAH for 2 h in the presence of CS (10 µmol/L); (D) by co-incubation with PMA (25 mg/L) and BFA (1 mmol/L), and then treated with CS for 2 h. Scale bar: 25 µm. (M) The semi-quantification of mean
pixel intensities obtained from the corresponding fluorescence images of (A)~(D).
Fig. 7. The fluorescence images of HeLa cells incubated with (A) Probe CS (10 µM) and NaClO (250 µM) for 30 min (blue channel: 405~640 nm, Ex = 365 nm); (B) LysoTracker Deep Red (50 nM) (red channel: 640~700 nm, Ex = 639 nm); (C) Merged images of (A) and (B) (purple); (D) Intensity profile of linear region of interest across HeLa cells incubated with LysoTracker Deep Red and blue channel of CS. Scale bar: 25 µm.
3.7 The bioassay of HClO in living cells Before used for cell imaging, the cell cytotoxicity assay of probe CS was investigated according to the MTT assay. After incubation with probe (0~30 µmol/L) for 12 h, the HeLa cell survival rate was more than 85%, indicating the low cytotoxicity and the favorable biocompatibility of this probe (Fig. S14). Subsequently, the cells were treated with probe (10 µmol/L) for 30 min and washed with PBS solution three times, and then incubated with various concentrations (0, 50, 100, 250 µmol/L) of NaClO. As is illustrated in Fig. S15A, HeLa cells gave almost no fluorescence without the addition of NaClO. However, the blue fluorescence from the cells increased remarkably with increasing concentration of NaClO (Fig. S15B~D). Besides, when the cells were pretreated with phorbol 12-myristate 13-acetate (PMA, as a ROS stimulant), HeLa cells displayed much stronger fluorescence (Fig. 6B), while the pretreatment of cells with 4-aminobenzoic acid hydrazide (ABAH, as a specific inhibitor of MPO) would lead to the extremely weak fluorescence (Fig. 6C). The results demonstrated that probe CS could penetrate cell membranes and detect exogenous and endogenous HClO in living cells. Additionally, in order to confirm that probes would only display the intracellular response in lysosome rather than cytoplasm and other neutral organelles, bafilomycin A1 (BFA) was selected to alkalinize the intracellular lysosomal pH values of the cells. By comparing with Fig. 6B and 6D, it is obvious that the fluorescence signals were suppressed severely by treatment with BFA, implying that the fluorescent response of probe CS was dependent on the lysosomal acid conditions. To further examine the capability of probe CS in imaging of HClO in the lysosomes, co-localization experiments were carried out. By pretreatments with LysoTracker Deep Red
DND-99 and probe CS, HeLa cells were then treated with NaClO (250 µmol/L). As illustrated in Fig. 7, the fluorescent signals of probe CS and LysoTracker Deep Red overlapped very well, with the Pearson's and overlap coefficients of 0.914, indicating that probe CS could be specifically targeted lysosomes in living cells. 4. Conclusion In this work, we have developed a lysosome-targetable fluorescent probe for detecting the lysosomal HClO in living cells by joining the dual-controlled molecular switches and coumarin fluorophores. The probe CS displayed high sensitivity, great selectivity and rapid response towards HClO, and could quantitatively detect HClO in the range of 0~80 µmol/L with a low detection limit of 24.3 nmol/L. According to the cellular imaging experiments, the probe exhibited excellent lysosome-targetable localization capability and good practicability for visualizing exogenous and endogenous HClO in the lysosomes of living cells, indicating its promising applications in biological systems. Acknowledgment We gratefully acknowledge financial support from the Research Project of Department of Education of Guangdong Province (2017GkQNCX003), the Science and Technology Planning Project of Guangzhou City (201904010250), the fund of Specialized Cultivation of Scientific and Technological Innovation for College Students in Guangdong Province (pdjh2019b0691), the Science and Technology Planning Project of Shenzhen City (JCYJ20180307164055935), Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources (2018KJY06), National Key R&D Program of China (2017YFC1600306). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.
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Supporting Information for A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes Lei Shi a, Sheng Yang b*, Hao-Jia Hong a, Yong Li c, Hui-Juan Yu c, Guang Shao c,d*, Kai Zhang e, Sheng-Zhao Gong a* a. Guangdong Engineering Technical Research Center for Green Household Chemicals, Guangdong Industry Polytechnic, Guangzhou, Guangdong, 510300, P. R. China. Email: 1996103022@ gdip.edu.cn. b. Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources, School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, Hunan 410114, P. R. China. Email: [email protected]. c. School of Chemistry, Sun Yat-sen University, Guangzhou, Guangdong, 510275, P. R. China. Email: [email protected]. d. Shenzhen Research Institute, Sun Yat-sen University, Shenzhen, 518057, P. R. China. e. College of Preclinical Medicine, Southwest medical university, Luzhou, Sichuan, 646000, P. R. China.
Table of Contents Description 1. The synthesis procedures of probe CS 2. The photographs of probe in the absence and presence of HClO 3. The pH effect study of compound 2, 3, 4 and 5
page 2-3 4 4-7
4. The recognition mechanism of probe for sensing HClO.
8
5. The absorption and fluorescent spectra of probe toward NaClO
8
6. Cytotoxic effect of probe CS
9
7. The fluorescent images of the probe after addition of NaClO
9
8. The NMR and MS spectra of compound 2 and CS
10 10
9. References 1
1. Synthesis of probe CS The coumarin 1 was prepared according to the procedure previously described[1,2].
Scheme S1. The synthetic route and structure of probe CS
1.1 Synthesis of compound 2 Compound
1
(0.60
g,
2.91
mmol),
DMAP
(14
mg,
0.11
mmol)
and
N-hydroxysuccinimide (0.43 g; 3.3 mmol) were dissolved in 7 mL of anhydrous DMF, and stirred at 0°C for 30 min. A solution of DCC (0.68 g, 4.6 mmol) in anhydrous CH2Cl2 (4 mL) was added dropwisely and stirred for 1 h at room temperature. Then the resulting mixture was added dropwisely into the solution of 4-(2-Aminoethyl)morpholine (0.44 g, 3.4 mmol) in anhydrous CH2Cl2 (10 mL) at 0 oC under N2, and the mixture was stirred at room temperature overnight. After that, the precipitates were filtrated, and the filtrate was concentrated under vacuum and redissolved in dichloromethane. The organic layers was washed by water and brine, dried over with Na2SO4 and evaporated under vacuum. The residue was purified on silica gel chromatography eluted with ethyl acetate: ethanol = 50:1 (v/v) to give compound 2 as a white solid (0.57 g, 62%). 1H NMR (400 MHz, d6-DMSO) δ = 8.85 (t, J = 6.4 Hz, 1H), 8.79 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 6.88 (dd, J = 8.8 Hz & J = 2.0 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 3.59 (t, J = 4.8 Hz, 4H), 3.40-3.46 (m, 2H), 3.34 (bs, 1H), 2.48 (t, J = 6.4 Hz, 2H), 2.40–2.44 (m, 4H); 13C NMR (100 MHz, d6-DMSO) δ = 163.6, 161.3, 160.9, 156.2, 147.9, 131.9, 114.3, 113.5, 111.0, 101.7, 66.2, 56.6, 53.0, 40.0 ppm. HRMS (ESI) Calcd for C16H19N2O5 [MH+] 319.1294, found 319.1300. 1.2 Synthesis of probe CS Under N2 in ice–water bath, a solution of dimethylthiocarbamoyl chloride (0.42 g, 3.4 mmol) in anhydrous CH2Cl2 (3 mL) were added dropwisely into the solution of compound 2 (0.32 g, 1.0 mmol) and DIPEA (1.0 mL) in THF/CH2Cl2 (10 mL, v/v, 1:1). After being cooled for 30 min, the resulting mixture was stirred at room temperature overnight. Then the solution was concentrated under vacuum and the residue was purified on silica gel chromatography eluted with ethyl acetate: ethanol = 50:1 (v/v) to give compound CS as a white solid (0.28 g, 2
70%). 1H NMR (400 MHz, CDCl3) δ = 9.13 (s, 1H), 8.91 (s, 1H), 7.16 (s, 1H), 7.14~7.16 (m, 2H), 3.78 (t, J = 4.8 Hz, 4H), 3.58-3.63 (m, 2H), 3.49 (s, 3H), 3.41 (s, 3H), 2.63 (t, J = 6.4 Hz, 2H), 2.51–2.60 (m, 4H); 13C NMR (100 MHz, CDCl3) δ = 186.3, 161.5, 161.0, 158.0, 155.0, 147.6, 130.1, 121.0, 117.9, 116.5, 111.5, 66.9, 56.7, 53.3, 43.4, 39.0 ppm. HRMS (ESI) Calcd for C19H24N3O5S [MH+] 406.1437, found 406.1436. 1.3 Synthesis of compound 3 The mixture of compound 2 (0.100 g, 0.31 mmol), K2CO3 (0.14 g; 1.0 mmol) and acetone (20 mL) were stirred at 50 oC for 30 min, and then a solution of iodomethane (0.14 g, 1.0 mmol) in acetone (2 mL) was added and heated under reflux for 6 h. The resulting mixture was filtered and concentrated under vacuum, and the residue was purified on silica gel chromatography eluted with CH2Cl2 : ethanol = 100:1 (v/v) to give compound 3 as a white solid (54 mg, 52%). 1H NMR (400 MHz, CDCl3) δ = 9.06 (s, 1H), 8.83 (s, 1H), 7.58 (d, J = 8.8 Hz, 1H), 6.95 (dd, J = 8.8 Hz & J = 2.0 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 3.92 (s, 3H), 3.75 (t, J = 4.8 Hz, 4H), 3.57-3.61 (m, 2H), 2.61 (t, J = 6.4 Hz, 2H), 2.54 (t, J = 4.4 Hz, 4H); 13
C NMR (100 MHz, CDCl3) δ = 166.2, 163.4, 163.0, 158.1, 149.5, 132.3, 116.3, 115.3, 113.8,
101.7, 68.3, 58.3, 57.4, 54.8, 37.9 ppm. MS(TOF) m/z [M+H+]: 333.1. 1.4 synthesis of compound 5 The compound 4 was prepared according to the previous procedures[3,4], and the preparation of compound 4 was similar to the previous report[5]. Briefly, The mixture of compound 4 (0.120 g, 0.51 mmol), K2CO3 (0.21 g; 1.5 mmol) and DMF (6 mL) were stirred at room temperature for 30 min, then a solution of iodomethane (0.21 g, 1.5 mmol) in DMF (0.5 mL) was added and stirred for further 5 h. Then, the reaction mixture was poured into water, and extracted with EtOAc. The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum, and the residue was purified on silica gel chromatography eluted with CH2Cl2 to give compound 3 as a white solid (83 mg, 65%). 1H NMR (400 MHz, CDCl3) δ = 8.53 (s, 1H), 7.52 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 8.4 Hz & J = 2.4 Hz, 1H), 6.84 (d, J = 2.4 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 3.93 (t, J = 7.2 Hz, 1H). MS(TOF) m/z [M+Na+]: 271.1.
3
Fig. S1. The photographs of probe CS (10 µmol/L) with or without addition of NaClO (10 eq) under the irradiation light of 365 nm.
Fig. S2. The UV absorption of compound 4 (A) and 5 (B) in solutions of different pH values.
Fig. S3. The fluorescence titration spectra of compound 4 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
4
Fig. S4. The variation of fluorescent intensity at 451 nm of compound 4 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
Fig. S5. The fluorescence titration spectra of compound 5 in solutions of different pH values under the irradiation light of 345 nm.
Fig. S6. The UV absorption of compound 2 (A) and 3 (B) in solutions of different pH values.
5
Fig. S7. The fluorescence titration spectra of compound 3 in solutions of different pH values under the irradiation light of 345 nm.
Fig. S8. The fluorescence titration spectra of compound 2 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
Fig. S9. The variations of fluorescent intensity at 451 nm of compound 2 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
6
Fig. S10. The fluorescent excitation spectra of compound 2 in solutions of different pH values (based on the λem = 451 nm).
Fig. S11. The fluorescent emission map spectra of Compound 2 in solutions of varied pH values.
7
Scheme S2. The possible recognition mechanism of probe CS for sensing HClO.
0.30
0.25
Absorption
0.20
0.15
0.10
0.05
0.00 260
280
300
320
340
360
380
400
420
440
460
480
500
Wavelength (nm)
Fig. S12. The absorption spectra of CS (10 µmol/L) toward varying concentrations of NaClO (0-10 eq) in acetate buffer (pH 5.0, containing 10% DMF) at 25 oC. Slit widths: 5 nm.
(A)1.8x10
(B) 1.8x10
6
6
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Fluorescence Intensity
1.6x10
1.4x106 6
1.2x10
6
1.0x10
5
8.0x10
5
6.0x10
5
4.0x10
pH 5.0
1.6x106
Fluorescence Intensity
6
5
2.0x10
0.0 360 380 400 420 440 460 480 500 520 540 560 580 600 620
1.4x106 1.2x106 1.0x106
pH 7.4
8.0x105 6.0x105 4.0x105 5
2.0x10
0.0 0
20
40
60
80
100
120
140
160
Concentration (µmol/L)
Wavelength (nm)
Fig. S13. (A) The fluorescence titration spectra of CS (10 µmol/L) toward varying concentrations of NaClO in PBS buffer (pH 7.4, containing 10% DMF) at 25 oC. Exited at 345 nm, slit widths: 0.5 nm/1.0 nm. (B) The dot plot between the concentrations of HClO and the fluorescence intensity at 451 nm of probe CS in the buffer solutions of pH 5.0 and pH 7.4.
8
Cell Viablity (100%)
100
80
60
40
20
0
10 20 2.5 5 Probe concentration (µmol/L)
0
30
Fig. S14. The cytotoxicity of probe CS at different concentrations for HeLa cells for 12 h.
(M) Piexl Intensity
8
6
4
2
0
0 µM
50 µM
100 µM
250 µM
Fig. S15. The Bright field and fluorescent images of the probe CS (10 µmol/L) in HeLa cells after addition of NaClO (0, 50, 100, 250 µmol/L). Scale bar: 25 µm. (M) The semi-quantification of mean pixel intensities obtained from the corresponding fluorescence images of (A)~(D).
9
Fig. S16. The mass spectrum for the reaction mixture of probe CS and HClO (5 eq) in MeOH/PBS buffer (pH 5.0, v/v = 1:1).
Fig. S17. 1H NMR of compound 2 in d6-DMSO 10
Fig. S18. 13C NMR of compound 2 in d6-DMSO.
Fig. S19. HRMS(ESI) spectrum of compound 2. 11
Fig. S20. 1H NMR of probe CS in CDCl3.
Fig. S21. 13C NMR of probe CS in CDCl3.
12
Fig. S22. HRMS(ESI) spectrum of probe CS.
Fig. S23. 1H NMR of compound 3 in CDCl3. 13
Fig. S24. 13C NMR of compound 3 in CDCl3.
Fig. S25. MS-TOF of compound 3. 14
Fig. S26. 1H NMR of compound 5 in CDCl3.
Fig. S27. MS-TOF of compound 5.
15
References [1] C. Gnaccarini; W. Ben-Tahar; A. Mulani; I. Roy; W.D. Lubell; J.N. Pelletier; J.W. Keillor. Site-specific protein propargylation using tissue transglutaminase. Org. Biomol. Chem. 2012, 5258-5265. [2] S. Shiota; S. Yamamoto; A. Shimomura; A. Ojida; T. Nishino; T. Maruyama. Quantification of Amino Groups on Solid Surfaces Using Cleavable Fluorescent Compounds. Langmuir, 2015, 8824-8829. [3] Mizukami, S.; Watanabe, S.; Hori, Y.; Kikuchi, K. Covalent protein labeling based on noncatalytic beta-lactamase and a designed FRET substrate. J. Am. Chem. Soc. 2009, 131(14):5016-7. [4] Starcevic, S.; Brozic, P.; Turk, S.; Cesar, J.; Rizner, T. L.; Gobec, S. Synthesis and biological evaluation of (6- and 7-phenyl) coumarin derivatives as selective nonsteroidal inhibitors of 17beta-hydroxysteroid dehydrogenase type 1. J. Med. Chem. 2011, 54(1):248-61. [5] Abdizadeh, T.; Kalani, M. R.; Abnous, K.; Tayarani-Najaran, Z.; Khashyarmanesh, B. Z.; Abdizadeh, R.; Ghodsi, R.; Hadizadeh, F. Design, synthesis and biological evaluation of novel coumarin-based benzamides as potent histone deacetylase inhibitors and anticancer agents. Eur. J. Med. Chem. 2017, 13242-62.
16
Highlights: 1. A novel dual-activatable fluorescent probe CS for hypochlorous acid and acidic condition was
rationally designed and synthesized. 2. The probe exhibited good sensitivity, high specificity and fast response towards hypochlorous
acid. 3. The probe was applied to detect exogenous and endogenous hypochlorous acid in living cells 4. The probe demonstrated an excellent lysosome-targetable ability.
Declaration of Interest Statement: All the authors approve that we do not have any commercial or associative interest in connection with the work submitted.
S0003-2670(19)31200-0
DOI:
https://doi.org/10.1016/j.aca.2019.10.004
Reference:
ACA 237138
To appear in:
Analytica Chimica Acta
Received Date: 10 May 2019 Revised Date:
26 September 2019
Accepted Date: 7 October 2019
Please cite this article as: L. Shi, S. Yang, H.-J. Hong, Y. Li, H.-J. Yu, G. Shao, K. Zhang, S.-Z. Gong, A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
Graphical Abstract Figure
Graphical Abstract A novel dual-activatable fluorogenic probe CS has been rationally designed. This probe exhibited good sensitivity, high specificity and fast response towards HClO under the lysosomal acidic conditions, and it has been successfully applied for visualizing exogenous or endogenous HClO in lysosomes of living cells.
A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes Lei Shi a, Sheng Yang b*, Hao-Jia Hong a, Yong Li c, Hui-Juan Yu c,d, Guang Shao c,d*, Kai Zhang e, Sheng-Zhao Gong a* a. Guangdong Engineering Technical Research Center for Green Household Chemicals, Guangdong Industry Polytechnic, Guangzhou, Guangdong, 510300, P. R. China. Email: 1996103022@ gdip.edu.cn. b. Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources, School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, Hunan 410114, P. R. China. Email: [email protected]. c. School of Chemistry, Sun Yat-sen University, Guangzhou, Guangdong, 510275, P. R. China. Email: [email protected]. d. Shenzhen Research Institute, Sun Yat-sen University, Shenzhen, 518057, P. R. China. e. College of Preclinical Medicine, Southwest medical university, Luzhou, Sichuan, 646000, P. R. China. *To whom correspondence should be addressed.
ABSTRACT: Hypochlorite (HClO) is involved in various physiological and pathological processes as well as regulation of lysosomal functions. Thus, it is appreciated to develop efficient molecule tools for precisely detecting HClO in lysosomes. Although several lysosomal-targetable fluorogenic probes for HClO have been developed to date, they still suffered from the discounted sensing performance under lysosomal acidic condition. Herein, on the basis of the “AND” logic gate strategy, a novel dual-activatable fluorogenic probe CS has been rationally designed by simultaneously incorporating HClO recognition site and pH-sensitive group with lysosomal-targetable characteristic into a coumarin fluorophore. Different from the single-activated ones previously reported, CS exhibited good sensitivity, high specificity and fast response towards HClO under the acidic conditions but was out of operation in the neutral or alkaline environment. Importantly, it had been successfully applied for spatial-resolution imaging of exogenous or endogenous HClO in lysosomes.
1. Introduction As one of the most important reactive oxygen species (ROS), hypochlorous acid (HClO) has essential roles in biological systems. The endogenous hypochlorite (HClO/ClO-) is generated from the biological oxidation reaction between hydrogen peroxide (H2O2) and chloride ion (Cl-) by the catalysis of myeloperoxidase (MPO)[1,2]. The HClO in vivo could act as a formidable oxidant and the pathogens-killer in the innate immune systems, and play a key part in many biologically vital processes[3-7]. However, misplaced or excessive HClO would result in serious damage to tissues and lead to various diseases, such as cardiovascular diseases, neuron degeneration, arthritis, and even cancers[8-10]. The lysosome as a kind of membrane-bound cytoplasmic organelles is generally recognized as the cellular “incinerators” for the degradation and recycling of cellular wastes. Recently, there is already compelling evidence that lysosome is also involved in many other fundamental processes, such as secretion,
plasma
membrane
repair,
signaling,
energy
metabolism,
and
lipid
degradation[11,12]. Furthermore, a tight relationship between the HClO level and the redox balance in lysosomes has been found, and aberrant endogenous or exogenous HClO level could lead to the dysfunction of lysosomes and might even cause apoptosis by the rupture of lysosomes[13,14]. Hence, it is very necessary to develop a facile and accurate analytical method for detecting HClO in lysosomes. Comparing with other analytical techniques, fluorimetry based on the organic fluorescent probes is regarded as an ideal tool for real-time monitoring biomolecules in living biosystem due to its high sensitivity, excellent selectivity, non-invasiveness and high spatiotemporal resolution[15-20]. In recent years, a large number of HClO fluorescence probes have been developed[21-24], but many of them were not very appropriate for HClO detection at subcellular organelles because of the deficiency in lysosome-specificity. To address this issue, some lysosome-targeting groups such as morpholine were incorporated into the skeleton of probes with the purpose of guiding the probes into lysosomes[25-31]. However, all of them were only able to show similar fluorescent responses in different pH conditions. Considering the imperfect targeting abilities of the guiding groups and the widespread existence of HClO in cells, lots of interfering signals would be caused in the neutral cytosol and other organelles. In order to solve this problem, we have previously proposed a new strategy for specifically visualizing the targeted analytes in a specific organelle by a fluorescent probe with the target and location dual-controlled molecular switches[32]. As a continuation of our research and considering the acidic environment of lysosomes (pH 4.0-6.0), we rationally designed a novel HClO and pH dual-activatable fluorescent probe CS. As depicted in Scheme
1 and Fig.1, N,N-dimethyl-thiocarbamate (DMTC) was separated due to the oxidation of HClO, resulting in the weak fluorescence (fluorescence quantum yield Φ = 6%) in neutral aqueous condition. By contrast, this probe would display much stronger blue fluorescence (Φ = 22%) under the acidic environment due to the protonation of phenolic anion and morpholine group, indicating its specific detection performance at lysosomal acidic condition. To the best of our knowledge, such a dual-locked model system that activates the fluorescent signals by the acidic condition and HClO remains unreported.
Scheme 1. The fluorescence response mechanisms of probe CS for sensing H+ and HClO alone or both of them.
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Fig. 1. The UV absorption and fluorescence spectra of probe CS (10 µmol/L) under different conditions (λex = 345 nm). (a) In buffer solution (10% DMF, pH 5.0); (b) with addition of NaClO (8 eq) in the same buffer solution; (c) in PBS buffer solution (10% DMF, pH 7.4); (d) with addition of NaClO (8 eq) in the same buffer solution.
2. Experimental section 2.1. Apparatus and chemicals 1
H NMR (400 MHz) and
13
C NMR (100 MHz) spectra were conducted by a Bruker
Avance 300 spectrometer. High-resolution mass spectrometry (HRMS) involved a Q-TOF6510 spectrograph (Agilent). UV–vis spectra were measured by a Hitachi U-4100 spectrophotometer. Quartz cuvettes used for all measurements were 1 cm path length and 3
mL volume. Perkin-Elmer LS-55 and Edinburgh F35 luminescence spectrophotometer were used to measure fluorescent spectra (slit widths: 0.5 nm/1.0 nm). A Mettler Toledo pH meter was used to test the pH values. The twice-distilled water was used throughout all experiments. All reagents were purchased from Energy Chemical and used without further purification unless otherwise stated. 2.2 General procedure for absorption and fluorescence analysis A stock solution (1.0 mmol/L) of probe CS was prepared in DMF, and its test solution (10 µmol/L) was prepared by the dilution of the stock solution with the buffer solution (containing 10% DMF) under an indicated pH value. Then various analytes were added separately, and the absorption and fluorescence measurements were carried out after being incubated for 10 min. For fluorescence measurements, the excitation wavelength was set at 345 nm with a slit width of 0.5×1.0 nm. Additionally, the absolute fluorescence quantum yield (Φ) values were determined by using an integrating sphere according to the previous report[33]. 2.3 Computational methods All the calculations were performed using density functional theory (DFT) and the Gaussian 09 program[34]. The ground state structures of the compounds were optimized by the density functional theory (DFT) using a B3LYP. The 6-31+G (d, p) basis sets were employed for CS and 2-O-, and 6-31G (d, p) basis sets were employed for CS+H+ and 2+H+. The excited-state calculations were carried out with the time-dependent density functional theory (TD-DFT) with the optimized structure of the ground state. The solvent effects were modeled with the polarizable continuum model (PCM). 2.4 Cell cytotoxic assays and imaging HeLa cells were seed in DMEM medium (Thermo Scientific HyClone) and cultured with a mixture of 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin and 100 U/mL gentamicin, Then the cells were grown at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The cytotoxic effects of the probe CS were tested by the MTT assay according to the previous report[27]. Before imaging, the HeLa cells were incubated with the probe CS (10.0 µmol/L) and washed three times with PBS buffer. Then the cells were treated by NaClO (0 ~ 250 µmol/L) and washed with PBS buffer. After that, the cells were sent to imaging analysis, and the fluorescent images (400-500 nm) were acquired by excitation with a multi Ar laser (345 nm). For monitoring endogenous HClO in the lysosomes, the cells were pretreated with probe CS (10 µmol/L) and lysosome tracker LysoTracker red (1.0 µmol/L) for 30 min, and then the
cells were incubated with phorbol 12-myristate 13-acetate (PMA) for 30 min. After rinsing cells with PBS buffer again, the imaging analysis was carried out by the confocal laser scanning microscopy.
3. Results and discussion 3.1. Design and synthesis of the probe CS In order to further understand the roles and functions of HClO in lysosomes, we hope to construct a novel fluorescent probe which could detect HClO precisely and specifically in lysosomes. With this goal in mind, we utilized the pH difference between the lysosome and other subcellular organelles, and developed a novel fluorescent probe dual-controlled by HClO and pH values. In this research, 7-hydroxy-coumarin was chosen as the fluorophore due to its high quantum yield, good stability, and biological compatibility. And the morpholine group was incorporated as a guiding group for the lysosome. More importantly, the phenolic hydroxyl group and the morpholine group were also acted as the binding sites for H+, leading to the changes
of
fluorescence
under
the
different
pH
conditions.
Additionally,
the
dimethylthiocarbamate (DMTC) was employed as the recognition group for HClO owing to its fast and selective oxidation reaction towards HClO[35-38]. Therefore, a novel lysosome-targetable fluorescent probe CS with the dual recognizing sites for HClO and H+ was designed and synthesized as shown in scheme S1. Besides, compound 3, 4 and 5 were also synthesized with the purpose of understanding the pH effects of this probe. All these compounds were characterized by NMR and HRMS spectra (Fig. S17-S27). (A) 3.0x10 (B) 1000 6
Probe Probe + 4 eq HClO Probe + 8 eq HClO Compound 2
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Fig. 2. (A) The fluorescence intensity variation of compound 2 and probe CS under different pH conditions (10 µmol/L, λex = 345 nm). (B) The Time-dependent fluorescence changes of probe CS (10 µmol/L) upon addition of NaClO (8 eq), NaOH (adjust to pH 10) and hydrochloric acid (adjust to pH 5), and the test was carried out under the excitation at 345 nm.
3.2. The pH effects of probe CS In this study, the hydroxyl and morpholine group were employed as the pH-sensing sites
and combined into the coumarin fluorophore, and we studied the pH effects of the two moieties, respectively. According to previous reports[39,40], the UV-absorbance and excitation spectra of 7-hydroxycoumarin would be red-shifted in the basic aqueous solution by comparison with that in the neutral or acidic solutions. Besides, the maximum fluorescent emission bands were almost the same during the pH range of 3~10 due to the rapid excited state acid-base equilibrium which took place during the singlet excited state lifetime of 10-8 seconds[39]. Therefore,
the
fluorescent
intensity
at
the
maximum
emission
wavelength
of
7-hydroxycoumarin would become pH-dependent by giving an excitation light at a fixed wavelength. In order verify the above hypothesis, the compound 4 and 5 were synthesized and investigated. As shown in Fig. S3 and S4, the fluorescent intensity at 451 nm of compound 4 enhanced greatly with increasing pH values under the irradiation light of 405 nm, but it became decreased when excited at 345 nm as a result of the red-shift on its UV-absorbance and excitation spectra. In contrast, the compound 5 as a hydroxyl-ether derivative of 4, emitted much stable fluorescent performance during the pH range of 3.2~9.2, illustrating the great pH-sensitive ability of the hydroxyl moiety. Subsequently, we determined the pH-dependent property of compound 3 to investigate the pH effect of the morpholine unit. Unlike compound 4, the fluorescence of 3 greatly enhanced when the pH value decreased from 8 to 4, which was due to the protonated nitrogen and blocked PET process from the morpholine moiety to the fluorophore[41]. By combining the two pH sensing sites of hydroxyl and morpholine group, compound 2 was also an excellent pH-sensitive probe. As the pH increased from 3 to 10, the UV-absorption spectra of 2 displayed a large red-shift, and its fluorescent intensity at 451 nm also showed greatly pH-dependent(Fig. S8-S11). In addition, the pKa values of compound 2 was calculated to be 6.06 according to the Henderson-Hasselbalch equation. As expected, the probe CS as a derivative of 2, displayed much stronger fluorescent responses to HClO in acidic conditions(Fig. 2A). The extraordinary fluorescent responses under acidic conditions provided a feasible way for constructing an ideal lysosome-targeted probe for visualizing HClO. Furthermore, the responses of CS to HClO and the pH variations were quite fast, which could be completed within a few seconds (Fig. 2B). The results suggested that probe CS would be very favorable for the real-time monitoring of HClO in lysosomal acid conditions. 3.3 The DFT/TDDFT calculations In order to gain a thorough comprehension of the changes in the photophysical properties
of compound 2 and CS, the theoretical calculations based on DFT and TDDFT were carried out. The pictorial drawing of their optimized geometries and the frontier molecular orbitals are shown in Fig 3, and the calculated data are summarized in Table 1.
Fig. 3. The frontier molecular orbital plots of compound 2 and CS in water (PCM model). Table 1. Frontier molecular orbital profiles of 2 and CS based on DFT/TD-DFT calculations. Compoun d
UV-vis Absorption
Emission
energy gap (nm)
fa
major contribution
CS
463.72 (2.67 eV) 390.38 (3.18 eV) 366.05 (3.39 eV) 317.68 (3.90 eV)
0.01 0.04 0.02 0.54
HOMO → LUMO (99.80%) HOMO-1 → LUMO (98.72%) HOMO-2 → LUMO (99.13%) HOMO-3 → LUMO (90.97%)
820.16 0.04 (1.51 eV)
HOMO → LUMO (99.84%)
CS+H+
387.91 (3.20 eV) 359.43 (3.45 eV) 320.05 (3.87 eV)
0.05 0.01 0.62
HOMO → LUMO (97.71%) HOMO-1 → LUMO (98.74%) HOMO-2 → LUMO (93.56%)
540.83 0.03 (2.29 eV)
HOMO → LUMO (99.51%)
2+H+
325.88 (3.80 eV)
0.63
HOMO → LUMO (96.72%)
368.20 0.83 (3.37 eV)
HOMO → LUMO (98.70%)
2-O
381.58 (3.25 eV)
0.74
HOMO → LUMO (96.96%)
433.67 0.87 (2.86 eV)
HOMO → LUMO (98.58%)
energy gap (nm)
fb
major contribution
fa is the oscillator strength of the absorption coefficient. fb is the oscillator strength of the emission coefficient.
As shown in Fig. 3, the HOMO and LUMO of compound 2-O and 2+H+ were localized on the coumarin fluorophore, which would facilitate the internal charge transfer (ICT) process upon excitation. It should be pointed out that the allowed HOMO → LUMO transition with the oscillator strength (f = 0.74 and 0.63) were observed for both derivatives (2-O and 2+H+) of compound 2. By comparing with 2+H+ (4.12 eV), a lower energy gap between the HOMO and LUMO was found for 2-O (3.49 eV). This might be the result of the relatively higher electron-releasing ability of the phenoxide anion in comparison with the phenol group[42]. As
a result, the ICT process of 2-O would be enhanced, leading to the redshift of its absorption spectra. This prediction has been proved by the experimental results as shown in Fig. S6. When the pH conditions varied from acid to base, the maximum absorption wavelength and the maximum excitation wavelength of compound 2 were red-shifted into a lower-energy region (345 nm to 405 nm). Consequently, when environmental pH value increased, a decrease of fluorescent intensities was observed under the irradiation light at 345 nm, while an enhancement of its fluorescence would appear with the excitation light source of 405 nm (Fig. S8~S11). Unlike compound 2, the HOMO of probe CS was localized on the morpholine moiety, and the HOMO of CS+H+ was mainly dominated by the DMTC group (Fig. 4). The occupation of HOMO by this group would prevent electronic relaxation from LUMO to HOMO, resulting in their fluorescence quenching[43,44]. Furthermore, for two forms of the probe (CS and CS+H+), the electron transitions of HOMO → LUMO were forbidden due to the extremely low oscillator strength (f = 0.01 and 0.05). Even though HOMO-3 → LUMO and HOMO-2 → LUMO might be the allowed transition, the emissive state of organic fluorophores was usually occurred during the HOMO → LUMO transition. Furthermore, considering the very low the oscillator strength of emission coefficient (f = 0.04 and 0.03), the radiative LUMO →HOMO transition of the probe was extremely unlikely to happen, implying that probe CS and CS+H+ were non-fluorescent according to the principle of Kasha's rule[45,46]. However, when the DMTC part was oxidized off by HClO, the electronic transition occurred completely in the coumarin moiety, and the oscillator strength of emission coefficient rose sharply (2+H+: f = 0.83; 2-O: f = 0.87), indicating the recovery of their strong fluorescence. The above predictions were in full agreement with the experimental results. 3.4 Proposed sensing mechanism of the probe According to the experimental results and the published reports[34,47,48], we proposed the possible mechanism of probe CS for detecting HClO as depicted in Scheme S2. The strong oxidation capability of HClO to sulfide moiety would induce the formation of unstable imine moiety via the release of SO2, and then the next hydrolysis reaction resulted in the generation of the coumarin fluorophore which would turn into the strong fluorescent compound 2 under the acidic condition. To prove this proposed sensing mechanism, the reaction mixture of probe CS and HClO was characterized by MS spectrum. After addition of NaClO, the peaks for probe CS (m/z 406.48, [M+H]+) lowered obviously, while a new peak for the predicted product (m/z 319.45, [M+H]+) emerged (Fig. S16). The MS analysis revealed the fact that the fluorescence turn-on
process of probe CS involved the oxidation and separation of DMTC group by HClO.
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y = 18216x+67995 R2 = 0.995
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Fig. 4. (A) The fluorescence titration spectra of CS (10 µmol/L) toward varying concentrations of NaClO in acetate buffer (pH 5.0, containing 10% DMF) at 25 oC. Exited at 345 nm. (B) Standard
curve for relationship between the concentration of HClO and the fluorescence intensity at 451 nm of probe CS in the buffer solutions of pH 5.0 and pH 7.4.
Fig. 5. Fluorescence responses of probe CS (10 µmol/L) towards various analytes (200 µmol/L except for 100 µmol/L HClO) in acetate buffer (pH 5.0, containing 10% DMF). Data shown in turn: (1) blank, (2) NaClO, (3) K+, (4) Na+, (5) Mg2+, (6) Zn2+, (7) Ca2+, (8) Cu2+, (9) Pb2+, (10) Al3+, (11) Fe3+, (12) NO3-, (13) NO2-, (14) I−, (15) SO42-, (16) PO43-, (17) S2O82- (18) H2O2, (19) ⋅OH, (20) TBHP,·(21) O2-, (22) ONOO−.
3.5 The spectroscopic properties of probe CS to HClO Encouraged by the above result, the fluorescent properties of probe CS were systematically analyzed. In the acidic condition, the probe displayed a fast and dramatic enhancement of its fluorescence around 451 nm upon addition of HClO, and its fluorescent intensity at 451 nm has a great linear relationship (R2 = 0.995) in the concentration range of 0~90 µmol/L (Fig. 4 and S13). However, a much milder enhancement of its fluorescence could be observed by replacement of the neutral buffer solution, suggesting that the fluorescent response of CS was really pH-dependent. Moreover, the quantum yield of the reaction
mixture of probe CS and HClO was up to 22% in comparison with probe CS and CS+H+ (Φ = 0.1% and 0.2%), indicating the huge enhancement of its fluorescence and extremely low background. Moreover, the limit of detection of probe CS was evaluated to be 24.3 nmol/L according to the formula of LOD = 3σ/Slope, implying the excellent sensitivity of probe CS for detecting HClO. 3.6 Selectivity of probe The selectivity is a very important parameter for evaluating the application performance of the fluorescent probe in the biological systems. Thus, we investigated the influence of representative metal cations (K+, Na+, Mg2+, et al), anions (NO3-, NO2-, et al), RNS (ONOO-), and other ROS (H2O2, ⋅OH, TBHP, et al) to probe CS. As shown in Fig. 5, no significant fluorescent response could be observed in the presence of the above analytes except for HClO, suggesting the excellent selectivity of probe CS towards HClO over other bioactive substances. Furthermore, the competition experiments revealed that the response of probe CS toward HClO would not be disrupted in the presence of these species, which is helpful for monitoring HClO in cells or actual samples.
(M) Piexl Intensity
8
CS+PMA
6
4
2 CS only
CS+ABAH CS+BMA/BFA
0
Fig. 6. The Bright field and fluorescent images of HeLa cells. (A) Cells only(control); (B) by incubation with PMA (25 mg/L) for 2 h in the presence of CS (10 µmol/L); (C) by incubation with ABAH for 2 h in the presence of CS (10 µmol/L); (D) by co-incubation with PMA (25 mg/L) and BFA (1 mmol/L), and then treated with CS for 2 h. Scale bar: 25 µm. (M) The semi-quantification of mean
pixel intensities obtained from the corresponding fluorescence images of (A)~(D).
Fig. 7. The fluorescence images of HeLa cells incubated with (A) Probe CS (10 µM) and NaClO (250 µM) for 30 min (blue channel: 405~640 nm, Ex = 365 nm); (B) LysoTracker Deep Red (50 nM) (red channel: 640~700 nm, Ex = 639 nm); (C) Merged images of (A) and (B) (purple); (D) Intensity profile of linear region of interest across HeLa cells incubated with LysoTracker Deep Red and blue channel of CS. Scale bar: 25 µm.
3.7 The bioassay of HClO in living cells Before used for cell imaging, the cell cytotoxicity assay of probe CS was investigated according to the MTT assay. After incubation with probe (0~30 µmol/L) for 12 h, the HeLa cell survival rate was more than 85%, indicating the low cytotoxicity and the favorable biocompatibility of this probe (Fig. S14). Subsequently, the cells were treated with probe (10 µmol/L) for 30 min and washed with PBS solution three times, and then incubated with various concentrations (0, 50, 100, 250 µmol/L) of NaClO. As is illustrated in Fig. S15A, HeLa cells gave almost no fluorescence without the addition of NaClO. However, the blue fluorescence from the cells increased remarkably with increasing concentration of NaClO (Fig. S15B~D). Besides, when the cells were pretreated with phorbol 12-myristate 13-acetate (PMA, as a ROS stimulant), HeLa cells displayed much stronger fluorescence (Fig. 6B), while the pretreatment of cells with 4-aminobenzoic acid hydrazide (ABAH, as a specific inhibitor of MPO) would lead to the extremely weak fluorescence (Fig. 6C). The results demonstrated that probe CS could penetrate cell membranes and detect exogenous and endogenous HClO in living cells. Additionally, in order to confirm that probes would only display the intracellular response in lysosome rather than cytoplasm and other neutral organelles, bafilomycin A1 (BFA) was selected to alkalinize the intracellular lysosomal pH values of the cells. By comparing with Fig. 6B and 6D, it is obvious that the fluorescence signals were suppressed severely by treatment with BFA, implying that the fluorescent response of probe CS was dependent on the lysosomal acid conditions. To further examine the capability of probe CS in imaging of HClO in the lysosomes, co-localization experiments were carried out. By pretreatments with LysoTracker Deep Red
DND-99 and probe CS, HeLa cells were then treated with NaClO (250 µmol/L). As illustrated in Fig. 7, the fluorescent signals of probe CS and LysoTracker Deep Red overlapped very well, with the Pearson's and overlap coefficients of 0.914, indicating that probe CS could be specifically targeted lysosomes in living cells. 4. Conclusion In this work, we have developed a lysosome-targetable fluorescent probe for detecting the lysosomal HClO in living cells by joining the dual-controlled molecular switches and coumarin fluorophores. The probe CS displayed high sensitivity, great selectivity and rapid response towards HClO, and could quantitatively detect HClO in the range of 0~80 µmol/L with a low detection limit of 24.3 nmol/L. According to the cellular imaging experiments, the probe exhibited excellent lysosome-targetable localization capability and good practicability for visualizing exogenous and endogenous HClO in the lysosomes of living cells, indicating its promising applications in biological systems. Acknowledgment We gratefully acknowledge financial support from the Research Project of Department of Education of Guangdong Province (2017GkQNCX003), the Science and Technology Planning Project of Guangzhou City (201904010250), the fund of Specialized Cultivation of Scientific and Technological Innovation for College Students in Guangdong Province (pdjh2019b0691), the Science and Technology Planning Project of Shenzhen City (JCYJ20180307164055935), Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources (2018KJY06), National Key R&D Program of China (2017YFC1600306). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.
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Supporting Information for A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes Lei Shi a, Sheng Yang b*, Hao-Jia Hong a, Yong Li c, Hui-Juan Yu c, Guang Shao c,d*, Kai Zhang e, Sheng-Zhao Gong a* a. Guangdong Engineering Technical Research Center for Green Household Chemicals, Guangdong Industry Polytechnic, Guangzhou, Guangdong, 510300, P. R. China. Email: 1996103022@ gdip.edu.cn. b. Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources, School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, Hunan 410114, P. R. China. Email: [email protected]. c. School of Chemistry, Sun Yat-sen University, Guangzhou, Guangdong, 510275, P. R. China. Email: [email protected]. d. Shenzhen Research Institute, Sun Yat-sen University, Shenzhen, 518057, P. R. China. e. College of Preclinical Medicine, Southwest medical university, Luzhou, Sichuan, 646000, P. R. China.
Table of Contents Description 1. The synthesis procedures of probe CS 2. The photographs of probe in the absence and presence of HClO 3. The pH effect study of compound 2, 3, 4 and 5
page 2-3 4 4-7
4. The recognition mechanism of probe for sensing HClO.
8
5. The absorption and fluorescent spectra of probe toward NaClO
8
6. Cytotoxic effect of probe CS
9
7. The fluorescent images of the probe after addition of NaClO
9
8. The NMR and MS spectra of compound 2 and CS
10 10
9. References 1
1. Synthesis of probe CS The coumarin 1 was prepared according to the procedure previously described[1,2].
Scheme S1. The synthetic route and structure of probe CS
1.1 Synthesis of compound 2 Compound
1
(0.60
g,
2.91
mmol),
DMAP
(14
mg,
0.11
mmol)
and
N-hydroxysuccinimide (0.43 g; 3.3 mmol) were dissolved in 7 mL of anhydrous DMF, and stirred at 0°C for 30 min. A solution of DCC (0.68 g, 4.6 mmol) in anhydrous CH2Cl2 (4 mL) was added dropwisely and stirred for 1 h at room temperature. Then the resulting mixture was added dropwisely into the solution of 4-(2-Aminoethyl)morpholine (0.44 g, 3.4 mmol) in anhydrous CH2Cl2 (10 mL) at 0 oC under N2, and the mixture was stirred at room temperature overnight. After that, the precipitates were filtrated, and the filtrate was concentrated under vacuum and redissolved in dichloromethane. The organic layers was washed by water and brine, dried over with Na2SO4 and evaporated under vacuum. The residue was purified on silica gel chromatography eluted with ethyl acetate: ethanol = 50:1 (v/v) to give compound 2 as a white solid (0.57 g, 62%). 1H NMR (400 MHz, d6-DMSO) δ = 8.85 (t, J = 6.4 Hz, 1H), 8.79 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 6.88 (dd, J = 8.8 Hz & J = 2.0 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 3.59 (t, J = 4.8 Hz, 4H), 3.40-3.46 (m, 2H), 3.34 (bs, 1H), 2.48 (t, J = 6.4 Hz, 2H), 2.40–2.44 (m, 4H); 13C NMR (100 MHz, d6-DMSO) δ = 163.6, 161.3, 160.9, 156.2, 147.9, 131.9, 114.3, 113.5, 111.0, 101.7, 66.2, 56.6, 53.0, 40.0 ppm. HRMS (ESI) Calcd for C16H19N2O5 [MH+] 319.1294, found 319.1300. 1.2 Synthesis of probe CS Under N2 in ice–water bath, a solution of dimethylthiocarbamoyl chloride (0.42 g, 3.4 mmol) in anhydrous CH2Cl2 (3 mL) were added dropwisely into the solution of compound 2 (0.32 g, 1.0 mmol) and DIPEA (1.0 mL) in THF/CH2Cl2 (10 mL, v/v, 1:1). After being cooled for 30 min, the resulting mixture was stirred at room temperature overnight. Then the solution was concentrated under vacuum and the residue was purified on silica gel chromatography eluted with ethyl acetate: ethanol = 50:1 (v/v) to give compound CS as a white solid (0.28 g, 2
70%). 1H NMR (400 MHz, CDCl3) δ = 9.13 (s, 1H), 8.91 (s, 1H), 7.16 (s, 1H), 7.14~7.16 (m, 2H), 3.78 (t, J = 4.8 Hz, 4H), 3.58-3.63 (m, 2H), 3.49 (s, 3H), 3.41 (s, 3H), 2.63 (t, J = 6.4 Hz, 2H), 2.51–2.60 (m, 4H); 13C NMR (100 MHz, CDCl3) δ = 186.3, 161.5, 161.0, 158.0, 155.0, 147.6, 130.1, 121.0, 117.9, 116.5, 111.5, 66.9, 56.7, 53.3, 43.4, 39.0 ppm. HRMS (ESI) Calcd for C19H24N3O5S [MH+] 406.1437, found 406.1436. 1.3 Synthesis of compound 3 The mixture of compound 2 (0.100 g, 0.31 mmol), K2CO3 (0.14 g; 1.0 mmol) and acetone (20 mL) were stirred at 50 oC for 30 min, and then a solution of iodomethane (0.14 g, 1.0 mmol) in acetone (2 mL) was added and heated under reflux for 6 h. The resulting mixture was filtered and concentrated under vacuum, and the residue was purified on silica gel chromatography eluted with CH2Cl2 : ethanol = 100:1 (v/v) to give compound 3 as a white solid (54 mg, 52%). 1H NMR (400 MHz, CDCl3) δ = 9.06 (s, 1H), 8.83 (s, 1H), 7.58 (d, J = 8.8 Hz, 1H), 6.95 (dd, J = 8.8 Hz & J = 2.0 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 3.92 (s, 3H), 3.75 (t, J = 4.8 Hz, 4H), 3.57-3.61 (m, 2H), 2.61 (t, J = 6.4 Hz, 2H), 2.54 (t, J = 4.4 Hz, 4H); 13
C NMR (100 MHz, CDCl3) δ = 166.2, 163.4, 163.0, 158.1, 149.5, 132.3, 116.3, 115.3, 113.8,
101.7, 68.3, 58.3, 57.4, 54.8, 37.9 ppm. MS(TOF) m/z [M+H+]: 333.1. 1.4 synthesis of compound 5 The compound 4 was prepared according to the previous procedures[3,4], and the preparation of compound 4 was similar to the previous report[5]. Briefly, The mixture of compound 4 (0.120 g, 0.51 mmol), K2CO3 (0.21 g; 1.5 mmol) and DMF (6 mL) were stirred at room temperature for 30 min, then a solution of iodomethane (0.21 g, 1.5 mmol) in DMF (0.5 mL) was added and stirred for further 5 h. Then, the reaction mixture was poured into water, and extracted with EtOAc. The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum, and the residue was purified on silica gel chromatography eluted with CH2Cl2 to give compound 3 as a white solid (83 mg, 65%). 1H NMR (400 MHz, CDCl3) δ = 8.53 (s, 1H), 7.52 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 8.4 Hz & J = 2.4 Hz, 1H), 6.84 (d, J = 2.4 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 3.93 (t, J = 7.2 Hz, 1H). MS(TOF) m/z [M+Na+]: 271.1.
3
Fig. S1. The photographs of probe CS (10 µmol/L) with or without addition of NaClO (10 eq) under the irradiation light of 365 nm.
Fig. S2. The UV absorption of compound 4 (A) and 5 (B) in solutions of different pH values.
Fig. S3. The fluorescence titration spectra of compound 4 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
4
Fig. S4. The variation of fluorescent intensity at 451 nm of compound 4 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
Fig. S5. The fluorescence titration spectra of compound 5 in solutions of different pH values under the irradiation light of 345 nm.
Fig. S6. The UV absorption of compound 2 (A) and 3 (B) in solutions of different pH values.
5
Fig. S7. The fluorescence titration spectra of compound 3 in solutions of different pH values under the irradiation light of 345 nm.
Fig. S8. The fluorescence titration spectra of compound 2 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
Fig. S9. The variations of fluorescent intensity at 451 nm of compound 2 in solutions of different pH values under the irradiation light of 345 nm (A) or 405 nm (B).
6
Fig. S10. The fluorescent excitation spectra of compound 2 in solutions of different pH values (based on the λem = 451 nm).
Fig. S11. The fluorescent emission map spectra of Compound 2 in solutions of varied pH values.
7
Scheme S2. The possible recognition mechanism of probe CS for sensing HClO.
0.30
0.25
Absorption
0.20
0.15
0.10
0.05
0.00 260
280
300
320
340
360
380
400
420
440
460
480
500
Wavelength (nm)
Fig. S12. The absorption spectra of CS (10 µmol/L) toward varying concentrations of NaClO (0-10 eq) in acetate buffer (pH 5.0, containing 10% DMF) at 25 oC. Slit widths: 5 nm.
(A)1.8x10
(B) 1.8x10
6
6
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Fluorescence Intensity
1.6x10
1.4x106 6
1.2x10
6
1.0x10
5
8.0x10
5
6.0x10
5
4.0x10
pH 5.0
1.6x106
Fluorescence Intensity
6
5
2.0x10
0.0 360 380 400 420 440 460 480 500 520 540 560 580 600 620
1.4x106 1.2x106 1.0x106
pH 7.4
8.0x105 6.0x105 4.0x105 5
2.0x10
0.0 0
20
40
60
80
100
120
140
160
Concentration (µmol/L)
Wavelength (nm)
Fig. S13. (A) The fluorescence titration spectra of CS (10 µmol/L) toward varying concentrations of NaClO in PBS buffer (pH 7.4, containing 10% DMF) at 25 oC. Exited at 345 nm, slit widths: 0.5 nm/1.0 nm. (B) The dot plot between the concentrations of HClO and the fluorescence intensity at 451 nm of probe CS in the buffer solutions of pH 5.0 and pH 7.4.
8
Cell Viablity (100%)
100
80
60
40
20
0
10 20 2.5 5 Probe concentration (µmol/L)
0
30
Fig. S14. The cytotoxicity of probe CS at different concentrations for HeLa cells for 12 h.
(M) Piexl Intensity
8
6
4
2
0
0 µM
50 µM
100 µM
250 µM
Fig. S15. The Bright field and fluorescent images of the probe CS (10 µmol/L) in HeLa cells after addition of NaClO (0, 50, 100, 250 µmol/L). Scale bar: 25 µm. (M) The semi-quantification of mean pixel intensities obtained from the corresponding fluorescence images of (A)~(D).
9
Fig. S16. The mass spectrum for the reaction mixture of probe CS and HClO (5 eq) in MeOH/PBS buffer (pH 5.0, v/v = 1:1).
Fig. S17. 1H NMR of compound 2 in d6-DMSO 10
Fig. S18. 13C NMR of compound 2 in d6-DMSO.
Fig. S19. HRMS(ESI) spectrum of compound 2. 11
Fig. S20. 1H NMR of probe CS in CDCl3.
Fig. S21. 13C NMR of probe CS in CDCl3.
12
Fig. S22. HRMS(ESI) spectrum of probe CS.
Fig. S23. 1H NMR of compound 3 in CDCl3. 13
Fig. S24. 13C NMR of compound 3 in CDCl3.
Fig. S25. MS-TOF of compound 3. 14
Fig. S26. 1H NMR of compound 5 in CDCl3.
Fig. S27. MS-TOF of compound 5.
15
References [1] C. Gnaccarini; W. Ben-Tahar; A. Mulani; I. Roy; W.D. Lubell; J.N. Pelletier; J.W. Keillor. Site-specific protein propargylation using tissue transglutaminase. Org. Biomol. Chem. 2012, 5258-5265. [2] S. Shiota; S. Yamamoto; A. Shimomura; A. Ojida; T. Nishino; T. Maruyama. Quantification of Amino Groups on Solid Surfaces Using Cleavable Fluorescent Compounds. Langmuir, 2015, 8824-8829. [3] Mizukami, S.; Watanabe, S.; Hori, Y.; Kikuchi, K. Covalent protein labeling based on noncatalytic beta-lactamase and a designed FRET substrate. J. Am. Chem. Soc. 2009, 131(14):5016-7. [4] Starcevic, S.; Brozic, P.; Turk, S.; Cesar, J.; Rizner, T. L.; Gobec, S. Synthesis and biological evaluation of (6- and 7-phenyl) coumarin derivatives as selective nonsteroidal inhibitors of 17beta-hydroxysteroid dehydrogenase type 1. J. Med. Chem. 2011, 54(1):248-61. [5] Abdizadeh, T.; Kalani, M. R.; Abnous, K.; Tayarani-Najaran, Z.; Khashyarmanesh, B. Z.; Abdizadeh, R.; Ghodsi, R.; Hadizadeh, F. Design, synthesis and biological evaluation of novel coumarin-based benzamides as potent histone deacetylase inhibitors and anticancer agents. Eur. J. Med. Chem. 2017, 13242-62.
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Highlights: 1. A novel dual-activatable fluorescent probe CS for hypochlorous acid and acidic condition was
rationally designed and synthesized. 2. The probe exhibited good sensitivity, high specificity and fast response towards hypochlorous
acid. 3. The probe was applied to detect exogenous and endogenous hypochlorous acid in living cells 4. The probe demonstrated an excellent lysosome-targetable ability.
Declaration of Interest Statement: All the authors approve that we do not have any commercial or associative interest in connection with the work submitted.