Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Open AccessCCS ChemistryRESEARCH ARTICLE1 Oct 2021Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells Rui-Ying Guo†, Yu-Tian Zhang†, Supphachok Chanmungkalakul, Hao-Ran Guo, Yongzhou Hu, Jia Li, Xiaogang Liu, Yi Zang and Xin Li Rui-Ying Guo† College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 †R.-Y. Guo and Y.-T. Zhang contributed equally to this work.Google Scholar More articles by this author , Yu-Tian Zhang† State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 University of Chinese Academy of Sciences, Beijing 100049 †R.-Y. Guo and Y.-T. Zhang contributed equally to this work.Google Scholar More articles by this author , Supphachok Chanmungkalakul Fluorescence Research Group, Singapore University of Technology and Design, Singapore 487372 Google Scholar More articles by this author , Hao-Ran Guo State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 University of Chinese Academy of Sciences, Beijing 100049 Google Scholar More articles by this author , Yongzhou Hu College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 Google Scholar More articles by this author , Jia Li State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 University of Chinese Academy of Sciences, Beijing 100049 Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory for Marine Science and Technology, Qingdao 266237 Google Scholar More articles by this author , Xiaogang Liu Fluorescence Research Group, Singapore University of Technology and Design, Singapore 487372 Google Scholar More articles by this author , Yi Zang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 Google Scholar More articles by this author and Xin Li *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202000501 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Nitric oxide (NO) participates in various pathways and revealing its dynamics is critical for resolving its pathophysiology. While there are methods available for detecting biological NO, few are capable of tracking NO dynamics. Herein, inspired by the cellular machinery of reversible thiol modification by NO, we have successfully designed a family of cysteine analogues tagged with fluorophores for visualizing cellular NO dynamics. Biocompatible probes 1a and 2a were found to be most tolerated by the cellular machinery to swiftly switch between their original fluorescent state and quenched S-nitrosated state in response to cellular NO dynamics. We showcased their applicability in various cell types and utility to track NO fluctuations in activated murine macrophages in response to an anti-inflammatory agent treatment. We expect that probes 1a and 2a will afford promising tools for studying NO pathophysiology by tracking its dynamics in health and disease. Download figure Download PowerPoint Introduction First being recognized as an endothelium-derived relaxing factor,1 nitric oxide (NO) is continually appreciated for its diverse roles in virtually every cell function. Synthesis and metabolism of NO are tightly regulated in living cells.2–4 Dysregulation of NO homeostasis is associated with various diseases, for example, cardiovascular diseases, inflammatory diseases, and cancers.5–7 However, the precise roles of NO in these diseases remain unanswerable, especially in the immune system, where both signaling and deleterious effects have been observed.8,9 This is largely due to the difficulty of monitoring NO signaling pathways given the diffusive and unstable nature of NO. Consequently, it is imperative to formulate strategies to enable real-time visualization of NO dynamics in its native environment to resolve its physiological and pathological roles in biological systems. NO is short-lived in vivo, and biological NO is often measured as the concentration of its metabolites.10 Griess assay is one of the most extensively used methods for in vitro NO detection (especially in the field of drug screening), due to its sensitivity and low cost.11 NO may also be measured as the conversion of stable isotope-labeled arginine to -labeled NO metabolites or -labeled citrulline.12,13 However, these methods are not applicable to live cells and are therefore not eligible for recording NO dynamics. While electron paramagnetic resonance (EPR) spectroscopy can be used to directly measure NO and this method is applicable for in vivo observation,14,15 the limited availability of EPR instrumentation limits the popularization of this technique. In contrast, the wide availability of various fluorescence microscopes has made fluorescent imaging a promising approach to interrogate NO pathways in native environments. To date, several fluorescent probes have been developed to image NO in live cells, mainly relying on the N-nitrosation reaction between the aniline moieties of the probes and NO to initiate the detection. Notably, the Nagano group16,17 first used an o-phenylenediamine moiety to trap NO after N-nitrosation to form a triazole, allowing the turn-on of fluorescent output. Ever since, the o-phenylenediamine moiety has been appended to various fluorophores, including fluorescein,18 Rhodamine,19–21 BODIPY,22,23 Cyanine,24 1,8-naphthalimide,25 and so forth, to develop probes with distinct emission properties for NO detection. Later on, the Ford and Lippard groups individually26–32 reported aniline copper complexes for directly imaging NO, and the mechanism was shown to involve a N-nitrosation step. The N-nitrosation reaction also inspired the development of probes trapping NO by forming diazo rings,33–35 and probes which solely rely on N-nitrosation for detection.36–38 These N-nitrosation-based probes have been thoroughly reviewed in several papers.39–42 Though most of these probes have realized the imaging of NO in live cells, proving probe-based fluorescent imaging as a powerful method for studying NO biology in its native biological context, all these probes unfortunately and irreversibly detect NO, because of their irreversible bonding with NO (Figure 1a). Therefore, these aforementioned probes are unsuitable for real-time monitoring of NO dynamics. Figure 1 | Design of biomimicking probes for tracking cellular NO dynamics. (a) Previous probes irreversibly bind NO and are unsuitable for NO dynamics tracking. (b) Mechanisms of reversible thiol nitrosation in live cells. (c) Design concept of reversible NO-bonding probes for tracking NO dynamics. (d) Molecular structures of biomimicking probes in this work. Download figure Download PowerPoint Herein, we report a bioinspired probe design strategy that enables the real-time and dynamic imaging of NO fluctuations in live cells. Our strategy takes advantage of the cellular machinery of reversible thiol modification by NO. By developing a small library of cysteine analogues tagged with fluorophores to mimic natural cellular thiols, and then performing cell-based screening, probes 1a and 2a were revealed to be mostly tolerated by the cellular machinery to respond to NO changes sensitively and dynamically in live cells. We believe that these probes will greatly aid the biochemical and biomedical research of NO in numerous biological processes. Experimental Methods Materials and instruments All reagents and solvents used for probe synthesis were from commercial suppliers and used without further purification unless otherwise indicated. Dichloromethane, methanol, ethyl acetate were obtained from Sinopharm Chemical Reagent Co.,Ltd, China. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, CAS NO. 7084-11-9), 1,2,3-Benzotriazol-1ol (HOBT, CAS NO. 2592-95-2), N,N-Diisopropylethylamine (DIPEA, CAS NO. 7087-68-5), Cystamine dihydrochloride (CAS NO. 56-17-7), L-Cystine (CAS NO. 56-89-3), L-Homocystine (CAS NO. 626-72-2) were obtained from Energy Chemical, China. NH4Cl, NaHCO3, Na2SO4 were obtained from Aladdin, China. Dry dichloromethane (DCM) was distilled from CaH2. 1H NMR spectra were obtained on a Bruker Fourier transform spectrometer (500 MHz) (Bruker Corporation, USA) at 25 °C. 13C NMR spectra were recorded on a Bruker Fourier transform spectrometer (125 MHz). All NMR spectra were calibrated using residual tetramethylsilane (TMS) as internal references, and all chemical shifts were reported in parts per million (ppm) and coupling constants (J) in Hz. High-resolution mass spectra (HRMS) were measured on an Agilent 6224 TOF LC/MS spectrometer (Agilent Technologies Inc, USA) using electrospray ionization time-of-flight (ESI-TOF). Liquid chromatography equipped with a mass detector (LC-MS) was used to analyze the reaction mechanism between 1a– 1c, 2a– 2c, and NO. The analysis was carried out with a Shimadzu LCMS-2020 mass spectrometer (Shimadzu Corporation, Japan) equipped with Shimadzu VP-ODS packed column (2.0 mm × 150 mm, 5 μm), eluted at 0.3 mL/min using gradient mobile phases [(A, H2O with 0.1% formic acid; B, MeOH): 0–3 min 20–95% B, 3–8 min 95% B, 8–8.01 min 95–20% B, 8.01–10 min 20% B]. High Performance Liquid Chromatography (HPLC) analysis was employed to monitor the reaction process between 1b and NO, or between 1b-SNO and glutathione (GSH). Analysis was performed on an Agilent 1260 infinity system (Agilent Technologies Inc, USA) equipped with Cosmosil 5C18-AR-II column (4.6 mm × 250 mm, 5 μm), eluting at 1.2 mL/min using gradient mobile phases [(A, H2O with 0.1% CF3COOH; B, MeCN): 0–2 min 80–95% B, 2–6.5 min 95% B, 6.5–6.51 min 95–80% B, 6.51–10 min 80% B]. Diethylamine NONOate (DEA·NONOate) was obtained from Santa Cruz Biotechnology (Texas, USA) (Lot nos. F1819 and F1719). Its stock solution was prepared by dissolving DEA·NONOate in a 0.1 M NaOH aqueous solution to make a 10 mM stock solution. Proline NONOate (PROLI·NONOate) was obtained from Santa Cruz Biotechnology (Lot no. I1819). Its stock solution was prepared by dissolving PROLI·NONOate in a 0.1 M NaOH aqueous solution to make a 10 mM stock solution. Fluorescence spectroscopy Fluorescence spectroscopic studies were conducted on a Cary Eclipse Fluorescence spectrophotometer (Agilent Technologies Inc, USA). The slit width was 5 nm for both excitation and emission. Probes were first dissolved in dimethyl sulfoxide (DMSO) to make stock solutions of 5 mM, which were then diluted with phosphate-buffered saline (PBS; with 20% acetonitrile, pH 7.4) to the desired concentrations. Cell lines and cell culture HeLa and Raw 264.7 cells were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher, USA) supplemented with 10% fetal bovine serum (Gibco, USA). Human umbilical vein endothelial cell (HUVEC;CRL-1730; ATCC, USA) was maintained in F12K complete growth medium as demanded. All cells were cultured in a humidified atmosphere at 37 °C and 5% CO2. Computational methods Quantum chemical calculations were carried out using density functional theory (DFT) and time-dependent DFT (TD-DFT) in Gaussian 16.43 All calculations were performed using M06-2X functional and def2SVP basis set in water.44 Positive values of frequencies were checked to validate geometric optimizations in the ground state. The solvation model based on density (SMD) model was chosen to account for the solvent effect.45 The molecular orbitals were visualized with Avogadro (Avogadro, USA) using result files from Gaussian 16 calculations.46 Live cell fluorescence imaging Cells (HeLa cells, 14,000 cells/well; Raw 264.7 cells, 25,000 cells/well; HUVEC cells, 8000 cells/well) were seeded on black 96-well microplates with optically clear bottoms (Greiner Bio-One, Germany) overnight. After cells were stimulated or pretreated with the compounds described below, probe 1a-SAc (1 μM) or 2a-SAc (1 μM) was added with serum-free medium for 30 min. Fluorescence images were recorded by Opera Phenix (PerkinElmer, USA) at an excitation wavelength of 408 nm. Fluorescence intensity was quantified by Columbus analysis system (PerkinElmer). Exogenous NO detection and GSH competition experiment Cells were incubated with DEA·NONOate (200 or 400 μM; Absin, China) for 15 min. GSH (400 μM; Sigma-Aldrich, USA) was added simultaneously in the competition experiment and probes were later added to detect exogenous NO. Endogenous NO detection To activate inducible nitric oxide synthase (iNOS) in macrophage, Raw264.7 cells were incubated with lipopolysaccharide (LPS; 1 μg/mL; Sigma-Aldrich, USA), interferon-γ (IFN-γ; 20 ng/mL; R&D Research, USA), and l-arginine (l-Arg; 0.5 mg/mL; Beyotime, China) for 16 h. At the same time, 1400w (100 μM; MCE) was added to inhibit iNOS. For modulating NO in endothelial cells, HUVEC was pretreated with Carboxy-PTIO (500 μM; Santa Cruz, USA), l-NAME (500 μM; Beyotime), or A23187 (5 μM; Abcam) for 2 h. Probes were then added to monitor endogenous NO level change in these situations. Real-time detecting NO dynamics HeLa cells were incubated with 1a-SAc or 2a-SAc or 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM DA; Meilunbio, China) and imaged with Opera Phenix in time series. Reversible cycles were generated by adding DEA·NONOate (400 μM) or GSH (400 μM) in turns (interval: 30 min). For real-time monitoring NO change in macrophage, Raw264.7 cells were pretreated with LPS, IFN-γ, and l-Arg, and then incubated with the probe. Next, N-acetyl-l-cysteine (NAC; 100 μg/mL; Beyotime) was added into the medium. Raw264.7 cells were immediately imaged with Opera Phenix in time series (interval: 10 min). Cell viability assay To assess the cytotoxicity of probes, cells were exposed to 1a-SAc, 1b-SAc, 1c-Sac, or 2a-SAc with increasing concentrations for 24 h. Then, the medium was changed to a fresh one for another 24 h and the cell viability was detected by Cell Counting Kit-8 (Dojindo, Dojindo Laboratorise, Japan) according to the manufacturer’s protocol. Statistical analysis An unpaired t-test was performed to analyze the results using GraphPad Prism software (GraphPad, USA). Results are presented as mean ± SEM. Statistical significance was determined at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Results and Discussion Biomimicking design of fluorescent probes for tracking NO dynamics, and their synthesis Imaging NO dynamics demands that the fluorescence signal of the probes can dynamically change in response to fluctuating NO levels. This necessitates a reversible bonding between the probes and NO so that the probes can switch between their original structures and their NO-bonding form. This proves challenging from a chemistry point of view given the radical nature of NO. However, NO levels in biological systems are finely tuned by a variety of cellular machinery. There are several reports that state NO is stored and transferred as S-nitrosated cysteine residues in cells,47,48 and the modification of cellular thiols by NO is dynamically modulated by S-nitrosation, S-transnitrosation, and S-denitrosation.49–51 Both chemical and enzymatic mechanisms are involved in these processes to maintain cellular NO signaling and NO homeostasis (Figure 1b).52–54 Therefore, we envisioned that a probe that can be prominently recognized by the cellular machinery of reversible S-nitrosation may facilitate NO dynamic imaging (Figure 1c). Based on this hypothesis, a panel of fluorescently labeled cysteine analogues were designed to mimic natural substrates of the cellular S-nitrosation machinery (Figure 1d). Their molecular structures are comprised of two parts (1) a cysteine analog with a free thiol as a potential S-nitrosation active site, and (2) a coumarin fluorophore as a signaling moiety. The cysteine analog part, including cysteamine, cysteine, and homocysteine, was chosen because they are natural biothiols in mammals and compatible with cellular S-nitrosation machinery. The coumarin fluorophores were used since the skeleton is natural product-like and privileged structure in medicinal chemistry demonstrating a variety of bioactivities,55 which may improve their chance of being recognized by the cellular enzymatic S-nitrosation machinery. These probes were readily synthesized by the condensation between coumarin acids and dimethyl cystinate, cystine, or dimethyl homocystinate, followed by the reduction of the disulfide bond with dithiothreitol (DTT) to expose free thiol groups ( Supporting Information Scheme S1). Detailed procedures for probe preparation and structure characterization are described in the Supporting Information. Evaluating probe capacity for tracking NO dynamics To test if the probes could be tolerated by the cellular S-nitrosation machinery to dynamically respond to fluctuating NO, cell-based screening was performed. Probes 1a, 1b, and 1c bearing different thiols but the same fluorophore were first tested. Though the probes were stable in aqueous solution for several hours without being oxidized ( Supporting Information Figure S1), for the sake of long-term storage of their stock solutions without the concern of oxidation, they were first derivalized to their S-acetylated proprobes. In live cells, the acetyl thioester is readily hydrolyzed by the ubiquitous esterase to free the thiol groups. First, the probes were confirmed to exhibit no potential toxicity in three different cell lines ( Supporting Information Figure S2), and then were tested in HeLa cells for dynamically imaging NO. Cells were first loaded with acetylated probe 1a, 1b, or 1c for 30 min, and then cellular NO levels were manipulated by sequential treatment of DEA·NONOate (a NO donor) and GSH (a robust cellular NO degradation agent and a S-nitrosothiol (RSNO) reductant).56 Interestingly, the probes demonstrated dramatically different responsive profiles (Figures 2a–2c). While all probes were active toward DEA·NONOate in HeLa cells with substantial decreases in their emission intensity, subsequent treatment of the cells with GSH restored only the quenched fluorescence of 1a but not 1b and 1c. We observed that probe 1a swiftly changed its emission intensity in response to DEA·NONOate-GSH cycles for at least two rounds. Nonetheless, the responsive profile of 1c toward the DEA·NONOate-GSH cycles was relatively similar to that of DAF-FM DA, a commercial and irreversible NO probe ( Supporting Information Figure S3). This result suggests that the cysteine thiol should be more tolerated by the cellular machinery of S-nitrosation. This hypothesis was further supported by the dynamic response of another cysteine thiol-bearing probe 2a to the DEA·NONOate-GSH cycles ( Supporting Information Figure S4). Figure 2 | Screening probes 1a–1c for those capable of tracking cellular NO dynamics. HeLa cells pretreated with (a) 1a-SAc, (b) 1b-SAc, or (c) 1c-SAc (each at 1 μM) were imaged in time series. Reversible cycles were generated by adding DEA·NONOate (400 μM) or GSH (400 μM) in turns (interval: 30 min). NONOate was applied at 0 and 60 min, and GSH was applied at 30 and 90 min, respectively. Quantified intracellular fluorescence intensity changes were shown in the right panel. (d–g) Sensing mechanism study. Fluorescence spectra of probes 1a–1c (each 10 μM) were individually collected in a time-lapsed way, during which aliquots of the solutions were analyzed by LC-MS at time points indicated by the arrows (in d), followed by rapid and sequential treatment with DEA·NONOate (10 equiv, black arrow in d) or GSH (10 equiv, red arrow in d) of the remaining solutions. The data shown are the time-course intensity changes at their maximum emission (d), and the LC traces (e–g) at the indicated time points. Full fluorescence spectra of the probes at indicated time points, and MS spectra of each peak in the LC traces are appended in the Supporting Information. Download figure Download PowerPoint Next, the underlying sensing mechanism was studied from a chemistry point of view. Probes 1a– 1c were treated with DEA·NONOate and GSH in a sequential way, respectively. Fluorescence spectrophotometer and LC-MS spectrometer were used to monitor the reactions. Specifically, an individual probe (10 μM) in PBS was treated with DEA·NONOate (10 equiv), and its fluorescence response was recorded in a time-lapsed way. After 15 min, an aliquot of the mixture was analyzed by LC-MS while the remaining was instantly treated with GSH (10 equiv) to degrade NO residual. The emission spectrum was continuously recorded for another 15 min, and then LC-MS analysis was repeated. It was observed that the probes responded to DEA·NONOate with decreased emissions (Figure 2d and Supporting Information Figures S5–S7), and LC-MS showed partial transformation to the S-nitrosated forms, accompanied by a trace of the disulfide forms (Figures 2e–2g and Supporting Information Figures S8–S10). This is in accord with previous reports that S-nitrosation may quench fluorophore emission.57,58 It should be noted that probe 1a bearing a cysteine thiol demonstrated the most dramatic response toward DEA·NONOate as judged by either spectra, while 1b and 1c were relatively inert. When the solutions were subsequently treated with GSH, a reduction of the S-nitrosated probes was observed, again with 1a-SNO being the most reactive. Notably, full reduction of 1a-SNO was observed, accompanied by the full restoration of its quenched fluorescence. These observations agreed with the most sensitive response of probe 1a toward DEA·NONOate-GSH cycles in cell imaging experiments. We also monitored the responses of probes 2a– 2c toward the sequential treatment of DEA·NONOate and GSH ( Supporting Information Figures S11–S14), and the cysteine thiol-bearing probe 2a was observed as the most sensitive. These results suggest that the cysteine thiol should be a desirable active unit for sensing NO, which is consistent with the cell imaging results. Though both S-nitrosated and disulfide probes were observed when treated with DEA·NONOate, S-nitrosation is recognized as the most favorable mechanism for the reaction. This could be concluded from the reaction between 1b and DEA·NONOate run at a larger scale. When 1b (50 μM) was treated with DEA·NONOate (10 equiv), almost full conversion to 1b-SNO was observed by HPLC analysis, accompanied by negligible amounts of 1b-disulfide ( Supporting Information Figures S15–S17). The resulting 1b-SNO could be fully reduced back to 1b by following GSH (10 equiv) treatment ( Supporting Information Figures S15 and S18). This observation suggests that S-nitrosation should have dominated the reaction between the thiol probes and DEA·NONOate. After confirming the S-nitrosation mechanism for the probes to reversibly sense NO, we then set out to discuss how this reaction proceeded in aqueous solution. Two chemistry mechanisms have been proposed for S-nitrosation as follows: (1) N2O3 nitrosates thiols, and (2) NO nitrosates thiyl radicals, which are formed via the one electron oxidation of the thiol by NO2.59 These two mechanisms are deduced to take place in parallel but are challenging to be dissected as either enhancing N2O3 hydrolysis or scavenging thiyl radicals would inevitably have complex effects on the reaction system.59 To check whether these mechanisms are also applicable to our thiol-based probes, the following experiments were performed. First, the fluorescent response of 1b toward DEA·NONOate under an anaerobic condition was examined where we observed that 1b demonstrated a dramatically decreased sensitivity toward NO ( Supporting Information Figure S19). This is in accord with the previous observation that S-nitrosation of GSH is much more efficient in the presence of air than in the absence of oxygen,59 and suggests that the oxidation of NO by O2 is essential for S-nitrosation to take place. Second, we used NOBF4 as a NO+ equivalent. When 1b was treated with NOBF4 in acetonitrile, a decrease of fluorescence intensity was observed. The emission intensity could be restored by the subsequent treatment of cysteamine, which acted as a surrogate of GSH due to the poor solubility of GSH in acetonitrile ( Supporting Information Figure S20). LC-MS analysis confirmed that treating 1b with NOBF4 caused its transformation to 1b-SNO ( Supporting Information Figure S21). Since NO+ exists in its N2O3 equivalent in aqueous solution, this result suggests the possible contribution of N2O3 to probe S-nitrosation. Third, we tested the sensitivity of 1b toward NO under aerobic and various pH conditions, and it was observed that the detection favored a more basic condition ( Supporting Information Figure S22). In basic solution, more 1b existed in its anionic form, which is more readily available for S-nitrosation by N2O3. These results confirmed the involvement of the N2O3 mechanism. Dose-dependent fluorescence responses of 1a and 2a to the sequential treatment of DEA·NONOate and GSH in aqueous solution Having confirmed the full reversible response of probes 1a and 2a to DEA·NONOate-GSH cycles in live cells and their sensing mechanism, we then tested their sensitivity toward NO in aqueous solution. First, their NO dose-dependent responses were studied. Probes 1a and 2a were individually treated with different doses of DEA·NONOate (a NO surrogate) and their fluorescence intensities were recorded in a time-lapsed way (Figures 3a and 3c and Supporting Information Figures S23a and S24a). Plotting the probe fluorescence intensity against DEA·NONOate doses gave sigmoidal dose–response curves (Figures 3e and 3g), according to which the DEA·NONOate concentration causing 50% maximal quenching effect (EC50) could be determined. When probes 1a and 2a were set at 2 μM, the calculated EC50 of DEA·NONOate to 1a and 2a was approximately 7.5 and 13 μM, respectively. Given the slow release of NO from DEA·NONOate,60 the actual EC50 of NO should be far below these values. When the nitrosated 1a or 2a was treated with GSH, a GSH dose-dependent fluorescence recovery was observed (Figures 3b and 3d and Supporting Information Figures S23b and S24b) which also fitted a sigmoidal correlation (Figures 3f and 3h), and the EC50 values of GSH to the resulting 1a-SNO and 2a-SNO were determined to be 498 and 661 μM, respectively. Figure 3 | Dose-dependent responses of probes 1a and 2a toward the sequential treatment of DEA·NONOate and GSH. (a and c) Time- and dose-dependent fluorescent intensity of 1a (2 μM) and 2a (2 μM) toward DEA·NONOate treatment. The fluorescent intensities were recorded for 30 min in kinetics mode (1 min/scan) and the data shown are normalized intensities. (b and d) GSH treatment recovered the fluorescence of nitrosated 1a and 2a. Probes (2 μM) were first treated with DEA·NONOate (40 μM) for 30 min, and then were treated with GSH (80–2000 μM), and the fluorescent intensities were recorded for 30 min in the kinetics mode (1 min/scan). λex: 418 nm for 1a and 447 nm for 2a. λem: 477 nm for 1a and 490 nm for 2a. (e and g) NO dose–response fitting curves of 1a and 2a which gave a sigmoidal fit with each R2 above 0.99. (f and h) GSH dose–response fitting curves of nitrosated 1a and nitrosated 2a which also gave a sigmoidal fit with each R2 above 0.99. Download figure Download PowerPoint In addition, although the response kinetics of 1a and 2a toward DEA·NONOate appeared slow (Figures 3a and 3c), this was due to the slow release of NO from DEA·NONOate. To recapitulate the sensing kinetics, a short-half-life (<2 s) NO donor PROLI NONOate was used to compare with DEA·NONOate,60 and rapid decay of probe fluorescence was observed ( Supporting Information Figures S25 and S26). Furthermore, the time courses of probe fluorescence decay and the NO release kinetics of the NO donors (by UV) were fitted to growth/sigmoidal curves ( Supporting Information Figure S27), suggesting that release of NO from the donors are actually the rate-limiting step for the detection. We also confirmed that the fluorescence turn-off respon