Development of a nitric oxide-responsive labeling reagent for

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Development of a nitric oxide-responsive labeling reagent for proteome analysis of live cells Yuki Nishikawa, Takayuki Miki, Masashi Awa, Keiko Kuwata, Tomonori Tamura, and Itaru Hamachi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01021 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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ACS Chemical Biology

Development of a nitric oxide-responsive labeling reagent for proteome analysis of live cells Yuki Nishikawa, 1 Takayuki Miki,1,2 Masashi Awa,1 Keiko Kuwata,3 Tomonori Tamura,1 Itaru Hamachi1*

1

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of

Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, JAPAN 2

Present address: School of Life Science and Technology, Tokyo Institute of Technology,

4259-B-40 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, JAPAN 3

Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Chikusa, Nagoya

464-8602, JAPAN *Correspondence should be addressed to I.H. ([email protected])

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ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Nitric oxide (NO) is a pleiotropic signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems. To understand the biological functions of NO in detail, comprehensive characterization of proteins found in high concentration NO environments is crucial. Herein, we describe the design of NO-responsive protein labeling reagents based on N-alkoxyacyl-o-phenylenediamine as an optimal reactive scaffold. The designed molecules can label proteins in murine macrophage cells in response to endogenously produced NO. The combination of NO-responsive protein labeling and liquid chromatography-tandem mass spectrometry (LC–MS/MS) technology allowed for characterization of the proteome under NO-generated conditions. Moreover, it was demonstrated that our reagent was able to selectively mark and be used to fluorescently visualize NO-producing cells in a mixed cell culture system.


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Main text: Introduction Nitric oxide (NO) is an important multifunctional biomolecule involved in a variety of physiological and pathophysiological processes, including regulation of blood vessel modulation, wound healing, and neuronal communication.1–6 Also, it is suggested that chronically elevated levels of NO are involved in the pathogenesis of some human pathological conditions, such as inflammatory bowel diseases,7 neurodegenerative disorders,8 and cancer.9,10 One of the physiological functions of NO is to modulate posttranslational modifications (nitrosation, nitration) of proteins via reactive nitrogen species.11,12 Therefore, NO is recognized as a modulator of many proteins. Several methods for monitoring endogenous NO, such as electron paramagnetic resonance spectroscopy13 and colorimetric,14 fluorometric,15–17 electrochemical,18 and chemiluminescence techniques,19 have been developed to understand the complicated functions of NO in living systems. However, to the best of our knowledge, there are no methods available to analyze the proteins in the environments where NO is produced. To achieve a better understanding of the biological functions of NO, it is important to clarify what kind of proteins are present in high concentration NO conditions. Chemical proteomics is now recognized as a powerful method to provide a large amount of information on the characteristics of various proteins in a particular context, since this approach allows us to take a snapshot of protein localization and activity in live cells.20,21 Various chemical tagging techniques, such as activity-based protein profiling (ABPP) pioneered by Cravatt and coworkers,22–25 and proximity labeling (such as BioID, APEX and TurboID),26–31 have been developed to classify and annotate proteins. Recently we reported a “conditional proteomics” approach using a Zn2+-responsive protein labeling reagent that can be used to identify which proteins localize under a high concentration of mobile Zn2+ in an effort to uncover zinc homeostasis in glioma cells in response to NO.32 Herein, we describe a novel design strategy to obtain NO-responsive protein labeling reagents that are activated only in the presence of a high concentration of NO. This 3 ACS Paragon Plus Environment


reagent labeled intracellular proteins in response to not only exogenously added but also endogenously produced NO, which enabled us to selectively mark and fluorescently visualize NO-producing cells. We also demonstrated the usefulness of this technique for proteome analysis of NO-rich conditions by combining NO-responsive protein labeling and liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis.

Results and discussion Molecular design of NO-responsive protein labeling reagents Previous studies on fluorescent NO chemosensors clearly showed that electron-rich o-phenylenediamine groups are selectively and efficiently converted to benzotriazole moiety in the presence of NO under aerobic conditions.15–17 This reaction might involve an oxidation product of NO, such as N2O3.33 It was also reported that an acyl benzotriazole formed from N-acylated o-phenylenediamine was spontaneously hydrolyzed in aqueous solution to release a benzotriazole motif.34 Also, N-acyl-benzotriazole formed from the reaction of a 3,4-diaminobenzoic acid (Dbz) linker with NaNO2 has been used as an active ester intermediate for native chemical ligation.35 Based on these findings, it was rational for us to design an N-acylated o-phenylenediamine derivative as a scaffold for a NO-responsive protein labeling reagent. This may be converted to an acyl benzotriazole in the presence of NO, which should enhance its reactivity for protein acylation as illustrated in Figure 1a. We initially designed three types of reagents 1–3 with different linkers between N-acyl-o-phenylenediamine and 7-diethylaminocoumarin (Dc) to examine whether acylated o-phenylenediamine derivatives can label proteins in the presence of NO (Figure 1b). HeLa cell lysates in the presence or absence of NO donor (NOC-7)36 (1 mM) were treated with reagents 1–3 (100 µM) for 1 h, followed by SDS-PAGE and in-gel fluorescence analysis (Figure 2a). Many fluorescence bands were detected when cell lysates were incubated with 2 or 3 only in the presence of NOC-7, while negligible fluorescence was observed upon incubation with 1. This result shows that N-acylated o-phenylenediamine derivatives are 4 ACS Paragon Plus Environment

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reactive groups activatable by NO. Among them, the NO-responsive labeling of 3 (6.6-fold) was greater than that of 2 (3.2-fold) in terms of the enhancement ratio, suggesting that the N-alkoxyacyl-o-phenylenediamine derivative was the most appropriate as a reactive group for NO-responsive labeling (Figure 2b).

Under NO-rich condition

(a)

NO-responsive reactive group NH2

H N

O2

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MeO

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Figure 1. (a) Schematic illustration of nitric oxide (NO)-responsive protein labeling in living cells. (b) Molecular structures of NO-responsive labeling reagents used in the present study.



Figure 2. In vitro evaluation of NO-responsive protein labeling. Reagent (100 µM) is added to HeLa cell lysates (1.0 mg/mL protein) in 100 mM HEPES buffer (pH 7.4). The mixture was incubated at 37°C in the presence or absence of 1 mM NOC-7 (NO donor) for 1 h. (a) SDS-PAGE and in-gel fluorescence analysis of protein labeling in HeLa cell lysates with 1–3. The right panel shows the Coomassie Brilliant Blue (CBB) stained gel image. (b) Relative fluorescent intensity of proteins labeled by 1–3 calculated from the data shown in (a). Error bars represent standard deviation (s.d.), n = 3. (c) In-gel fluorescence analysis of protein labeling in HeLa cell lysates with 3–7. (d) Selectivity of 6 (NOAR-1) against several reactive oxygen/nitrogen species (ROS/RNS). NOAR-1 (100 µM) was added to PBS buffer (pH 7.4) containing BSA (100 µM). The mixture was incubated at 37°C for 60 min in the presence or 6 ACS Paragon Plus Environment

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absence of various ROS/RNS (1 mM). O2– was generated from KO2. 1O2 was generated from endoperoxide reagent. •OH was generated from H2O2 and FeSO4. ONOO– was generated from SIN-1 (the detailed procedures are shown in Supporting Information). Error bars represent s.d., n = 3.



We sought to further improve the NO-response ability and reactivity of the labeling reagent by varying the substituent of the benzene ring of o-phenylenediamine. We prepared 4–7 with a nitro or fluorine group as the electron withdrawing group, and a methyl or methoxy group as the electron donating group (Figure 1b), and then examined NO-responsive protein labeling using the HeLa cell lysates. As shown in Figure 2c, many labeled bands were observed by 3, 5, 6 and 7 only in the presence of NOC-7, whereas labeling by 4 proceeded even in the absence of NOC-7. Thus it is likely that the introduction of the strong electron withdrawing nitro group enhanced the inherent reactivity of the acyl group of o-phenylenediamine and concurrently suppressed the reactivity of the amine moiety of o-phenylenediamine for the benzotriazole formation to decrease its response. The derivative 5 bearing a fluorine group, a moderately electron withdrawing group, showed insufficient labeling in response to NO compared with that of 3 because of the suppressed reactivity of the amine group similar to the case of 4. In contrast, incorporation of an electron donating group increased the reactivity of the amine moiety of o-phenylenediamine (in 6 and 7), so that the NO-responsive protein labeling via the benzotriazole proceeded effectively. In particular, 6 with a methyl group most efficiently labeled proteins included in the HeLa cell lysates in response to NO. It was plausible that the benzotriazole formation was also improved in the case of 7 with a methoxy group, whereas the reactivity of the resulting acyl benzotriazole might be decreased relative to the case of 6. Overall, it was clear that reagent 6 (NOAR-1, NO-activatable reagent-1), which contains o-phenylenediamine with a methyl group derived from a urethane bond, was optimal as an NO-responsive labeling reagent. The selectivity of NOAR-1 for NO among the reactive oxygen/nitrogen species (ROS/RNS) was then evaluated using bovine serum albumin (BSA) labeling experiments. As shown in Figure 2d, almost no labeled BSA was observed when NOAR-1 was incubated with ROS/RNS species other than NO, demonstrating that NOAR-1 exhibits a high selectivity to NO. By changing the concentration of NOC-7 in the BSA labeling experiment, it was confirmed that NOAR-1 can respond to 10 µM of NOC-7 under our experimental conditions (Figure S1). As a result of evaluating the pH effect on BSA labeling, it turned out that the labeling proceeded most 8 ACS Paragon Plus Environment

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efficiently at neutral pH 7.4 among pH 6.0–8.0 (Figure S2).

Protein labeling mechanism of NOAR-1 in response to NO and its labeling kinetics The reaction mechanism of NO-responsive protein labeling by NOAR-1 was validated using HPLC analysis (Figure 3a). When NOAR-1 and NOC-7 were incubated in PBS buffer (pH 7.4), we observed the decrease of the peak attributed to NOAR-1 at 20 min and the appearance of two new peaks at 18 and 34 min (Figure 3b, d, Figure S3). Based on mass spectrometry analysis (Figure 3c, Figure S4), the peak at 34 min was identified as N-acyl-benzotriazole, an assumed intermediate of NO-triggered reaction, and the peak at 18 min as its hydrolyzed product (Dc-OH). It was also confirmed that the increase of this intermediate was attenuated in the presence of BSA (Figure 3e, Figure S5) and the corresponding fluorescent band of labeled BSA was observed in the in-gel fluorescence from SDS-PAGE analysis (Figure S6). These results revealed that the acyl benzotriazole intermediate was indeed formed according to the scheme of Figure 3a and was directly involved in the protein labeling. We next examined the labeling kinetics of NOAR-1 using the HeLa cell lysates. As shown from the time course data, the fluorescence intensity reached a plateau within 30 min upon addition of NOC-7, indicating its half-life was 10–15 min (Figure S7)37. Additionally, the labeled proteins in the HeLa cell lysates were digested and the Dc-modified peptides were detected by LC–MS/MS, which indicated that Lys, Ser, Thr, His, Cys, and Tyr, almost all of the nucleophilic amino acid residues of natural proteins, were acylated by the NO-activated NOAR-1 (Figure 3f, Figure S8, Supplementary Data 1). Among them, Lys, Ser, and Thr which are likely to undergo acylation were predominantly detected (83% in these three amino acids).



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(a) NH2

O H N

O O

O

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O

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NO

O

N N N

O2

O

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NOAR-1 ( )

O

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triazole intermediate ( ) Exact Mass: 507.2118

Dc-OH ( ) Nu O

BSA BSA

(b) 0 min

O

H N

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N

O

[M+Na]+

(c)

60 min

O

H N

530.2024

[M+H]+ 508.2205

(d)

(e)

(f) Cys (3) 6%

Tyr (1) 2%

His (4) 8% Lys (16) 33% Thr (12) 25% Ser (12) 25%

Figure 3. NO-responsive reaction mechanism of NOAR-1. (a) Scheme of the putative NO-mediated reaction mechanism of NOAR-1 and subsequent hydrolysis or reaction with BSA. Dc-OH indicates the hydrolysis product. (b) HPLC analysis of NOAR-1 (100 µM) before (0 min) and after (60 min) treatment with NOC-7 (1 mM) in PBS buffer (pH 7.4) at 37°C. (c) Mass spectrum of the peak at 34 min. (d, e) Time-course product analyses of the reaction of NOAR-1 with NOC-7 in PBS buffer in the (d) absence or (e) presence of 100 µM BSA. Error bars represent s.d., n = 3. (f) Numbers of Dc-labeled amino-acid residues. Labeled proteins in HeLa cell extracts were digested by trypsin and the resultant peptides were analyzed by LC–MS/MS to determine the Dc-labeled amino acids.


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NO-responsive protein labeling in living cells For NO-responsive labeling in living cells, we prepared NOAR-2 with diacetyl fluorescein (Figure 4a). HeLa cells were incubated with NOAR-2 (10 µM) and observed by confocal laser scanning microscopy (CLSM). Intracellular fluorescence reached a plateau within 10 min and NOAR-2 was uniformly distributed throughout the cells including the nuclei (Figure 4b, Figure S9). To evaluate the NO-responsive labeling, we exogenously added NOC-7 (1 mM) to live HeLa cells that were pre-treated with 10 µM of NOAR-2 for 10 min. The broad fluorescence bands attributed to labeled proteins were detected only in the presence of NOC-7 by SDS-PAGE analysis of the lysis samples, implying that NOAR-2 can be activated in live cells in response to NO. It was also noteworthy that there was negligible background labeling even inside cells (lane 5 of Figure 4c). We subsequently investigated whether NOAR-2 responds to endogenously generated NO. It is known that the expression level of inducible nitric oxide synthase (iNOS) in murine macrophage RAW264.7 cells increases after addition of lipopolysaccharide (LPS) and interferon-γ (IFN-γ), which results in the production of a high concentration of NO.38 Indeed, we confirmed the induction of iNOS expression in the RAW264.7 cells after treatment with LPS and IFN-γ by western blotting (Figure S10) and also confirmed the uniform distribution of NOAR-2 in RAW264.7 cells by CLSM observation (Figure S11). The stimulated cells were incubated with NOAR-2 for 1 h followed by cell lysis and SDS-PAGE analysis. As shown in Figure 4d, the labeled bands were remarkably enhanced after LPS/IFN-γ stimulation, which clearly revealed that NOAR-2 can be activated by NO endogenously produced inside cells and be used to label intracellular proteins. CLSM imaging experiments also supported these results. We observed RAW264.7 cells after fixation with cold methanol and confirmed that unreacted NOAR-2 was removed by this fixation process. Strong fluorescence was detected in the intracellular regions in the stimulated cells (Figure 4e), whereas there was negligible fluorescence in the nonstimulated cells (Figure 4f) and cells treated with selective iNOS inhibitor (1400W) during the stimuli (Figure S12). These results indicated that, (i) NOAR-2 was activated by NO produced from iNOS and could label 11 ACS Paragon Plus Environment


proteins in NO-producing cells, and (ii) the labeled proteins were trapped inside the cells and could be visualized by the fluorescein (Fl) fluorescence of NOAR-2. Given the trappable feature of the proteins labeled in response to NO, it may be expected that NOAR-2 is able to selectively tag NO-producing cells. As a proof of principle experiment, we used a mixed cell culture system of RAW264.7 cells and HeLa cells. We separately confirmed that the substantial increase of labeled proteins was not detected in HeLa cells in the presence of the LPS/IFN-γ stimuli and the intracellular fluorescence did not increase (as shown in Figure S13). In this mixed cell culture system, the cell surface of HeLa cells was biotinylated and fluorescently marked by an Alexa647–Streptavidin (SAv) conjugate to distinguish them from RAW264.7 cells. After the mixed cells were stimulated by LPS and IFN-γ, we added NOAR-2 and conducted CLSM imaging of the methanol-fixed cells. As shown in Figure 4g, strong green fluorescence was observed only from RAW264.7 cells, while negligible fluorescence was detected from HeLa cells. This demonstrated that NOAR-2 can selectively label and fluorescently visualize NO-producing cells with the single cell level of the spatial resolution.


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Figure 4. NO-responsive protein labeling in living cells. (a) Chemical structure of NOAR-2 13 ACS Paragon Plus Environment


for intracellular protein labeling. (b) CLSM images of HeLa cells treated with NOAR-2 (10 µM) for 10 min. (c) SDS-PAGE and in-gel fluorescence imaging of exogenous NO-responsive labeling in living HeLa cells. HeLa cells were pre-treated with NOAR-2 (10 µM) for 10 min, followed by incubation with 1 mM NOC-7 for 5, 15, 30 or 60 min. (d) SDS-PAGE and in-gel fluorescence imaging of endogenous NO-responsive labeling in living RAW264.7 cells. After stimulation with LPS (0.5 µg/mL) and IFN-γ (7.5 ng/mL) for 20 h, RAW264.7 cells were treated with NOAR-2 (10 µM) for 1 h. (e, f) CLSM images of RAW264.7 cells that were (e) treated or (f) not treated with LPS and IFN-γ followed by incubation with NOAR-2 (10 µM) for 1 h and cold methanol fixation. Scale bar: 20 µm. (g) CLSM analysis of NO-generating cell-specific labeling by NOAR-2. The cell mixture (RAW264.7 cells and surface-biotinylated HeLa cells) was stimulated by LPS and IFN-γ, and subsequently incubated with NOAR-2 (10 µM) for 1 h. After reaction, HeLa cells in the mixture were selectively labeled by Alexa647–SAv, and the cell mixture was then fixed using cold methanol and analyzed by CLSM.


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Proteomic analysis of labeled proteins in iNOS-induced RAW264.7 cells The proteome of the stimulated RAW264.7 cells was next characterized using a mass fingerprinting technique. The proteins labeled with the Fl moiety were enriched by immunoprecipitation using an anti-fluorescein antibody, tryptically digested in-gel and subjected to LC–MS/MS measurements. To clearly evaluate the dynamic changes of labeled proteins brought about by the LPS/IFN-γ stimuli, we conducted quantitative mass analysis using tandem mass tagging (TMT) technology.39 Digested peptides were modified with heavy-(H-) or light-(L-) TMT reagents for stimulated or nonstimulated samples, respectively, and then were mixed in a 1:1 ratio for LC–MS/MS (Figure 5a). We performed three independent biological replicates in which proteins detected at least twice were selected as identified proteins, resulting in a total of 379 proteins identified and quantified (Supplementary Data 2). Comparing the proteome of the stimulated cells with those of nonstimulated cells, 310 proteins (82%) showed more than zero in log2(H/L) values, indicating that the protein labeling was greatly facilitated with NO produced by iNOS in live RAW264.7 cells (Figure 5b). We focused on 20 proteins having significant enrichment by the stimuli (log2(H/L) > 1.0 (corresponding to 2.0-fold); P < 0.05) and analyzed the gene ontology (GO) term enrichment (Table S1).40 Cellular component analysis showed that the proteins located in the endoplasmic reticulum (ER) were highly enriched (Figure 5c). The co-localization experiment of labeled proteins with ER-tracker Red also demonstrated by CLSM that the NOAR-2-labeled proteins predominantly localized in ER (Figure S14). This may be attributed to the fact that iNOS is reported to be localized in the regions associated with the cytoplasmic face of the ER membrane in fibroblast-like cells,41 and localized in a vesicle population associated with the ER in macrophages, particularly at the trans face of the Golgi apparatus.42 Additionally, biological process analysis clearly revealed that the proteins implicated in cell redox homeostasis and protein folding were enriched (Figure 5d). The ER plays several important roles in the folding, export, and processing of newly synthesized proteins. According to previous

reports,

NO

produced

from

iNOS

may


inhibit

the

activity

of

the


sarcoplasmic/endoplasmic reticulum Ca2+–ATPase (SERCA) by nitration of tyrosine of the channel-like domain, which causes ER stress.6 Under such ER stress conditions, protein folding in the ER is facilitated by ER chaperone proteins (such as BiP and GRP94 (endoplasmin)) and ER oxidoreductases (such as protein disulfide isomerase (PDI)), many of which were indeed enriched in our proteomics data. We subsequently performed western blotting analysis on these proteins to examine if the elevated labeling was due to changes in their expression levels. As shown in Figure S15, cyclooxygenase-2 (COX-2) was increased by LPS/IFN-γ stimuli, while the expression of PDI and GRP94 did not substantially change. Furthermore, we evaluated the genome-wide changes in mRNA expression after the LPS/IFN-γ stimuli by use of DNA microarrays (Figure 5e, Supplementary Data 3). The microarray data showed that 182 genes were largely up regulated (> 10-fold, highlighted in red in Figure 5e) and 232 genes were largely down regulated (> 10-fold, highlighted in blue in Figure 5e). In term of 20 proteins that were significantly enriched by our NOAR-2-based proteomics, the 4 proteins (colored in red) were positively correlated with increase of the mRNA level, while the other 16 proteins did not show any clear correlation with the change of mRNA (Figure 5f). These results strongly suggested that our chemical proteomics data reflected not only the expression levels of proteins modulated by NO-induced ER stress, but the environment surrounding the NO. Overall, we concluded that our reagent NOAR-2 is capable of accurately reporting the intracellular events of the proteome that are associated with NO endogenously produced from LPS/IFN-γ stimuli in RAW264.7 cells.


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Figure 5. Proteomic analysis of labeled proteins in iNOS-induced RAW264.7 cells. (a) Workflow for TMT-based quantitative analysis of labeled proteins using LC–MS/MS. After in-gel digestion, the peptides obtained from iNOS-induced and noninduced cells were labeled by heavy (H) and light (L) TMT reagents, respectively, and mixed in a 1:1 ratio. The mixture was analyzed by LC-MS/MS. (b) Volcano plot summarizing NO-responsive protein labeling in RAW264.7 cells. The protein group showing log2(H/L) > 1.0 and P < 0.05 is highlighted in red. (c,d) GO cellular component (c) and biological process (d) analyses of highly enriched proteins in iNOS-induced RAW264.7 cells (log2(H/L) > 1.0; P < 0.05). (e) Volcano plot of DNA microarray data, comparing iNOS-induced and noninduced RAW264.7 cells. Up 17 ACS Paragon Plus Environment


regulated genes (> 10-fold) are highlighted in red, and down regulated genes (> 10-fold) are highlighted in blue. (f) Correlation of mRNA expression changes and NO-responsive protein labeling by the LPS/IFN-γ stimuli. Positively correlated 4 proteins and noncorrelated 16 proteins are colored in red and gray, respectively.

Conclusion In summary, we found that N-alkoxyacyl-o-phenylenediamine is a novel NO-responsive reactive moiety for labeling proteins under NO-rich conditions. Benefiting from low background labeling even inside living cells, our NO-responsive labeling reagent is applicable for labeling proteins in murine macrophage cells that endogenously produce NO, allowing for proteome analysis of NO-rich conditions by LC–MS/MS techniques. We also demonstrated the applicability of this reagent to the selective labeling and fluorescent visualization of NO-producing cells in a mixed cell culture system. Taken together, this indicates that our NO-responsive labeling reagent can be a potentially powerful tool for selective imaging and analysis of NO-rich environments in more complex systems such as tissues.


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Methods Details of the experimental procedures are provided in the Supporting Information.

Associated context Supporting Information Supplementary figures and tables, experimental procedures, synthesis and characterization of compounds.

Author information Corresponding Author *E-mail: [email protected].

ORCID Tomonori Tamura: 0000-0003-1648-9296 Itaru Hamachi: 0000-0002-3327-3916

Notes The authors declare no competing financial interests. The MS raw data and analysis files have been deposited to the ProteomeXchange Consortium (http://proteomecentral.

proteomexchange.org)

via

the

jPOST

partner

repository

(http://jpostdb.org)43 with the data set identifier PXD012310.

Acknowledgment We thank K. Nishimura (Kyoto University) for experimental support of MS measurements. We also thank R. Mosi from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was funded by a Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists to Y.N. and T.M., and the Japan Science and Technology Agency (JST) ERATO Grant No. JPMJER1802 to I.H. This work 19 ACS Paragon Plus Environment


was also supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” (JSPS KAKENHI Grant No. 17H06348).


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TOC 80x32mm (300 x 300 DPI)

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Development of a nitric oxide-responsive labeling reagent for

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