Naphthalimide Scaffold Provides Versatile Platform for Selective Thiol

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Naphthalimide Scaffold Provides Versatile Platform for Selective Thiol Sensing and Protein Labeling Pengcheng Zhou, Juan Yao, Guodong Hu, and Jianguo Fang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00856 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on January 31, 2016

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Naphthalimide Scaffold Provides Versatile Platform for Selective Thiol Sensing and Protein Labeling Pengcheng Zhou, Juan Yao, Guodong Hu, and Jianguo Fang* State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China E-mail: [email protected]

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Abstract: Reversible thiol modifications are fundamental of cellular redox regulation. Specific thiol detection, including thiol sensing and protein thiols labeling, is critical to study such

modifications.

We

reported

the

discovery

of

4-methylsulfonyl-N-n-butyl-1,

8-naphthalimide (MSBN), a highly selective fluorogenic probe for thiols based on the 1, 8-naphthalimide scaffold. Thiols react with MSBN nearly quantitatively via nucleophilic aromatic substitution to replace the methylsulfonyl group and restore the quenched fluorescence (>100-fold increase). MSBN was employed to selectively image thiols in live cells, and specifically label protein thiols with a turn-on signal to determine diverse reversible protein thiol modifications. In addition, we introduced a bulky group into the MSBN as a mass tag to create a probe MSBN-TPP, which readily discriminates the reduced thioredoxin from the oxidized one. The specific reaction of MSBN with thiols and the easy manipulation of the naphthalimide unit enable MSBN a versatile scaffold in developing novel probes for thiol-based protein bioconjugation and studying various thiol modifications.

Keywords: Fluorescent probe • Protein labeling • Thiol sensing • Naphthalimide • Nucleophilic aromatic substitution

TOC



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Introduction Biological thiols, including small molecule thiols, such as cysteine (Cys) and glutathione (GSH), and protein thiols (the sulfhydryl group in Cys residues), play pivotal roles in maintaining cellular redox homeostasis and regulating diverse redox signaling pathways involved in cell proliferation, differentiation and death.1,2 The reversible oxidative modifications of thiols, such as forming disulfides (RS-SR’), nitrosothiols (RS-NO) and sulfenic acids (RSOH), form the fundamental of redox regulation.3-6 Among various assays in unveiling the molecular basis of redox signaling, fluorescent dyes are powerful and popular tools in determining multiple biological events due to their high sensitivity, good biocompatibility, diverse selection and convenient operation. Thus, numerous selective thiol probes have emerged in the literature.7-15 In contrast to the ease of measurement of small molecule thiols, it possesses difficulties and challenges to concisely detect a certain type of protein thiol modifications. Nevertheless, the strong nucleophilic property of the sulfhydryl group opens a window to chemically label protein thiols with specific thiol-reactive agents, and hence enables reliable and robust detection of protein thiol modifications. 4,6,16-27

Scheme 1. Design of NAS-type thiol fluorogenic probes.

Molecules with the structure of electron withdrawing group (EWG)-π system-electron donating group (EDG) are generally fluorescent due to the internal charge transfer (ICT) effect, while those with EWG- π system-EWG or EDG- π system-EDG usually display weak emission because of the blocked ICT (Scheme 1).28 Conversion of EWG- π system-EWG or EDG- π system-EDG to EWG- π system-EDG often accompanies with the change of the absorbance/fluorescence profiles, thus providing a strategy for design of optical probes. The high nucleophilicity of thiols allows them reacting with electron-deficient aromatic compounds via nucleophilic aromatic substitution (NAS).20,22,29 As the aromatic system 3 ACS Paragon Plus Environment

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within the EWG-aromatic system-EWG is electron-deficient, we reasoned that thiols (RSH) may attack the aromatic system via NAS to generate RS-aromatic system-EWG, forming the EDG-aromatic system-EWG structure, and hence switch on the fluorescence (Scheme 1). Consequently, molecules with the EWG-aromatic system-EWG motif are potential NAS-type thiol probes. In addition, as thiols are covalently linked to the fluorophores, the Scheme 1 also enables turn-on label of protein thiols. In the continuation of our interests in developing small molecule modulators of the biological redox system,7,30-34 we reported the synthesis of EWG-substituted naphthalimides and evaluation of their ability for thiol sensing and protein thiol labeling. Twelve

n-butylnaphthalimides bearing diverse substituents with varying leaving character and electron-withdrawing capacity were prepared (Scheme 2). The EWGs at the 4-position and the imide group make the naphthalene ring electron deficient, thus forming the EWG-aromatic system-EWG motif. The naphthalimide unit not only provides a suitable fluorescent scaffold, but permits attaching other tags of interest via replacing the butylamine with various functionized amines. After screening the response of the substituted naphthalimides to Cys, we discovered 4-methylsulfonyl-N-n-butyl-1, 8-naphthalimide (MSBN, 5a) as a highly selective fluorogenic probe for thiols. Thiols react quantitatively with MSBN to replace the methylsulfonyl group and switch on the fluorescence (>100-fold increase) via NAS. MSBN was employed to selectively image thiols in live cells, and specifically label protein thiols with turn-on signal to determine diverse thiol modifications. In addition, a bulky group was introduced into the MSBN as a “mass tag” to create a protein redox states probe MSBN-TPP (20), which readily discriminates the reduced thioredoxin (Trx) from the oxidized one. The selective conjugation of MSBN with thiols and the easy derivatization of the naphthalimide unit enable MSBN a universal platform in developing novel agents for studying protein thiol modifications.



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Scheme 2. Synthesis of the substituted naphthalimides.


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Scheme 3. Synthesis of MSBN-TPP. COOH

COOH

O

O

O

NH

O N

O

O

O

O

N

a

O

N

N

O

b

Br

O

O

O

O d

c

Br

N

O HOOC

S

S

14

O

15

17 S

16

O NH HN O O

N

P+

O HOOC

e

NH

g

Br

-

O O

18 O HBr H2N

Br

N

S O

O

Br

f

P O S O

H2N 19

20 (MSBN-TPP) MW: 805

Reaction condition: (a) 6-aminohexanoic acid, EtOH, reflux, 10 h, 80%; (b) CH3SNa, DMF, 60 oC, 8 h, 75%; (c) Nhydroxysuccinimide, EDAC, rt, 12h, 70%; (d) 6-aminohexanoic acid, TEA, rt, 70%; (e) m-CPBA, DCM, reflux, 5 h, 80%; (f) triphenylphosphine, MeCN, reflux, 24 h, 46%; (g) EDAC, DMAP, rt, 12 h, 65%.

Results and Discussion Chemical synthesis The target probes were synthesized from the readily available starting materials (Scheme 2).7,29,32 Briefly, probe 2 was prepared from 5-nitroacenaphthene (Scheme 2A). Different thiols react with 2 to afford the thioether intermediates 3a-3c, which were further oxidized by meta-chloroperoxybenzoic acid (m-CPBA) at room temperature or refluxing in dichloromethlene (DCM) to give the probes 4a-4c or 5a-5c, respectively. Probe 6 was synthesized from 4-bromo-1, 8-naphthalic anhydride via one step of amine insertion (Scheme 2B). Substitution of the bromo in 6 with cyanide or methoxide furnishes probe 10 and intermediate 7. Demethylation of 7 affords 8, which was further reacted with sulfonyl 6 ACS Paragon Plus Environment


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chlorides to yield probes 9a and 9b. Probe 13 was synthesized from the starting material acenaphthene via 3 steps of acetylation, oxidation and amine insertion (Scheme 2C). The Trx redox states probe 20 was prepared as illustrated in Scheme 3.29,35-37 All final probes were fully characterized by 1H NMR,

13

C NMR and MS, and some compounds were further

analyzed by elemental analysis. The original NMR and MS spectra were included in the Supporting Information (Supplementary Figures S2-82).

Probes screening Initially, we screened the response of the different naphthalimides towards Cys. As expected, several probes, such as 2, 4a-c and 5a-c, exhibited turn-on signal upon addition of Cys (Figure 1A) in 0.1 M phosphate buffer solution, pH 7.4 (PBS) at 37 oC. Among all the tested compounds, 4-methylsulfonyl-N-n-butyl-1, 8-naphthalimide (MSBN, 5a) gives the most remarkable fluorescence increment (~100-fold increase after 60 min). MSBN has weak fluorescence at ~490 nm (λex=390 nm, φ90%) of NAS product 4-ethylthio-N-n-butyl-1, 8-naphthalimide (3b). However, it takes 30 min for 4a to react with EtSH affording 3b (87%), and a minor reduction product 3a (2%). Further analysis of the reaction between 4c and EtSH confirms this observation, which gives the NAS product 3b (20%), and the reduction product 3a (58%). This is not surprising as sulfoxides are prone to be reduced by thiols.38,39 The fast and quantitative reaction with a thiol by MSBN could attribute to the strong electron-withdrawing and good leaving characters of the methylsulfonyl group. No reaction was observed for 6, 10 and 12, and only trivial reactions took place for 9a and 9b. The reaction details of all naphthalimides with EtSH were summarized in the Supplementary Table S1. Since MSBN shows a clean reaction with thiols to give a remarkable turn-on fluorescence signal, it was selected for the follow-up studies. 7 ACS Paragon Plus Environment

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Figure 1. Recognition of thiols by MSBN. (A) Response of the probes (5 µM) to Cys (1 mM). The fluorescence intensity at 490 nm (λex=390 nm) was read at 0, 30 and 60 min. The inset shows the samples illuminated at 365 nm after 1 h incubation. Time-dependent absorbance (B) and emission (C) spectra of MSBN (5 µM) to Cys (1mM). The absorbance and emission spectra (λex=390 nm) were recorded. The insets show the dynamic changes of the absorbance at 390 nm and emission at 490 nm. (D) Fluorescence spectra of MSBN (5 µM) to varying concentrations of Cys. The emission spectra (λex=390 nm) were recorded after 1 h-incubation. The inset shows the dose-dependent changes of the emission. (E) Image of thiols in live cells. The bright field (top panel) and fluorescence (bottom panel) images were shown. Scale bars: 20 µm.

Selective Response to thiols by MSBN MSBN responds to thiols exclusively, while other 20 non-thiol amino acids, cystine and inorganic ions give no response (Supplementary Figure S1). MSBN has a maximum absorbance at ~335 nm. Upon addition of Cys, a new absorbance peak centered at ~390 nm appeared gradually with an isosbestic point at ~360 nm (Figure 1B), supporting a clean reaction occurs. The inset shows the time-dependent changes of the absorbance at 390 nm. 8 ACS Paragon Plus Environment


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Addition of Cys also yields a time-dependent increment of the emission spectra centered at ~490 nm (Figure 1C). The inset shows the time-dependent changes of the fluorescence at 490 nm. Both the absorbance and emission spectra reach a plateau within ~60 min, suggesting the completion of the reaction. The fluorescence intensity at varying Cys concentrations (0-4 mM) was also determined, and >100-fold increase of the intensity could be achieved (Figure 1D). The inset shows the dose-dependent changes of the emission. As MSBN selectively responds to thiols, we then applied MSBN to image the cellular thiols in the cultured cells. The cytotoxicity of MSBN was evaluated before we started the live cell imaging experiments. MSBN, with the concentrations less than 10 µM, appears no toxicity to either HeLa cells or HepG2 cells after 24 h treatment (Supplementary Figure S83). As expected, the live HeLa cells treated with MSBN give brilliant blue fluorescence (Figure 1E), which could be suppressed by pretreatment with the thiol blocking agent N-ethylmaleimide (NEM), indicating the specific recognition of cellular thiols by MSBN. The turn-on character of MSBN allows taking the images without additional wash to remove the probe. The formation of covalent bond between the fluorophore and the cellular biomolecules is also preferred for live cell imaging as this would significantly prevent the leakage of the dye from the cells, and thus minimizing the background signal and facilitating the downstream studies.

Figure 2. Specific label of protein thiols by MSBN. (A) Label of BSA thiols by MSBN. BSA was reduced with TCEP (lanes 2 & 3), and blocked with NEM (lane 3). Then MSBN was added to label the protein. Quantification of the in-gel fluorescence intensity was performed by ImageJ. The relative intensity for lanes 1, 2 and 3 is 1, 23.3 and 0 respectively. (B) Interference of TCEP for the label of BSA. BSA samples were reduced with TCEP followed by MSBN labeling. (C) Label of BSA thiols by different probes. BSA was reduced with DTT. Then, samples (lanes 4, 7 & 10) were blocked with NEM. Probe 2 (lanes 2-4),


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MSBN (lanes 5-7) and 5b (lanes 8-10) were added. All samples were separated by SDS-PAGE and visualized by UV illumination (top) and coomassie blue (CB) staining (bottom).

Turn-on label of protein thiols by MSBN Chemical derivatization of proteins with different tags is increasingly used in protein research. As EtSH attacks MSBN to form fluorescent NAS product quantitatively, we hypothesized that MSBN could be a novel turn-on protein thiol labeling reagent. The bovine serum albumin (BSA) was used as a model protein to address this issue. MSBN efficiently labels BSA (Figure 2A) under denatured conditions with (lane 2) or without the reducing agent Tris-(2-carboxyethyl)-phosphine (TCEP) (lane 1). The band in lane 2 is 23.3-fold brighter than that in lane 1 by quantification of the band intensity with the ImageJ software. This is due to that BSA has only one free sulfhydryl group and 17 disulfides. TCEP reduces the disulfides to free sulfhydryl groups, thus giving stronger signal. As the quantification of the band intensity in the gel gives only semi-quantitative results, we then titrated the sulfhydryl

groups

in

BSA

with

5,5’-dithiobis(2-nitrobenzoic

acid)

(DTNB).

After

TCEP-reduction, the sulfhydryl content in BSA is 32.9-fold higher than that in the protein without TCEP pretreatment. This result supported full reduction of the disulfides in BSA by TCEP under our experimental conditions. Blocking the protein thiols by NEM prior to the MSBN treatment completely vanished the fluorescence, suggesting the specific targeting of sulfhydryl groups in BSA by MSBN (Figure 2A, lane 3). The slight shift of the lane 2 to the higher molecular weight position is caused by the binding of MSBN molecules to the protein. TCEP is an alternative reagent to keep the protein thiols in reduced state. TCEP is superior to dithiothreitol (DTT) in some experiments as it has better reducing ability, and the presence of low concentration of TCEP usually does not interfere with the following protein thiol labeling operations. We next determined the tolerance of TCEP in labeling BSA by MSBN. BSA was incubated with varying concentrations of TCEP (0.5-10 mM), and MSBN was added to label the sulfhydryl groups without removal of TCEP. There is no significant interference if the TCEP concentration no higher than 7 mM (Figure 2B, lanes 1-6). Compared to the general thiol labeling reagents such as iodoacetamide derivatives and 10 ACS Paragon Plus Environment


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NEM derivatives,40 MSBN displays better TCEP tolerance. Other substituted naphthalimides, such as 2 and 5b, also label the DTT-reduced BSA specifically, but with less in-gel fluorescence compared to MSBN (Figure 2C), consistent with the highest response of MSBN to thiols (Figure 1A).

Figure 3. Detection of reversible protein thiol modifications by MSBN. (A) The principle of detection of RPTM. (B) Determination of BSA disulfides. BSA was reduced with TCEP followed by MSBN labeling. (C) Detection of PSNO in vitro. BSA was reduced with TCEP followed by SNOC treatment. After the samples were blocked by NEM, ascorbate (VC) and MSBN were added. Samples from (B) and (C) were separated by SDS-PAGE, and visualized by UV illumination (top) and CB staining (bottom). Decrease of cellular thiols (D) and elevation of protein disulfides (E) after H2O2 insult in HeLa cells. (F) Detection of PSNO in HeLa cells. The bright field (top) and fluorescence (bottom) images were shown. Scale bars: 20 µm.

Determination of reversible protein thiol modifications (RPTM) by MSBN As we have demonstrated that MSBN is an efficient and specific protein thiol labeling agent, we next applied MSBN to determine the RPTM in vitro and in cells. The principle of the assay was illustrated in Figure 3A.16,17,41 Briefly, the target protein was first denatured to expose the buried groups, and the free thiol groups were blocked by blocking regents (BR). Then, the modified thiol groups were treated with appropriate agents to liberate the sulfhydryl groups, which were specifically labeled by MSBN to give the fluorescence. We 11 ACS Paragon Plus Environment

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demonstrated that two common RPTM, i. e., disulfide formation and S-nitrosylation, are readily analyzed by MSBN. This assay could be easily extended to determine other RPTM. BSA contains 35 Cys residues that are present as 17 disulfide bonds and 1 free sulfhydryl group. According to the principle in Figure 3A, the presence of disulfide bonds in BSA was confirmed (Figure 3B). The increase of the band intensity indicates more sulfhydryl groups, from the reduction of disulfide bonds by TCEP, were labeled by MSBN. Next, we determined the formation of protein disulfides in cells. The HeLa cells were treated with H2O2 to induce formation of disulfides. The cells were fixed and the remaining free thiols were blocked by NEM. After reduction with TCEP, the nascent sulfhydryl groups were subsequently labeled by MSBN. The cellular thiol level was directly visualized by addition of MSBN to the cells, and gradually decreased after H2O2 treatment (Figure 3D), indicating the oxidation of the thiols. By adopting the procedures in Figure 3A, the presence of protein disulfides and other TCEP-reducible species was demonstrated (Figure 3E). The control cells had marginal fluorescence, indicating that the majority of cellular thiols are present in the reduced form. After H2O2 treatment and then TCEP reduction, the cells gave remarkable fluorescence signal, and this fluorescence intensity is more pronounced with increased amounts of H2O2, suggesting the formation of protein disulfides or other reversibly oxidized thiol species. Protein S-nitrosylation (PSNO) has emerged as an important mechanism for regulation of many classes of proteins.42,43 Following the same principle (Figure 3A), we showed MSBN could detect PSNO both in vitro and in fixed cells. The S-nitrosylated BSA was generated by treatment of the pre-reduced BSA with S-nitrosocysteine (SNOC).43

The remaining

sulfhydryl groups were blocked by NEM, and the protein nitrosothiols were reduced by ascorbate to free thiols, which were synchronously labeled by MSBN. The decomposed SNOC (old SNOC) was used as a negative control. SNOC induces BSA S-nitrosylation dose-dependently (Figure 3C). Next, we determined the PSNO in the cells (Figure 3F). The HeLa cells were treated with SNOC to induce PSNO.43 The positive control (cells directly stained by MSBN) gives the strongest fluorescence signal, indicating most thiols in unstressed cells are in reduced form. The negative control (cells treated with the old SNOC) 12 ACS Paragon Plus Environment


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gives negligible background signal. In contrast, the SNOC-treated cells give the clear fluorescence, and the intensity is dependent on the SNOC concentration, indicating the formation of PSNO in cells.

Determination of Trx redox states by probe 20 (MSBN-TPP) Trx is a highly conserved small redox protein in all cells. Trx interacts with its target proteins via a dynamic thiol-disulfide exchange reaction,44 and the redox states of Trx, i. e., the reduced form where the Cys residues present as free thiols and the oxidized form where the Cys residues present as disulfide, arbitrate the function of Trx. In healthy cells, Trx is predominantly present as a reduced form to participate a variety of cellular processes ranging from antioxidation to apoptosis inhibition. It is thus of great importance to assay the redox states of Trx. We further incorporated a bulky group into MSBN as a “mass tag” to determine the Trx redox states. The triphenylphosphonium-conjugated MSBN, termed as MSBN-TPP (20), was prepared as depicted in Scheme 3. The principle of detection of Trx redox states was illustrated in Figure 4A. The reduced Trx contains free sulfhydryl groups, while the oxidized Trx contains no sulfhydryl groups. MSBN-TPP selectively reacts with the sulfhydryl groups enabling it bind to the reduced Trx but not to the oxidized Trx. The molecular weights (MW) of Trx and MSBN-TPP are ~12 kDa and ~0.8k Da, respectively. Attachment of MSBN-TPP to the reduced Trx remarkably increases the latter apparent MW, while there is no change on the MW of the oxidized Trx after reacting with MSBN-TPP. Based on the MW difference, the reduced Trx could be easily separated from the oxidized one by the conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).


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Figure 4. Assay of Trx redox states by MSBN-TPP. (A) The principle of discrimination of the reduced Trx from the oxidized one. Separation of the reduced form of purified E. Coli Trx (B) or human Trx (C) from the oxidized one by MSBN-TPP labeling. All samples were separated by SDS-PAGE, and were visualized by CB staining. M: MW marker. (D) Detection of Trx redox states in HeLa cell lysate by Western blots.

The E. coli Trx has 2 Cys residues, and the reduced Trx binds 2 molecules of MSBN-TPP to give the protein adduct with retarded mobility in gel. The oxidized Trx, having no free sulfhydryl groups, could not bind MSBN-TPP. Thus, the oxidized Trx could be easily separated from the reduced one by SDS-PAGE (lane 2 in Figure 4B). We further employed mass spectrometer to characterize the modification of the E. coli Trx by MSBN-TPP (Supplementary Figure S84). The MW of the protein was determined to be 12790.46. Bind of 2 molecules of MSBN-TPP to the protein would be expected to give a MW of 14239.3. The adduct of the protein with MSBN-TPP was found to be 14239.7, which matches well with the expected value. Both the sulfhydryl groups in the E. coli Trx were efficiently labeled by MSBN-TPP, as only trace mono-labeled protein was identified (Data not shown). The human cytosolic Trx contains 5 free sulfhydryl groups in its reduced form. Similarly, the reduced human Trx is also readily distinguished from the oxidized form (lane 2 in Figure 4C). The mobility shift between the oxidized and reduced human Trxs is larger than that between the

E. coli ones. This should be due to the bind of more MSBN molecules to the reduced human Trx. There are two well-established assays to determine the redox states of Trx based on 14 ACS Paragon Plus Environment


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the alteration of protein electrophoretic mobility by introducing additional mass or negative charges.45-47 Iodoacetic acid (IAA) is generally applied to bind to the free thiol groups and introduce additional one negative charge per sulfhydryl group. It requires to run urea-PAGE or native PAGE to separate the IAA labeled Trx from the nonmodified Trx. AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonic acid, MW: 490) is a commercially available agent to alkylate the free thiols, which increases the protein molecular weight by ~0.5 kDa per sulfhydryl group. The AMS-modified Trx is then separated from the nonmodified Trx by the regular SDS-PAGE. Compared to the AMS, MSBN-TPP has a molecular weight of 804, which would give better separation of the reduced Trx from the oxidized one after chemical derivatization of the protein. Coupled with the western blots, this assay is readily extended to detect the redox states of Trx in the HeLa cell lysate (Figure 4D). The lysate was treated with excessive diamide or TCEP, which gives the fully oxidized or reduced Trx, respectively. Based on this procedure, we expect that MSBN-TPP could distinguish different oxidation states of Trx in biological samples.

The sulfhydryl group of Cys is considered the strongest nucleophile of all amino acid side chains under physiological conditions. This property allows selective recognition of the biological thiols employing NAS or thiol-addition reaction.12,48 For creation of NAS type probes, the highly electron-deficient 2, 4-dinitrophenyl moiety is generally adopted as an electron sink to quench the emission of various fluorophores.7,9,49-51

Nevertheless, these

probes are not eligible for labeling protein thiols as the thiol-2, 4-dinitrobenzene adducts give little detectable signal. The recent reports using heterocyclic system to construct NAS probes achieve great success to label protein thiols,20,22,52 but there is still lack of convenient readout signal of the protein-probe adducts. The thiol-addition reaction is another strategy to sense thiols via forming covalent adducts, but there is limited application in studying protein thiol modifications.12,26 Popik et al. engaged the photochemical derivatization of Cys residues to label proteins and further demonstrated that many tags could be incorporated into target proteins.19 However, this strategy can only give on-on label of proteins. Although the recent work regarding MSBN (the authors termed as Me-SO2-Naph in the paper) in 15 ACS Paragon Plus Environment

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sensing thiols has been reported, the application of the probe is quite limited and the further modification of the probe was not explored.53 In contrast, we demonstrated here that MSBN selectively conjugates with thiols giving the turn-on fluorescence signal, making it a suitable reagent for not only thiol sensing but also protein thiol labeling. Furthermore, two common RPTM, i. e. formation of disulfides and nitrosothiols, were readily revealed both in vitro and in cells. Moreover, the mass tag probe MSBN-TPP, from the derivatization of MSBN, successfully distinguishes the reduced Trx from the oxidized one. The easy manipulation of the naphthalimide scaffold and the specific reaction of MSBN with thiols provide a versatile platform for constructing thiol-specific probes to study the diverse modification of protein thiols. One major drawback of MSBN is its slow reaction with thiols. In the absorbance and fluorescence spectra (Figure 1), it takes ~60 min for MSBN to complete the reaction with the excessive Cys. It should be noted that although the reaction is slow, the conversion of MSBN to the corresponding NAS product is almost quantitative (Supplementary Table S1). We expected that using large excess of the probe could compensate the slow reaction and achieve complete label of biomolecules, which is supported by the results from the label of BSA and Trx proteins (Figures 2 & 4).

Conclusions In conclusion, we have rationally designed a series of potential thiol-reactive probes based on the NAS reaction and discovered MSBN as a novel turn-on thiol sensing and protein thiol labeling agent. The convenient image of thiols in live cells and determination of RPTM demonstrated the broad applications of MSBN. Furthermore, based on the specific reaction of MSBN with thiols, we constructed a mass-tagged probe MSBN-TPP to assay the redox states of Trx. We expect that MSBN would provide a universal platform for further development of novel molecules with diverse tags for studying protein thiol modifications and thiol-based protein bioconjugation.



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Acknowledgements The financial supports from the Lanzhou University (lzujbky-2014-56), Natural Science Foundation of China (21572093), and Natural Science Foundation of Gansu Province (145RJZA225) are greatly acknowledged.

Supporting Information

Supporting Information Available: Materials and Methods, compounds characterization data, original NMR and MS spectra of compounds, selectivity of MSBN towards various analytes, mass spectra of proteins, and cytotoxicity of MSBN to HeLa cells and HepG2 cells. This material is available free of charge via the Internet.

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