Naphthalimide Scaffold Provides Versatile Platform for Selective Thiol Sensing and Protein Labeling

Article abstract of DOI:10.1021/acschembio.5b00856

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.

Full text of DOI:10.1021/acschembio.5b00856

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Article
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
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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: fangjg@lzu.edu.cn
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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.
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Keywords: Fluorescent probe • Protein labeling • Thiol sensing • Naphthalimide •
Nucleophilic aromatic substitution
TOC
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Introduction
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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
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Scheme 1. Design of NAS-type thiol fluorogenic probes.
Molecules with the structure of electron withdrawing group (EWG)-
donating group (EDG) are generally fluorescent due to the internal charge transfer (ICT)
effect, while those with EWG- system-EWG or EDG- system-EDG usually display weak
emission because of the blocked ICT (Scheme 1).28 Conversion of EWG-
system-EWG or
EDG- system-EDG to EWG- system-EDG often accompanies with the change of the
π system-electron
π
π
π
π
π
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
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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.
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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.
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O
COOH
COOH
O
N
O
NH
O
9
O
d
O
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O
O
O
O
N
O
N
O
N
O
O
O
N
O
a
b
c
HOOC
Br
Br
S
S
S
17
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O
NH
HN
O
P+
O
N
O
O
-
Br
NH
HOOC
g
e
O
O
N
O
S
O
18
Br
f
P
HBr
H2N
Br
S
O
O
H2N
20 (MSBN-TPP)
MW: 805
19
Reaction condition: (a) 6-aminohexanoic acid, EtOH, reflux, 10 h, 80%; (b) CH3SNa, DMF, 60 oC, 8 h, 75%; (c) N-
hydroxysuccinimide, 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
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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, 13C 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).
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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-
most remarkable fluorescence increment (~100-fold increase after 60 min). MSBN has weak
fluorescence at ~490 nm ( ex=390 nm, <0.001). Cys raises the emission intensity
drastically ( =0.078). The inset shows the picture of samples illuminated at 365 nm. Next,
n-butyl-1, 8-naphthalimide (MSBN, 5a) gives the
λ
φ
φ
we investigated the reaction of the naphthalimides with 10 eq. of a model thiol compound,
ethanethiol (EtSH). Due to the solubility of the reactants, the reactions were carried out in
ethanol/PBS (50/50) mixed solution. The reaction of MSBN and EtSH completes within 15
min to give almost quantitative yield (>90%) 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.
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Figure 1. Recognition of thiols by MSBN. (A) Response of the probes (5 ꢀM) 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 ꢀM) 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 ꢀM) 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 ꢀm.
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.
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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
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imaging experiments. MSBN, with the concentrations less than 10 ꢀM, 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
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visualized by UV illumination (top) and coomassie blue (CB) staining (bottom).
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Turn-on label of protein thiols by MSBN
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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
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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).
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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 ꢀm.
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
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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.
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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)
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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.
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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
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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.
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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
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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).
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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
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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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