A thiol-thiosulfonate reaction providing a novel strategy for turn-on thiol sensing

Article abstract of DOI:10.1039/c5cc05390k

The first thiol-specific turn-on probe, BODIPY-TS, utilizing a thiosulfonate scaffold as the thiol recognition unit was reported. BODIPY-TS displays low toxicity, and features high sensitivity, fast response and quantitative reaction towards thiols. The structural novelty of BODIPY-TS would guide the development of novel thiol probes.

Full text of DOI:10.1039/c5cc05390k

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DOI: 10.1039/C5CC05390K
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Thiol-Thiosulfonate Reaction Providing Novel Strategy for Turn-on
Thiol Sensing
Received 00th January 20xx,
Accepted 00th January 20xx
Chunpo Ge, Hao Wang, Baoxin Zhang, Juan Yao, Xinming Li, Weimin Feng, Panpan Zhou, Yawen
Wang, and Jianguo Fang a *
DOI: 10.1039/x0xx00000x
www.rsc.org/
the absorbance/fluorescence profiles through the alteration of the
intramolecular charge transfer (ICT) or photo-induced electron
transfer (PET) process. The specific reaction of thiols and
thiosulfonates (Scheme 1A) has long been known,16, 17 and led to
the development of several thiol-reactive agents. For example, S-
methyl methanethiosulfonate (MMTS) is a classic thiol-targeted
molecule widely used to block protein thiols.18, 19 However, this
reaction has never been applied to design optical probes. In this
reaction, replacement of the sulfinate moiety by the sulfide also
inverts the group from an EWG to EDG. Thus, we speculated that
attachment of the thiosulfonate unit to certain fluorophores might
generate novel turn-on probes for thiols (Scheme 1B).
The first thiol-specific turn-on probe, BODIPY-TS, utilizing the
thiosulfonate scaffold as a thiol recognition unit was reported.
BODIPY-TS displays low toxicity, and has high sensitivity, fast
response and quantitative reaction towards thiols. The structural
novelty of BODIPY-TS would guide the development of novel thiol
probes.
Small molecule thiols, such as cysteine (Cys) and glutathione (GSH),
and protein thiols, play vital roles in regulating multiple redox-
mediated signaling pathways. Alternations of the cellular thiol
homeostasis have been implicated into a number of diseases,
including cancer, neurodegenerative disorders and cardiovascular
diseases.1-3 Among various methods in detecting thiols,
fluorescence-based assays are powerful and popular due to their
high sensitivity, operational simplicity and good biocompatibility.
Thus, numerous thiol-responsive small molecule fluorescent probes
have been rationally developed in the past years.4-15
Scheme 1 Thiol-thiosulfonate reaction.
Fig. 1 Chemical structures of potential probes.
As part of our continuing interest in discovering and
developing novel small molecule regulators of cellular redox
systems,20-24 we reported, for the first time, the strategy for thiol
probes by employing the specific reaction between thiols and
thiosulfonates. The thiosulfonate scaffold was introduced into
different fluorophores to construct the potential probes (Fig. 1).
The results demonstrate that BODIPY-TS was exclusively switched
on by thiols with ~13-fold increase of the fluorescence intensity.
Analysis of the reaction between Cys and BODIPY-TS revealed that
nearly quantitative conversion of the probe to the corresponding
disulfide product achieved. The response of BODIPY-TS toward
thiols is fast (~5 min) and the detection limit could be down to a few
nanomolar. Submicromolar of BODIPY-TS efficiently images the
thiols in live cells within a few minutes.
Manipulation of the electronic characters of the substituents
attached to a fluorophore from an electron donating group (EDG) to
an electron withdrawing group (EWG) or vice versa usually changes
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S2). Accordingly, BODIPY-TS was selected for studyinVgieiwtsArrteicslepOonnlisnee
DOI: 10.1039/C5CC05390K
to Cys in detail.
BODYPY-TS has maximum absorbance at ~510 nm. After
addition of Cys, its maximum absorbance is blue-shifted to ~495 nm
(Fig. 2A). The response of BODIPY-TS to Cys is fast, and the reaction
completes within 5 min. The inset shows the time-dependent
changes of absorbance at 498 nm. Usually, BODIPY dyes display
narrow Gaussian-shaped absorption bands.29, 30 We noticed that
BODIPY-TS exhibits a broad and structureless band. This is probably
due to the electron-withdrawing capacity of the sulfonyl group.
After addition of Cys, the absorbance was restored to a sharp and
narrow band. The fluorescence spectra were shown in Fig 2B.
BODYPY-TS gives a weak emission centered at 515 nm when excited
at 490 nm (=0.051, absolute quantum yield). The fluorescence
intensity increased ~13-fold after addition of Cys ( =0.356,
absolute quantum yield) (Fig. 1B). The inset shows the dynamic
changes of the emission, and the fluorescence intensity reaches a
plateau after 5 min, consistent with the observation from the
absorbance spectra. The fluorescence intensity of the probe at
varying Cys concentrations (0-1 equiv. of BODYPY-TS) was also
determined (Fig. 2C). The response of BODIPY-TS towards Cys is
linear within the range of 0-1 equiv. of Cys (R2=0.9944, Fig. 2D). The
experimental detection limit of Cys could be down to 39 nM, which
displays
a ~1.2-fold increase of the intensity with good
reproducibility (R2=0.9902, the inset in Fig. 2D). Increasing the Cys
concentration to >1 equiv. of BODYPY-TS causes gradual decreases
of the fluorescence (Fig. 2 E & F). This is probably due to the
decomposition of the disulfide product of the reaction (vide infra)
by the excessive Cys.
Fig. 2 Response of BODIPY-TS to Cys. Time-dependent absorbance
(A) and fluorescence (B) spectra of BODIPY-TS towards Cys. The
spectra were scanned every 1 min for 10 min after mixing the probe
(5 M) with Cys (5 M) in PBS. The insets show the dynamic
changes of absorbance at 498 nm and emission at 515 nm. (C)
Dose-dependent emission spectra of BODIPY-TS toward Cys.
BODIPY-TS (5 M) was incubated with varying concentrations of Cys
in PBS. The emission spectra were recorded after 5 min. (D) Plotting
the fold of fluorescence increase (F/F0) at 515 nm versus the
concentrations of Cys from (C). The inset shows the detection limit
of BODIPY-TS could be down to 39 nM of Cys. (E) Dose-dependent
quench of fluorescence by excessive Cys. BODIPY-TS (5 M) was
incubated with increasing concentrations of Cys (5-60 M) for 5 min,
and the emission spectra were recorded. (F) Plotting the F/F0 at 515
nm versus the increasing ration of Cys from (E). For all fluorescence
spectra, ex=490 nm.
Fig. 3 (A) Selective recognition of thiols by BODIPY-TS. The
fluorescence change at 515 nm (ex=490 nm) was determined after
mixing the probe (5 M) with various analytes for 5 min in PBS. The
concentrations of TGA, GSH and Cys are 5 M, while others are 50
M. The photo of the inset was taken under UV illumination (365
nm). (B). Influence of pH on the response of BODIPY-TS to Cys. The
fluorescence change at 515 nm (ex=490 nm) was determined after
mixing the probe (5 M) with Cys (5 M) for 5 min in buffers with
the pH ranging from 2.0 to 12.0.
The thiosulfonate unit was constructed via coupling sodium
methanesulfinate with the corresponding thiols in acetonitrile as
illustrated in Scheme S1.20, 22, 25-28 The original spectra (1H NMR, 13C
NMR and MS) of the final probes were included in the Supporting
Information (Fig. S3-14). We initially examined the absorbance and
fluorescence spectra of the thiosulfonate probes in responding to
Cys. Upon addition of Cys (1 equiv.), the absorbance spectra of all
compounds were changed distinctly (Fig. S1). The maximum
absorbance of Nap-TS, MC-TS and TFMC-TS was all red-shifted,
while that of BODIPY-TS was blue-shifted. The fluorescence spectra
revealed that Nap-TS and MC-TS gave the on-off signal, while no
significant alteration of the emission spectra for TFMC-TS. In
contrast, BODIPY-TS showed >10-fold increment of the intensity (Fig.
We then evaluated the selectivity of BODIPY-TS towards thiols.
As shown in Fig. 3A, amino acids without a sulfhydryl group give no
response to the probe. NADH, ascorbate and inorganic salts are also
negative to the probe. However, the fluorescence could be
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switched on with >10-fold increment of the intensity by thioglycolic
acid (TGA), GSH or Cys, indicating the exclusive recognition of the
probe by thiols. The photo in Fig. 3A was acquired under UV
illumination (365 nm) after mixing BODIPY-TS (5 M) with the
control buffer (left) or Cys (5 M, right) for 5 min at room
temperature. The pH-dependent response of BODIPY-TS was shown
in Fig. 3B. The emission intensity increases as the pH rises, and
reaches a maximal value at ~7.0. This may be attributed to that the
higher pH facilitates the ionization of Cys to thiolate, and hence
promotes the reaction. As the pH further increases from 8.0 to 12.0,
the emission intensity decreases sharply, which could be due to the
instability of the disulfide product under alkaline circumstance.
BODIPY-TS exhibits stable response to Cys at the pH range of 6.0 to
8.0, suggesting that it is suitable for applying under physiological
environments.
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DOI: 10.1039/C5CC05390K
Fig. 4 Imaging thiols in live HepG2 cells. The cells were treated with
vehicle or IAM followed by incubation with BODIPY-TS (0.5 M),
and the bright-field (top panel) and fluorescence (bottom panel)
images were acquired. Scale bars: 20 m.
With these favorable properties of BODIPY-TS in hands, we
then performed experiments to demonstrate the practical
applications of the probe in thiol sensing in live cells (Fig. 4). The
control cells without the probe treatment show non-detectable
fluorescence (left panel). As expected, the live HepG2 cells stained
with submicromolar of BODIPY-TS for 10 min give bright green
fluorescence (middle panel). Pretreatment of the cells with a thiol-
specific alkylating agent iodoactamide (IAM) completely suppresses
the fluorescence (right panel), supporting the specific recognition of
cellular thiols by the probe.
Scheme 2 Reaction of BODIPY-TS with Cys.
As BODIPY-TS has been demonstrated exclusive response to
thiols, we then employed Cys as a model thiol to study the reaction
details. Slight excess of BODIPY-TS (0.07 mmol) was mixed with Cys
(0.065 mmol) in ethanol/H2O. The reaction runs smoothly at room
temperature, and completes within 5 min monitored by TLC. The
only product was identified as the mixed disulfide compound (Cys-
S-S-BODIPY, Scheme 2) with 85% isolated yield, indicating a clear
reaction of BODIPY-TS with Cys. We also run a reaction of BODIPY-
TS with excess Cys (5 eq.), and the product was identified as
BODIPY-SH in 78% yield (Scheme 2). The original spectra (NMR and
MS) of Cys-S-S-BODIPY and BODIPY-SH were included in the
Fig. 5 Cytotoxicity of BODIBY-TS and the general thiol-reactive
agents to HeLa (A) and HepG2 (B) cells. The cells were treated with
different compounds at indicated concentrations for 24 h, and the
cell viability was determined by the MTT assay. Data were
expressed as the mean±SD.
Supporting Information (Fig. S15-19).
It was reported that
Most of the thiol probes contain a thiol-reactive motif that
selectively and irreversibly reacts with the sulfhydryl group,6-10 thus
inevitably depleting cellular thiols. As thiols are vital for cell
function, these probes might elicit potential toxicity. In contrast,
BODIPY-TS reacts with thiols to form mixed disulfide compounds,
which are physiologically reversible for regeneration of the thiols.
We thus hypothesized that BODIPY-TS might have lower toxicity.
The MTT assay was adopted to determine the cytotoxicity of
BODIPY-TS and some commercially thiol-reactive agents, i. e., N-
ethylmaleimide (NEM), 2, 4-dinitro-1-choloro-benzene (DNCB) and
IAM. Compared to NEM, DNCB and IAM, BODIPY-TS exhibits much
less cytotoxicity (Fig. 5). As the core structure of NEM, DNCB or IAM
attachment of 2, 4-dinitrobenzene moiety to the BODIPY scaffold
quenches the later fluorescence via a PET process.31, 32 In analogy to
this, we reasoned that the weak fluorescence of BODIPY-TS is likely
due to the PET process as the benzene ring in the molecule is more
electron-deficient compared to that in Cys-S-S-BODIPY. Conversion
of BODIPY-TS to Cys-S-S-BODIPY increases the electron density of
the benzene ring and blocks the PET process, thus enhancing the
emission intensity.
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is commonly employed in a majority of thiol probes,6-10 the low
cytotoxicity of BODIPY-TS provides another advantage for biological
thiol sensing.
19 S. R. Jaffrey, H. Erdjument-Bromage, C. D. Ferris, P. Tempst
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In summary, the thiosulfonate scaffold was employed to
create a turn-on thiol probe BODIPY-TS for the first time. The
probe has low toxicity, and features high selectivity, low
detection limit, fast response and quantitative reaction to
thiols. The structural novelty of BODIPY-TS would guide the
development of novel thiol sensors and protein thiol labeling
agents.
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ACKNOWLEDGEMENTS
The financial supports from the Lanzhou University (lzujbky-
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