A highly selective sulfinate ester probe for thiol bioimaging

Article abstract of DOI:10.1039/c4cc05462h

We describe here hitherto unexplored chemistry of the sulfinate ester functional group as being highly selective towards nucleophilic substitution by thiols at physiological pH. Using this cleavable trigger, an optical thiol probe that is suitable for thi

Full text of DOI:10.1039/c4cc05462h

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A highly selective sulfinate ester probe for
thiol bioimaging†
Cite this: Chem. Commun., 2014,
50, 11533
Satish R. Malwal, Ajay Labade, Abhijeet S. Andhalkar, Kundan Sengupta and
Harinath Chakrapani*
Received 15th July 2014,
Accepted 6th August 2014
DOI: 10.1039/c4cc05462h
www.rsc.org/chemcomm
We describe here hitherto unexplored chemistry of the sulfinate ester
functional group as being highly selective towards nucleophilic substitu-
tion by thiols at physiological pH. Using this cleavable trigger, an optical
thiol probe that is suitable for thiol bioimaging has been developed.
Thiols are present in nearly all cells and mediate numerous cellular
processes1–3 including cellular signaling, protection from cytotoxic
agents, and maintenance of homeostasis.4 Levels of thiols are thus
tightly regulated in cells and variations are associated with disease
states including diabetes, arteriosclerosis, neurodegenerative dis-
orders, certain cancers and acquired immunodeficiency syndrome
(AIDS).5 Hence, reliable tools to detect and image thiols inside cells
have become essential for diagnostics.6 Among available options,
optical probes, which upon entry into cells undergo an attack or
cleavage by the sulfhydryl group to produce a highly fluorescent
molecule, are desirable due to their simplicity as well as conveni-
ence.7,8 The aforementioned sensor or probe consists of two portions,
first, the thiol cleavable or the reactive group and second, the latent
fluorophore. While numerous classes of latent fluorophores are
reported, the number of distinct thiol-selective activation strategies
remains few.7,9,10 Here, we report a novel sulfinate ester probe that is
selectively cleaved by thiols at physiological pH and this functional
group is suitable for use as a thiol selective cleavable trigger for
sensing biological thiols in physiological media as well as within cells.
Esters of sulfinic acids have long been used in asymmetric
synthesis11,12 and previous investigations have revealed that these
groups can be cleaved by nucleophiles to produce an alcohol and a
thiosulfinate.13,14 The thiophilicity of sulfur-based functional groups
is well established15 and we therefore anticipated that such sulfinate
esters might be reactive with biological thiols as well (Scheme 1).
Upon reaction with a thiol and cleavage, an alcohol is liberated; the
choice of an aryl alcohol which can fluoresce or develop color upon
release might allow us to develop novel thiol probes. To our knowl-
edge, such sulfinate ester-based sensors are not reported.
Scheme 1 Design of a sulfinate ester as a thiol probe: the sulfinate ester
attached with a latent fluorophore is expected to be cleaved by a thiol to
produce a thiosulfinate and a fluorescent alcohol. Inset: compounds 1 and 2.
We considered 1 which contains a tert-butyl sulfinyl functional
group as a possible thiol probe (Scheme 2).16 Resorufin (2), a highly
fluorescent molecule, is routinely used as a fluorescent as well as a
colorimetric probe (Scheme 2).17 When alkylated at the phenolic
position, the resulting ether is weakly fluorescent and upon
cleavage it releases the free phenolate 2. When 1 reacts with thiols,
the liberated phenol would undergo self-immolation to generate 2,
which is highly fluorescent at physiological pH (Scheme 1).
The sulfinate ester 1 was prepared from commercially available
4-hydroxybenzaldehyde in 4 steps (ESI,† Scheme S1). The sulfinate
ester 1 was found to be stable in buffer and weakly fluorescent
(Fig. 1A). Upon addition of cysteine (10 equiv.), we find a significant
Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road,
Pashan Pune 411 008, Maharashtra, India. E-mail: harinath@iiserpune.ac.in
† Electronic supplementary information (ESI) available: Preparative procedures, char-
acterization data, assay protocols and spectral data. See DOI: 10.1039/c4cc05462h
Scheme 2 Proposed mechanism for thiol-mediated cleavage of sulfinate
esters.
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hypochlorite and the hydroxyl radical, we found no significant
increase in fluorescence (Fig. 1C) suggesting that this probe was not
cleaved by common ROS and would hence be suitable for study of
cellular events related to thiol response during elevated ROS as well.
Another important reactive species that is generated in cells is nitric
oxide (NO) and associated reactive nitrogen species (RNS) such as
peroxynitrite (PN). When 1 was exposed to these RNS, we found no
evidence for an increase in fluorescence, again, suggesting that the
sulfinate ester was stable when exposed to these RNS. Finally, we
found no increase in fluorescence when 1 was reacted with Fe(II), a
metal ion that mediates a number of biological processes. Together
our data suggest that probe 1 is inert to conditions frequently
encountered during cellular stress.
Next, cysteine (10 eq.) was added to a solution of 1 and
fluorescence spectra were periodically recorded (Fig. 1D). Curve
fitting gave a pseudo first order rate constant of 0.0414 minÀ1
.
During the time course of the thiol reaction, the sulfinate ester
1 in the absence of a thiol was found to be stable at pH ranging
from 5–9 suggesting the stability in the pH range encountered
under physiological conditions (see ESI†).13,14 Next, 1 was reacted
with increasing equivalents of cysteine and fluorescence emission
was recorded after 30 min. A concentration-dependent increase
in fluorescence response was recorded (Fig. 1E). Based on these
data, we estimated the detection limit for cysteine to be 28 nM
(3Â standard deviation/slope; for GSH, the detection limit was
estimated to be 33 nM) supporting the potential use of this probe
for detecting thiols at low concentrations (see ESI†).
Having established that 1 was an excellent fluorogenic probe
for thiols the capability of 1 to permeate cells to detect thiols
was evaluated. Human adenocarcinoma DLD-1 cells were first
treated with 1 and viable cells were estimated after 24 h using a
standard cell viability assay. We found that DLD-1 cells tolerated 1
well (495% viable cells) at 20 mM (ESI,† Fig. S3). Live cell imaging
of DLD-1 cells treated with 1 using a confocal microscope revealed
that this probe permeated cells and fluoresced upon reaction with
thiols (Fig. 2A). Next, DLD-1 cells were incubated with 1 mM
N-phenylmaleimide (NPM) following which these cells were trea-
ted with 1; NPM is reactive to thiols and has been previously used
to block free thiols inside cells.18 During this experiment, we
found no fluorescence signal attributable to the formation of 2.
Fig. 1 (A) Fluorescence spectra recorded with 1 (10 mM) alone in pH 7.4
buffer and upon reaction with cysteine (100 mM) after 30 min; inset: a
photograph of the cuvette after addition of cysteine. (B) Absorbance
spectra recorded with 1 (50 mM) alone in pH 7.4 buffer and upon reaction
with cysteine (500 mM) after 30 min; inset: a photograph of the cuvette
after addition of cysteine. (C) Fold-increase in fluorescence after exposure
of 1 (10 mM) to 10 eq. of various glutathione (GSH), amino acids, ROS, RNS
and Fe(II) in pH 7.4 buffer after 30 min. (D) fluorescence spectra recorded
when 1 (10 mM) was reacted with cysteine (100 mM) in pH 7.4 buffer.
(E) Thiol probe 1 (10 mM) was independently exposed to various equivalents
of cysteine and fluorescence spectra were recorded after 30 min. Inset: a
plot of fluorescence intensity and concentration. Linear regression analysis
(R2 = 0.9949) gave a slope of 21380.35. The detection limit was estimated
to be 2.77 Â 10À8 M at a signal to noise ratio of 3 : 1.
increase in fluorescence after 30 min (Fig. 1A). When the UV-visible
spectrum was recorded, similarly we found that 1 did not show
significant absorbance in the visible region (Fig. 1B) and upon
addition of cysteine, a significant color change is recorded (Fig. 1B).
This probe is hence suitable for visual detection of thiols as well.
This probe was found to fluoresce only when exposed to free
thiol containing biomolecules such as glutathione; none of the
other biological nucleophiles including amino acids and the
disulfide cysteine were capable of eliciting a significant increase
in fluorescence (Fig. 1C).
Biological thiols are first responders during oxidative stress
caused by elevated levels of reactive oxygen species (ROS) in
cells and hence, detecting thiols during elevated levels of ROS
assumes importance. When 1 was reacted with various biolo-
gically relevant ROS including superoxide, hydrogen peroxide,
the organic peroxides (tert-butyl hydroperoxide, TBHP), sodium
Fig. 2 Live cell imaging carried out with DLD-1 cells: (A) 1, red channel;
(B) 1, Hoechst33258; (C) overlay; (D) 1 co-treated with N-phenylmaleimide
(NPM), a competitive thiol-reactive electrophile; (E) 1 + NPM, Hoechst33258;
and (F) overlay. Probe: excitation 561 nm and emission 568–797 nm;
Hoechst33258: excitation 360 nm and emission 426–797 nm.
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to a diverse array of potential interfering agents including ROS,22
RNS and metal ions. To our knowledge, this is the first report of
a thiol probe that uses a sulfinate ester as the reactive func-
tional group. This probe was found to be cell permeable and
useful for bioimaging biological thiols in live cells. Sulfinate
esters have been viewed thus far as interesting intermediates
for asymmetric synthesis and their reactions with amine
nucleophiles have received considerable attention.11,12 We
report new chemistry of this functional group that might find
applications in thiol-based bioimaging and can, in principle, be
extended to covalent modification of biomacromolecules and
thiol-induced drug delivery.23,24
The authors thank IISER Pune, the Department of Biotechnology
(Grant number BT/05/IYBA/2011), India and Council for Scientific
and Industrial Research (CSIR) for financial support.
Fig. 3 HPLC traces for authentic 3, 4, 5 and reaction mixtures of 3 and
PhSH (0.2 equiv.) and 3 and PhSH (1 equiv.) recorded after 1 h.
Notes and references
Taken together, these results suggest that 1 is highly selective to
activation by thiols and when free thiols within live cells are
unavailable no signal is recorded which support the use of 1 for
thiol bioimaging.
1 D. M. Townsend, K. Tew and H. Tapiero, Biomed. Pharmacother.,
2003, 57, 145.
2 A. Meister, J. Biol. Chem., 1988, 263, 17205.
3 B. M. Lomaestro and M. Malone, Ann. Pharmacother., 1995, 29, 1263.
4 V. Gupta and K. S. Carroll, Biochim. Biophys. Acta, 2014, 1840, 847.
5 A. Bachi, I. Dalle-Donne and A. Scaloni, Chem. Rev., 2012, 113, 596.
6 C. T. Dooley, T. M. Dore, G. T. Hanson, W. C. Jackson, S. J. Remington
and R. Y. Tsien, J. Biol. Chem., 2004, 279, 22284.
7 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120.
8 X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2014, 114, 590.
9 B. Zhu, X. Zhang, Y. Li, P. Wang, H. Zhang and X. Zhuang, Chem.
Commun., 2010, 46, 5710.
10 V. Hong, A. A. Kislukhin and M. G. Finn, J. Am. Chem. Soc., 2009,
131, 9986.
11 J. Drabowicz, A. Zajac, P. Lyzwa, P. J. Stephens, J.-J. Pan and
F. J. Devlin, Tetrahedron: Asymmetry, 2008, 19, 288–294.
12 M. Mikolajczyk, J. Drabowicz and P. Kielbasinski, Chiral Sulfur
Reagents: Applications in Asymmetric and Stereoselective Synthesis,
CRC Press, 1997.
Next, in order to study the mechanism of cleavage of the
sulfinate ester in the presence of thiols, the model sulfinate
ester 3 (Fig. 3) was synthesized by reacting para cresol 4 with
t-butylsulfinyl chloride (see ESI†). A model thiosulfinate 5
(Scheme 2) was similarly synthesized by reacting thiophenol
with t-butylsulfinyl chloride (see ESI†). The sulfinate ester 3 was
treated with sub-stoichiometric amounts of thiophenol (0.2 equiv.)
and HPLC analysis of the reaction mixture (Fig. 3) revealed the
generation of 5 and para-cresol 4 along with unreacted 3. This
observation suggested that a thiol-mediated displacement of the
phenol occurred. When equimolar amounts of 3 and thiophenol
were reacted, we found a significant decrease in 3 with concomitant 13 T. Okuyama, Bull. Chem. Soc. Jpn., 1996, 69, 3281–3287.
14 T. Okuyama, H. Takano and K. Senda, Bull. Chem. Soc. Jpn., 1996,
formation of 5 and 4 suggesting that the thiosulfinate ester was an
intermediate during reaction of sulfinate esters with thiols.
69, 2639.
15 N. Kharasch, S. J. Potempa and H. L. Wehrmeister, Chem. Rev., 1946,
Based on the aforementioned results, a mechanism for
reaction of sulfinate esters was proposed (Scheme 2). Reaction
of a thiol with the sulfinate ester produces intermediate I.13,14
39, 269.
16 Attempts to directly derivatize resorufin with t-butylsulfinyl chloride
failed to give the desired product and we therefore revised our
strategy and introduced a self-immolable linker.
Previous studies have shown that such a hypervalent sulfur 17 K. Cui, Z. Chen, Z. Wang, G. Zhang and D. Zhang, Analyst, 2011,
136, 191.
intermediate was likely when sulfinate esters were reacted with
18 S. Girouard, M.-H. Houle, A. Grandbois, J. W. Keillor and
nucleophiles.6,14,19 Decomposition of I gives a thiosulfinate and the
S. W. Michnick, J. Am. Chem. Soc., 2004, 127, 559.
phenolate II. When the thiol probe 1 was used, the phenolate II 19 T. Okuyama, Chem. Lett., 1995, 997.
could rearrange to produce the fluorophore 2. Previous reports4,20
20 (a) P. Nagy and M. T. Ashby, J. Am. Chem. Soc., 2007, 129, 14082;
(b) P. Nagy, K. Lemma and M. T. Ashby, J. Org. Chem., 2007, 72, 8838.
21 M. H. Lee, Z. Yang, C. W. Lim, Y. H. Lee, S. Dongbang, C. Kang and
indicate that the thiosulfinate ester might further react with a thiol
to produce a disulfide.4 When 5 was treated with excess of
thiophenol, indeed, we find the production of PhS–SPh (see ESI,†
Fig. S1). Similarly, when N-(4-nitrobenzoyl)-cysteine methyl ester
J. S. Kim, Chem. Rev., 2013, 113, 5071.
22 (a) A. T. Dharmaraja, M. Alvala, D. Sriram, P. Yogeeswari and
H. Chakrapani, Chem. Commun., 2012, 48, 10325; (b) A. T. Dharmaraja
and H. Chakrapani, Org. Lett., 2014, 16, 398.
(10 equiv.) was reacted with 3, we found complete decomposition 23 (a) A. T. Dharmaraja, T. K. Dash, V. B. Konkimalla and H. Chakrapani,
Med. Chem. Commun., 2012, 3, 219; (b) H. Chakrapani, A. E. Maciag,
M. L. Citro, L. K. Keefer and J. E. Saavedra, Org. Lett., 2008, 10, 5155;
(c) D. Andrei, A. E. Maciag, H. Chakrapani, M. L. Citro, L. K. Keefer and
within 30 min with concomitant formation of the corresponding
disulfide (see ESI,† Fig. S2).21
Taken together, we report a novel sulfinate ester based ‘‘turn
on’’ fluorescence as well as colorimetric probe for selective
detection and estimation of thiols. This probe can be synthe-
sized with ease from commercial starting material and was inert
J. E. Saavedra, J. Med. Chem., 2008, 51, 7944.
24 (a) S. R. Malwal, D. Sriram, P. Yogeeswari, V. B. Konkimalla and
H. Chakrapani, J. Med. Chem., 2012, 55, 553; (b) S. R. Malwal,
D. Sriram, P. Yogeeswari and H. Chakrapani, Bioorg. Med. Chem.
Lett., 2012, 22, 3603.
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