A highly selective fluorescent probe for thiol bioimaging

Article abstract of DOI:10.1021/ol702539v

A new fluorescent turn-on probe (3) for the selective sensing and bioimaging of thiols is reported. In aqueous buffer solutions at physiological pH, thiols cleave the 2,4-dinitrobenzenesulfonyl group to release the red-emissive donor-acceptor fluorophore

Full text of DOI:10.1021/ol702539v

ORGANIC
LETTERS
2008
Vol. 10, No. 1
37-40
A Highly Selective Fluorescent Probe
for Thiol Bioimaging
Jean Bouffard,† Youngmi Kim,‡ Timothy M. Swager,† Ralph Weissleder,‡ and
Scott A. Hilderbrand*,‡
Center for Molecular Imaging Research, Massachusetts General Hospital, HarVard
Medical School, Charlestown, Massachusetts 02129, and Department of Chemistry,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
scott_hilderbrand@hms.harVard.edu
Received October 18, 2007
ABSTRACT
A new fluorescent turn-on probe (3) for the selective sensing and bioimaging of thiols is reported. In aqueous buffer solutions at physiological
pH, thiols cleave the 2,4-dinitrobenzenesulfonyl group to release the red-emissive donor acceptor fluorophore (4). The probe displays excellent
immunity to interference from nitrogen and oxygen nucleophiles and the imaging of thiols in living cells is demonstrated.
−
The rapid, sensitive, and selective sensing of thiols is of
significant interest in areas ranging from the petrochemical
industry,1 to food quality control2 and medicine. The optical
sensing of thiols is particularly relevant in biological systems.
Intracellular thiols such as glutathione (GSH), cysteine (Cys),
and homocysteine (HCys) play a crucial role in maintaining
biological redox homeostasis through the equilibrium estab-
lished at a given electrical potential between reduced free
thiols (RSH) and oxidized disulfides (RSSR). Thiols are also
active in the catalytic sites of enzymes, and play important
roles in metabolic pathways.3 The levels of certain thiols,
such as homocysteine, have been linked to a number of
diseases, including cancer, Alzheimer’s, and cardiovascular
disease.4 Thiol levels may also be affected in response to
the oxidative stress that has been associated with some of
these conditions.5 Furthermore, thiol probes are desirable for
assaying enzymes that release a thiol upon reaction with a
substrate (e.g., thioesterases) and for finding and evaluating
new inhibitors of these enzymes.6
Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)) is the
classical probe for the determination of sulfhydryl groups
by UV-vis absorption spectrophotometry.7 A large number
(4) (a) Nekrassova, O.; Lawrence, N. S.; Compton, R. G. Talanta 2003,
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254. (b) Ohshima, H.; Tatemichi, M.; Sawa, T. Arch. Biochem. Biophys.
2003, 417, 3-11. (c) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-
247.
† Massachusetts Institute of Technology.
‡ Harvard Medical School.
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1425. (b) Earle, T. E. Anal. Chem. 1953, 25, 769-771.
(2) (a) Scampicchio, M.; Lawrence, N. S.; Arecchi, A.; Cosio, S.;
Mannino, S. Electroanalysis 2007, 19, 85-90. (b) Whitman, D. W.;
Whitney, R. M. Anal. Chem. 1953, 25, 1523-1527.
(3) (a) Finkelstein, J. D.; Martin, J. J. Int. J. Biochem. Cell Biol. 2000,
32, 385-389 (b) Meister, A. Methods Enzymol. 1995, 251, 3-7.
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Cre´minon, C.; Grassi, J.; Mioskowski, C. J. Am. Chem. Soc. 2002, 124,
4874-4880. (b) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki,
K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922-2925. (c) Maeda, H.;
Katayama, K.; Matsuno, H.; Uno, T. Angew. Chem., Int. Ed. 2006, 45,
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10.1021/ol702539v CCC: $40.75
© 2008 American Chemical Society
Published on Web 12/07/2007

Scheme 1. Synthesis of Fluorescent Thiol Probe 3
of chromophores and fluorophores bearing electrophilic
is detailed. Probe 3’s design is based on a classical donor-
π-acceptor architecture. Thiol-mediated cleavage of the
electron-withdrawing sulfonyl group releases an aniline
donor, increasing the push-pull character of the dye and
resulting in a higher quantum yield and in large bathochromic
shifts in the absorption and emission spectra. The choice of
the arenesulfonamide13 protecting group improves the probe’s
resistance toward oxygen and nitrogen nucleophiles, and
increase selectivity toward thiols in comparison to arene-
sulfonate-based probes.6b,c Substitution of the sulfonamide
N-H with a side chain extends the pH range in which probe
3 can be used compared to the corresponding acidic
secondary 2,4-dinitrobenzenesulfonamides. The latter de-
compose to the corresponding 2,4-dinitroanilines after extru-
sion of sulfur dioxide under basic conditions.10,13 Further-
more, the side chain influences the properties of the probe.
The triethyleneglycol methyl ether chain confers higher water
solubility to probe 3. The synthesis of probe 3 is presented
in Scheme 1.
Probe 3 is prepared in moderate yield in three steps starting
from an acetal-protected 4-aminobenzaldehyde. The modular
design allows for the independent variation of each compo-
nent (arenesulfonyl group, side chain, π-conjugated bridge,
electron acceptor) and facilitates the tailoring of probes for
specific applications. For instance, side chains bearing
molecular recognition groups or surface anchors may be
introduced without the need to redesign the probe’s synthesis.
The validity of the proposed sensing mechanism was
confirmed by the preparative-scale synthesis of the free dye
4 from 3 and thioglycolic acid (Scheme 2).
groups (e.g., iodoacetamides, maleimides, and benzyl halides)
to which thiols may be covalently attached have been
developed for thiol bioconjugation and are commercially
available. However, these often suffer from low on/off signal
ratios, necessitating washing and isolation steps, thus pre-
cluding rapid quantification.8
The area of thiol-sensitive sensors and imaging agents is
undergoing a renaissance that is largely driven by the
development of selective fluorescent turn-on probes. In one
of these approaches, the reaction between a thiol and a
maleimide interrupts an intramolecular PET (photoinduced
electron transfer) process, converting the quenched probe into
an efficient fluorophore.9 In an alternative strategy, the use
of a thiol nucleophile as a reagent for the deprotection of an
electron-poor arenesulfonate ester10 results in the release of
a fluorophore.6b,c,11 Many of these probes still suffer from
poor solubility in aqueous media, requiring the use of organic
cosolvents.12 Arenesulfonate protecting groups are also
susceptible to attack by oxygen or nitrogen nucleophiles.
These parasitic side reactions (e.g., unselective hydrolysis)
lower both sensitivity (higher blank signals) and selectivity
for thiol analytes. Finally, many of these probes are excited
and/or emit in the near-UV to green region of the spectrum.
However, long-wavelength probes with emission in the red
or near-infrared are optimal for biological imaging applica-
tions due to decreased light scattering, increased optical
transparency, reduced autofluorescence, and greater photo-
stability of tissues at these wavelengths.
In this report, the design and synthesis of a new fluorescent
probe for thiols (3) that addresses some of these drawbacks
Scheme 2. Cleavage of Sulfonamide 3 with Thiols
(8) For surveys of thiol probes: (a) Wang, W.; Rusin, O.; Xu, X.; Kim,
K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry, M.;
Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.;
Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949-15958. (b) Haughland,
R. P. Handbook of Fluorescent Probes and Research Products, 9th ed.;
Molecular Probes, Inc.: Eugene, OR, 2002; p 79.
(9) (a) Matsumoto, T.; Urano, Y.; Shoda, T.; Kojima, H.; Nagano, T.
Org. Lett. 2007, 9, 3375-3377. (b) Langmuir, M. E.; Yang, J.-R.; Moussa,
A. M.; Laura, R.; LeCompte, K. A. Tetrahedron Lett. 1995, 36, 3989-
3992.
(10) Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan, T.
Tetrahedron Lett. 1997, 38, 5831-5834 and references cited therein.
(11) For a related approach: Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu,
F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666-11667.
(12) Zhang, M.; Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.; Liu,
Z.; Zhang, J.; Zhang, D.; Yi, T.; Huang, C. J. Am. Chem. Soc. 2007, 129,
10322-10323.
The sensory response of probe 3 is exemplified by its
reaction with cysteine (Figure 1). Upon addition of cysteine
(13) During the preparation of this manuscript, a first thiol probe based
on the deprotection of a sulfonamide has been reported: Jiang, W.; Fu, Q.;
Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed. 2007, 46, 8445-8448.
38
Org. Lett., Vol. 10, No. 1, 2008

Figure 1. (a) Absorption and (b) emission spectra (λex ) 560 nm) of probe 3 before (dotted line) and after (solid line) addition of cysteine
in HEPES buffer solution (10 mM, pH 7.4). The spectra were obtained immediately after addition of cysteine. The final concentrations of
probe and cysteine are 10 µM and 1 mM, respectively.
to a solution of 3 in HEPES buffer (10 mM, pH 7.4), the
solution instantly turns from yellow to pink, and the
absorption spectrum shows the development of the charac-
teristic absorption band of 4 that is red-shifted by 158 nm
compared to that of 3. A remarkable increase in the
fluorescence intensity of up to 120-fold, depending on the
concentrations of the reagents, is also observed.
The selectivity of probe 3 for thiols was confirmed by
screening its response to biologically relevant analytes under
physiological conditions (37 °C, 10 mM HEPES buffer, pH
7.4) (Figure 2). Probe 3 shows a positive fluorescence
increases above blank levels were observed in the presence
of amines (lysine), reactive oxygen species (tert-butylhy-
droperoxide, hydrogen peroxide, sodium hypochlorite), or
reducing agents (ascorbic acid). Probe 3 also remains
essentially unaffected by a hydrolytic enzyme (porcine liver
esterase). It is worth noting that probe 3 is responsive to
thiols at a pH lower than their pKa (∼10-11), indicating
that complete deprotonation of the thiols is not required for
their detection.
In aqueous media (10 mM HEPES buffer, pH 7.4), probe
3 has an absorption maximum at 405 nm (ꢀ ) 21 200 mol
L-1 cm-1) and is essentially nonfluorescent with a trace
emission centered at λex ) 560 nm (ΦF ) 0.0008). The
quenched fluorescence is attributed to the absence of charge-
transfer character in the protected probe and to an efficient
donor-excited photoinduced electron transfer (PET) to the
dinitrobenzenesulfonyl group. Upon deprotection, free dye
4 exhibits a significant bathochromic shift in its absorption
spectrum, with an absorption maximum at 563 nm (ꢀ )
35 100 mol L-1 cm-1), indicative of the more efficient
delocalization in the push-pull chromophore.
The absorption and emission spectra of 3 and 4 remain
essentially unchanged over a wide pH range (5.6-9.5) that
is compatible with most biological applications. Furthermore,
in aqueous buffer solution at pH 7.4, no hydrolysis of probe
3 is detected over 12 h at 37 °C.
In aqueous solution, the fluorescence quantum yield of 4
remains modest with ΦF ) 0.01 (λem ) 623 nm). However,
like other donor-acceptor fluorophores bearing dicyanom-
ethylene-based acceptors,14 the fluorescence quantum yield
increases in a more viscous or rigid environment. In 90:10
glycerol:methanol (v/v), the quantum yield reaches a value
of 0.18. This dependence on media can be advantageous for
Figure 2. Response of probe 3 to different analytes. Relative
fluorescence intensity of 10 µM probe 3 (HEPES buffer, pH 7.4)
at 627 nm (λex ) 565 nm) before (white bars, left) and after (black
bars, right) incubation at 37 °C for 15 min in the presence of
100 µM (final concentrations) analytes. Porcine liver esterase was
added to a concentration of 1.4 units/mL.
(14) (a) Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg, R. J.; Moerner,
W. E. J. Am. Chem. Soc. 2003, 125, 1174-1175. (b) Willets, K. A.;
Nishimura, S. Y.; Schuck, P. J.; Twieg, R. J.; Moerner, W. E. Acc. Chem.
Res. 2005, 38, 549-566 and references cited therein. (c) Nesterov, E. E.;
Skoch, J.; Hyman, B. T.; Klunk, W. E.; Bacskai, B. J.; Swager, T. M. Angew.
Chem., Int. Ed. 2005, 44, 5452-5456. (d) Haidekker, M. A.; Theodorakis,
E. A. Org. Biomol. Chem. 2007, 1669-1678.
response only in the presence of thiols (cysteine, reduced
glutathione, dithiothreitol), for which fluorescence signals
were increased 60- to 120-fold. No significant signal
Org. Lett., Vol. 10, No. 1, 2008
39

Figure 3. Fluorescence images of 3T3 cells: (A) brightfield image of cells incubated with probe 3 (25 µM) for 10 min at 37 °C, and
stained with a nucleus-staining dye, Hoechst 33342; (B) fluorescence image collected with a Hoechst dye filter set; (C) fluorescence image
collected with a Cy3 dye filter set; (D) overlay image of B and C; and (E-H) corresponding control images of cells pretreated with
N-methylmaleimide (1 mM) for 60 min at 37 °C and then incubated with 3 as above.
biological imaging. Upon interaction or adsorption with
macromolecules and surfaces in cellular environments, the
ΦF of 4 increases, while probe 3 remains essentially
nonemissive. The photophysical properties of the free dye 4
in the presence of human serum albumin (HSA) illustrate
this effect. The addition of 1% HSA to a solution of 4 in
HEPES buffer increased its ΦF to 0.24. Small bathochromic
and hyperchromic shifts are also observed upon addition of
HSA. The beneficial influence on the fluorescence quantum
yield of interactions between dye 4 and biological surfaces
and macromolecules suggests that turn-on fluorescence signal
improvements greater than the 120-fold on/off ratios ob-
served in ordinary aqueous solutions may be achieved in a
number of biosensing schemes. Furthermore, the photophysi-
cal properties of fluorophores closely related to 4 suggest
that this thiol probe could be adequate for single-molecule
or 2-photon sensing schemes.14b
The monitoring of thiols in living cells by probe 3 was
undertaken. Albino Swiss mouse embryo fibroblast cells (3T3
cell line) were incubated with a solution of probe 3 (25 µM
in 1:100 DMSO-PBS v/v, pH 7.4) for 10 min at 37 °C.
Probe 3 was found to be cell-permeable and to react with
intracellular thiols, resulting in strong fluorescence emission
as observed by fluorescence microscopy (Figure 3C). In a
control experiment, cells that were pretreated with an excess
of the thiol-reactive N-methylmaleimide, which consumes
all of the free thiols within the cell, and then incubated with
probe 3 do not show a significant fluorescence signal (Figure
3G). This confirms the specificity of probe 3 for thiols over
other analytes in living cells.
In conclusion, probe 3 has been shown to be a thiol-
selective turn-on fluorescence probe with high on/off ratios
(up to 120-fold) with emission in the red region of the
spectrum. Probe 3 shows good resistance toward unselective
hydrolysis. The emissive free dye 4 released after reaction
with a thiol exhibits large batho- and hyperchromic shifts
with respect to 3, and features a fluorescence quantum
efficiency that is greatly enhanced in the presence of
biological macromolecules. The application of probe 3 for
the bioimaging of thiols in live cells has been demonstrated,
and shows potential for in vivo small animal imaging.15 The
modular nature of the probe’s design opens the possibility
of tailored probes for specific applications.
Acknowledgment. This work has been supported at MIT
in part by a NSERC Fellowship to J.B. and by NIH grant
1-U01-HL080731.
Supporting Information Available: Experimental pro-
cedures and full spectroscopic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL702539V
(15) Ntziachristos, V.; Ripoll, J.; Wang, L.; Weissleder, R. Nat. Bio-
technol. 2005, 23, 313-320.
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