Highly fluorescent and specific molecular probing of (homo)cysteine or superoxide: Biothiol detection confirmed in living neuronal cells

Article abstract of DOI:10.1021/ol401480w

Chemodosimetric action in detection of cysteine and homocysteine (310- and 290-fold) and superoxide inputs (336-fold increase) gives significant fluorescence intensity increases. Detection limits of 2.13 × 10 ^-5 M, 1.37 × 10^-5 M, and 2.71 × 10 ^-5 M, respectively, are biorelevant and are consistent with OR logic gating, demonstrated in intracellular biothiol detection in neuronal cells by way of novel fluorescein derivatization. As per our knowledge, this is the first example of a novel fluorescent probe based on the nucleophilic substitution reaction of biothiols and superoxide through ester cleavage.

Full text of DOI:10.1021/ol401480w

ORGANIC
LETTERS
2013
Vol. 15, No. 14
3630–3633
Highly Fluorescent and Specific Molecular
Probing of (Homo)Cysteine or Superoxide:
Biothiol Detection Confirmed in Living
Neuronal Cells
Dhiraj P. Murale,† Hwajin Kim,‡ Wan Sung Choi,‡ and David G. Churchill*,†
Molecular Logic Gate Laboratory, Department of Chemistry, Korea Advanced Institute
of Science and Technology (KAIST), 373À1 GuseongÀdong, YuseongÀgu,
Daejeon, 305À701, Republic of Korea, and Department of Anatomy and Neurobiology,
Medical Research,Center for Neural Dysfunction, Institute of Health Science,
School of Medicine, Gyeongsang National University, 92 ChilamÀdong, Jinju,
Gyeongnam 660À751, Republic of Korea
dchurchill@kaist.ac.kr
Received May 27, 2013
ABSTRACT
Chemodosimetric action in detection of cysteine and homocysteine (310- and 290-fold) and superoxide inputs (336-fold increase) gives significant
fluorescence intensity increases. Detection limits of 2.13 Â 10À5 M, 1.37 Â 10À5 M, and 2.71 Â 10À5 M, respectively, are biorelevant and are
consistent with “OR” logic gating, demonstrated in intracellular biothiol detection in neuronal cells by way of novel fluorescein derivatization. As
per our knowledge, this is the first example of a novel fluorescent probe based on the nucleophilic substitution reaction of biothiols and
superoxide through ester cleavage.
Selective and sensitive detection of biothiols such as
cysteine (Cys), homocysteine (Hcy) and glutathione (GSH)
has became an important issue because of the vital role of
these analytes in maintaining various biological processes.1
Cys and Hcy are very important small biomolecules, essential
for growth of cells and tissues in living organisms. Among
other considerations, the imbalance in the levels of these
biothiols are thought to lead to many serious disease path-
ways. A decreased level of Cys can cause retardation in
growth, hair pigmentation, edema, lethargy, liver damage,
muscle and fat loss, skin lesions and weakness.2 Increased
levels of Hcy in blood plasma may lead to Alzheimer’s disease
and cardiovascular disease.3 GSH deficiency will lead to
(2) Shahrokhian, S. Anal. Chem. 2001, 73, 5972.
† Korea Advanced Institute of Science and Technology (KAIST).
‡ Gyeongsang National University.
(1) Chen, X.; Zhou, Y.; Peng, X. J.; Yoon, J. Chem. Soc. Rev. 2010,
39, 2120.
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I. H.; D’Agostino, R. B.; Wilson, P. W. F.; Wolf, P. A. N. Engl. J. Med.
2002, 346, 476. (b) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E.
Annu. Rev. Med. 1998, 49, 31.
r
10.1021/ol401480w
Published on Web 06/28/2013
2013 American Chemical Society

oxidative stress, which is considered among the possible
causes of Alzheimer’s disease.4 Thus, the selective detection
of biothiols is a very important issue. For the detection of
biothiols, previously reported chemodosimeters,5 based on
disulfide cleavage,6 cyclization of aldehydes,7 nucleophilic
additions,8 ligand reactions9 and Michael additions,10 have
been reported, among other modes.
and mass spectrometry (ESI). We obtained selective sensing
for biothiols and superoxide through a nucleophilic additionÀ
elimination reaction at the benzoyl position (Scheme 1)
releasing unsubstituted fluorescein as the final product.
Scheme 1. Synthesis of Probe 1 and Mechanism
Reactive oxygen species (ROS) are thought to play a decisive
role in neurodegenerative disease disorders (Alzheimer’s and
Parkinson’s disease) and in environmental chemistry.11 Re-
active oxygen species (ROS) include superoxide, hypochlo-
rite, hydrogen peroxide, hydroxyl radical, nitric oxide and
peroxynitrite. Superoxide (O2À) has a short half-life; real-
time detection is an important task. O2À is involved in a range
of physiological processes in living organisms, such as aging,
muscle fatigue, ischemiaÀreperfusion and inflammation.12,13
The detection of superoxide with high selectivity and sensi-
tivity continues to be a significant challenge.
Selectivity and sensitivity are currently important ad-
vantages of chemodosimeters over chemosensors; in recent
times, a lot of research is being conducted in the develop-
ment of new chemodosimeters. Recently, in our research
group, we have developed a novel rechargeable mesoÀaryl
BODIPY-based chemodosimeter for the selective detec-
tion of cysteine over other biothiols (Hcy and GSH).14
Herein, we report a novel fluorescein-based probe for
selective detection of biothiols and superoxide with a
dramatic change in fluorescence intensity.
Spectroscopic properties of probe 1 were determined
under physiological conditions (20 mM HEPES, pH 7.4).
First, the probe was dissolved in DMSO and then subse-
quently diluted in 80% 10 mM HEPES, pH 7.2. The
detection properties of our new probes can be assessed
via UVÀvis absorption and emission spectroscopy.
We designed our probe via the concept of simple benzoyl
protection of the hydroxyl groups of fluorescein (Scheme 1).
With this in mind, we started our synthesis from fluorescein
and commercially available 3,5-dinitrobenzoyl chloride in a
facile one-step reaction with good yield and purity. The probe
The presence of the benzoyl group encouraged us to check
probe reactivity with different amino acids. When test-
ing the probe with sulfur-containing amino acids (L-Cys,
Hcy, N-acetyl-L-Cys, Met, GSH) and non-sulfur-containing
amino acids (Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile,
Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val) as aqueous
solutions (∼ 10 equiv), changes in the color of the probe
solution were found with Cys and Hcy only. In these assays,
we used 3 mL of probe solution (4 Â 10À6 M, buffered H2O/
DMSO 80:20; pH 7.2; 10 mM HEPES) and incubated with
1
was first characterized by H and 13C NMR spectroscopy
(4) (a) Wu, G. Y.; Fang, Y. Z.; Yang, S.; Lupton, J. R.; Turner, N. D.
J. Nutr. 2004, 134, 489. (b) Townsend, D. M.; Tew, K. D.; Tapiero, H.
Biomed. Pharmacother. 2003, 57, 145. (c) Herzenberg, L. A.; eRosa,
S. C.; Dubs, J. G.; Roederer, M.; Anderson, M. T.; Ela, S. W.;
Deresinski, S. C.; Herzenberg, L. A. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 1967. (d) Huber, W. W.; Parzefall, W. Curr Opin. Pharmacol.
2007, 7, 404.
(5) (a) Yang, Y. M.; Zhao, Q.; Feng, W.; Li, F. Y. Chem. Rev. 2013,
113, 192. (b) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev.
2010, 39, 2120. (c) Das, P.; Mandal, A. K.; Chandar, N. B.; Baidya, M.;
Bhatt, H. B.; Ganguly, B.; Ghosh, S. K.; Das, A. Chem.;Eur. J. 2012,
18, 15382.
(6) Cao, X. W.; Lin, W. Y.; Yu, Q. X. J. Org. Chem. 2011, 76, 7423.
(7) Tanaka, F.; Mase, N.; Barbas, C. F. Chem. Commun. 2004, 1762.
(8) Zhang, M.; Yu, M. X.; Li, F. Y.; Zhu, M. W.; Li, M. Y.; Gao,
Y. H.; Li, L.; Liu, Z. Q.; Zhang, J. P.; Zhang, D. Q.; Yi, T.; Huang, C. H.
J. Am. Chem. Soc. 2007, 129, 10322.
(9) Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H. Science
1994, 266, 776–779.
(10) Jung, H. S.; Ko, K. C.; Kim, G. H.; Lee, A. R.; Na, Y. C.; Kang,
C.; Lee, J. Y.; Kim, J. S. Org. Lett. 2011, 13, 1498.
(11) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem.
Rev. 1997, 97, 1515. (b) Gunnlaugsson, T.; Glynn, M.; Tocci, G. M.;
Kruger, P. E.; Pfeffer, F. M. Coord. Chem. Rev. 2006, 250, 3094. (c) Que,
E. L.; omaille, D. W.; Chang, C. J. Chem. Rev. 2008, 108, 4328.
(12) Chaudhury, S.; Sarkar, P. K. Biochim. Biophys. Acta 1983, 763,
93.
(13) Kozumbo, W. J.; Trush, M. A.; Kensler, T. W. Chem. Biol.
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Figure 1. (Left) Emission spectra of probe 1 (4.0 Â 10À6 M,
buffered H2O/DMSO 80:20; pH 7.2; 10 mM HEPES) with
amino acids L-Cys, Hcy, N-acetyl-L-Cys, Met, GSH, Ala, Arg,
Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,
Trp, Tyr, Val) (3.3 Â 10À3 M in water) incubated for 20 min at RT.
(Right) Time-dependent emission spectra of probe 1 (4.0 Â 10À6 M,
buffered H2O: DMSO 80:20; pH 7.2; 10 mM HEPES) with Cys and
Hcy (∼10 equiv in water).
Org. Lett., Vol. 15, No. 14, 2013
3631

10 equiv of amino acid. These two analytes also gave
dramatic increases in fluorescence intensity: 310- and
290-fold, respectively (Figure 1). Also when testing with
other competing amino acids under analogous conditions,
no changes in emission intensity were found, suggesting no
participation of other amino acids in ester hydrolysis
(Figures S5, S6, Supporting Information).
Since superoxide is well-known as a “super nucleophile,”15
the probe was tested with superoxide: the same response as
that for biothiols was found via ester cleavage (Figure 2).
When other reactive oxygen species were assayed, there was
no response; also, there was no interference or competition of
other ROS with superoxide activity (Figure S7, Supporting
Information). As per the above studies, we obtained two
inputs for this sensing as biothiols and superoxide; when
implicated, the logic gate was found to be an “OR” gate, as
shown in Figure 3.
Figure 3. “OR” logic gating with biothiols and superoxide along
with truth table for the substrate and probe 1.
assay of probe 1 with SHÀSY5Y cells (Figure 4). Cells were
first treated with probe 1 (10 μM) for 30 min before being
washed with PBS buffer. Then, incubation with Mitotracker
Red (100 nM) was performed for 10 min, followed by
washing before imaging (probe 1, λexci = 490 nm;
Mitotracker, λexci = 590). For the “NEM experiment,” cells
were pretreated with N-ethylmaleimide (NEM) (1.0 mM)
for 30 min; this consumes all the cysteine residues in the cells.
Washing with PBS buffer preceded incubation with probe 1
(10 uM) for 30 min. Cells were washed, incubated with
Mitotracker Red (100 nM) for 10 min, and washed again
before imaging was acquired. Mitotracker Red confirmed
cell viability and intracellular fluorescence of Probe 1.
Figure 2. (Left) Emission spectra of probe 1 (4.0 Â 10À6 M,
buffered H2O/DMSO 80:20; pH 7.2; 10 mM HEPES) with
(ROS) KO2, H2O2, NaOCl, tBuOOH, •OH, •OtBu, mÀCPBA.
(3.3Â 10À3 M in water) incubated for 20 min at RT. λexci =490nm,
slit width Ex, Em = 1.5. (Right) Time-dependent emission spectra
of probe 1 (2.0 Â 10À6 M, (buffered H2O/DMSO 80:20; pH 7.2;
10 mM HEPES) with KO2 (∼10 equiv in water).
From the above emission studies, two input logic gate
“OR” systems can be represented. Here, the probe has
been established as a molecular system that exhibits com-
binational logic gate properties based on two biologically-
important inputs: biothiols and superoxide. The optical
output completes the OR logic gate. We plot the “truth
table” for the response of these inputs in which output “1”
is fluorescence enhancement (@ 522 nm) and “0” is no
change in emission intensity (Figure 3). Because of the
selectivity, analytes give no intermediate signals between a
strong signal or no signal.
To support the proposed sensing mechanism, we per-
formed a small scale reaction of probe 1 with 2 equiv. of
LÀCys. LÀCys (3.0 mg) was added to a solution of the probe
1 (20 mg) in DMSO: D2O (9:1), triethyl amine (100 μL) and
was left for 45 min at RT. After this time reaction mixture was
filtered, and the filtrate was subjected to ESI mass spectro-
metry. The filtrate is seen as a fluorescein, resulting in fluores-
cence intensity increase (Figure S4, Supporting Information).
To evaluate the practicality of Probe 1 as a real biological
sensor for intracellular biothiol detection, we performed an
Figure 4. Fluorescence microscopic images of living SHÀSY5Y
cells. (A) SHÀSY5Y cells were incubated with probe 1 (10 μM)
for 30 min and incubated with Mitotracker Red (100 nM) for
10 min. (B) SHÀSY5Y cells were preÀincubated with NEM
(1.0 mM) for 30 min, incubated with probe-1 (10 μM) for 30 min,
and then incubated with Mitotracker Red (100 nM) for 10 min.
(C) SHÀSY5Y cells were incubated with vehicle (DMSO) for
30 min. 1, bright-field images; 2, fluorescence images (490 nm);
3, fluorescence images (590 nm) [Scale bar length = 10 μm].
Excitation laser source is Mercury lamp.
As a result of these SHÀSY5Y cell assays, the probe was
found to be cell permeable and selective for the detection of
intracellular cysteine which was also supported by the
“NEM” experiments. The Mitotracker Red experiment
(15) Afanas’ev, I. B. Med. Hypotheses 2005, 64, 127.
3632
Org. Lett., Vol. 15, No. 14, 2013

at the same time serving as a superoxide input (336-fold
increases). Fluorescence intensity and “turn-on” responses
at 522 nm is consistent with liberation of free fluorescein
and can be interpreted as novel “OR” logic gating with
biothiols and superoxide as two inputs. Also, this probe is
useful in detecting intracellular biothiols. To our knowl-
edge, this is the first probe for biothiols and superoxide
detection throughester hydrolysis. Inthe future, variations
of this probe may be used for site-specific drug delivery.
Acknowledgment. D.G.C. acknowledges research sup-
port from the National Research Foundation (NRF)
(Grant # 2010À0013660 & 2011À0017280) and Mr. Hack
Soo Shin is acknowledged for his help in acquiring NMR
spectroscopic data. The research support staff at KAIST
facilitating the acquisition of MS data.
Figure 5. Magnified images of SHÀSY5Y cells incubated with
probe 1 (left) and Mitotracker Red (right). Scale bar 10 μm.
showed the permeability of probe inside the cells along
with entry into the mitochondria (Figure 5) to allow for
neurological detection of cysteine.
In conclusion, a novel fluorescein-based probe has been
developed that acts as a fluorescent chemodosimeter for
the detection of biologically important biothiols (cysteine
and homocysteine: 310- and, 290-fold, respectively), while
Supporting Information Available. Methods, experimen-
tal procedures, additional spectral data. This material is
available free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 14, 2013
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