Highly selective excited state intramolecular proton transfer (ESIPT)-based superoxide probing

Article abstract of DOI:10.1021/ol4017222

Two novel fluorescent probe conjugates of 2-(benzothiazol-2-yl)-phenol (HBT) enable ratiometric and selective superoxide detection by an established excited state intramolecular proton transfer (ESIPT) mechanism giving a 60-fold intensity increase.

Full text of DOI:10.1021/ol4017222

ORGANIC
LETTERS
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Vol. XX, No. XX
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Highly Selective Excited State
Intramolecular Proton Transfer
(ESIPT)-Based Superoxide Probing
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), 373-1 Guseong-dong, Yuseong-gu, Daejeon,
305-701, 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 Chilam-dong, Jinju,
Gyeongnam 660-751, Republic of Korea
dchurchill@kaist.ac.kr.
Received June 18, 2013
ABSTRACT
Two novel fluorescent probe conjugates of 2-(benzothiazol-2-yl)-phenol (HBT) enable ratiometric and selective superoxide detection by an
established excited state intramolecular proton transfer (ESIPT) mechanism giving a 60-fold intensity increase.
Reactive oxygen species (ROS) play a decisive role in
neurodegenerative disorders (e.g., Alzheimer’s disease and
Parkinson’s disease) and also widely in biological systems
and in nature. Because of these significant issues related to
health and the environment, selective and sensitive detec-
tion of these species has drawn much recent research
attention.1 Along with the neurodegenerative diseases,
reactive oxygen species (ROS)2 are also a key factor in
other pathologies including cancer, diabetes, and simply
aging.3 Reactive oxygen species (ROS) include superoxide,
hypochlorite, hydrogen peroxide, hydroxyl radical, nitric
oxide, and peroxynitrite. Superoxide (O2•À) is a fascinating
analyte in that it is both a radical and anion. It has a
† Korea Advanced Institute of Science and Technology (KAIST), Repub-
lic of Korea.
short half-life and can combine easily with NO to give
3
peroxynitrite. Real-time detection of superoxide is vital in
revealing the origin of a range of physiological processes in
living organisms, such as aging, muscle fatigue, ischemia-
reperfusion, and inflammation.4,5 The detection of super-
oxide with high selectivity and sensitivity is an ongoing
challenge. Different kinds of fluorescent molecular probes
have been synthesized previously; most of them reported
to date are BODIPY-, fluorescein-, and cyanine-based
species involving cleavable groups.6
‡ Gyeongsang National University, Republic of Korea.
(1) (a) De Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
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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.; Domaille, D. W.; Chang, C. J. Chem. Rev. 2008, 108, 4328.
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r
10.1021/ol4017222
XXXX American Chemical Society

[P(O)Ph2] and 4-nitro ether involves the detachment via
hydrolytic action by superoxide through an additionÀ
elimination reaction (Figure 3) giving free 2-(benzothiazol-
2-yl)-phenol (HBT) as a final fluorescent product.
2-(Benzothiazol-2-yl)-phenol (HBT) is very well-known
as an intramolecularly hydrogen bonded molecule
exhibiting excited state intramolecular proton transfer
(ESIPT)11 in which rapid photoinduced proton transfer
results in tautomerization. The tautomerization depicted
in Figure 3 affords a large bathochromic shift. Also it
resembles THT (Thioflavin-T), a well-known dye to aid in
visualizing plaques formed by misfolding of the amyloid
protein.
The probes were obtained by commercially available
diphenylphosphinic chloride (Ph2P(O)Cl) and 1-chloro-4-
nitrobenzene with HBT, in one convenient step under
facile reaction conditions; probes 1 and 2 were obtained
in excellent purity and good yield (68% and 54%). These
probes were characterized by 1H and 13C NMR spectros-
copy and mass spectrometry (Supporting Information).
2-D NMR spectroscopic data were also obtained to allow
for more definitive atomic assignments on the solution
structure of probe 1 (Supporting Information).
Figure 1. Previously known HBT probes based on ESIPT.7
In this report, we present two novel flurescent probes for
superoxide detection based on phosphinate and ether
hydrolysis. Recent reportshaveinvolved chemodosimeters
based on superoxide-induced oxidation reactions,8aÀc
reactions with nitroxide,8d and deprotection of the 2,4-
dinitrobenzenesulfonyl8e and phosphinate groups.9,10 As
per our knowledge, this is the first example of (i) a super-
oxide sensor based on excited state intramolecular proton
transfer (ESIPT),11 and (ii) one involving the reaction of
superoxide with an ethereal group. Some of the reported
probes based on ESIPT are shown in Figure 1. Superoxide
has been coined a super nucleophile12 which permits hydro-
lysis of phophinates and the 4-nitro ether by way of
nucleophilic addition reactions. Also, fluorescein-based
phophinates have been used as probes for the detection
of superoxide by this same principle.9,10 Herein, we have
chosen a probing modality based on a simple deprotection
phenomenon: the hydroxyl groupof 2-(benzothiazol-2-yl)-
phenol (HBT)13 (Figure 2) in which the phosphinate group
Figure 2. Structures of probes 1 and 2.
(7) (a) Santra, M.; Roy, B.; Ahn, K. H. Org. Lett. 2011, 13, 3422. (b)
Hu, R.; Feng, J. A.; Hu, D. H.; Wang, S. Q.; Li, S. Y.; Li, Y.; Yang, G. Q.
Angew. Chem., Int. Ed. 2010, 49, 4915. (c) Kim, T. I.; Kang, H. J.; Han,
G.; Chung, S. J.; Kim, Y. Chem. Commun. 2009, 5895. (d) Xu, Z.; Xu, L.;
Zhou, J.; Xu, Y.; Zhu, W.; Qian, X. Chem. Commun. 2012, 48, 10871.
(e) Chen, S.; Hou, P.; Wang, J.; Song, X. RSC Adv. 2012, 2, 10869.
(8) (a) Zhao, H. T.; Kalivendi, S.; Zhang, H.; Joseph, J.; Nithipatikom,
ꢀ
K.; Vasquez Vivar, J.; Kalyanaraman, B. Free Radical Biol. Med. 2003, 34,
1359. (b) Zhao, H. T.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine,
R. L.; Vasquez Vivar, J.; Kalyanaraman, B. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 5727. (c) Kundu, K.; Knight, S. F.; Willett, N.; Lee, S.; Taylor,
W. R.; Murthy, N. Angew. Chem., Int. Ed. 2009, 48, 299. (d) Medvedeva,
N.; Martin, V. V.; Weis, A. L.; Likhtenshten, G. I. J. Photochem. Photobiol.
A. 2004, 163, 45. (e) Maeda, H.; Yamamoto, K.; Nomura, Y.; Kohno, I.;
Hafsi, L.; Ueda, N.; Yoshida, S.; Fukuda, M.; Fukuyasu, Y.; Yamauchi,
Y.; Itoh, N. J. Am. Chem. Soc. 2005, 127, 68.
(9) Xu, K. H.; Liu, X.; Tang, B. ChemBioChem 2007, 8, 453.
(10) Xu, K. H.; Liu, X.; Tang, B.; Yang, G. W.; Yang, Y.; An, L. G.
Chem.;Eur. J. 2007, 13, 1411.
(11) Sytnik, A.; Kasha, M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
8627.
Figure 3. Synthesis of probe 1 from 2-(benzothiazol-2-yl)-phenol
(HBT), its deprotonation, and a depiction of the ESIPT mechan-
ism involving plausible ROS-based byproducts.
(12) Afanas’ev, I. B. Med. Hypotheses 2005, 64, 127.
(13) (a) Lim, S. J.; Seo, J.; Park, S. Y. J. Am. Chem. Soc. 2006, 128,
14542. (b) Qian, Y.; Li, S. Y.; Zhang, G. Q.; Wang, Q.; Wang, S. Q.; Xu,
H. J.; Li, C. Z.; Li, Y.; Yang, G. Q. J. Phys. Chem. B 2007, 111, 5861.
(c) Guo, H. Y.; Li, J. C.; Shang, Y. L. Chin. Chem. Lett. 2009, 20, 1408.
Spectroscopic properties of probes 1 and 2 were ob-
tained under physiological conditions. First, the probe was
dissolved in DMSO14 and subsequently diluted in 50%
B
Org. Lett., Vol. XX, No. XX, XXXX

Figure 4. Emission spectra of (a) Probe 1 (5 Â 10À6 M, buffered
H2O/DMSO 50:50; pH 7.2; 10 mM HEPES buffer) and (b)
probe 2 (5 Â 10À6 M, H2O/DMSO 50:50) with (ROS) KO2,
H2O2 (30%inwater), NaOCl(30%inwater),tBuOOH (5.0À6.0 M
in decane), •OH (10 mg of FeSO4 in 0.1 M H2O2 solution), •OtBu
(10 mg of FeSO4 in 0.1 M tBuOOH solution), m-CPBA
(∼10 equiv) incubated for 10 min at rt. λexci = 310 nm, slit width
Ex, Em = 5.
Figure 5. Emission spectra of (a) probe 1 (5.0 Â 10À6 M,
buffered H2O/DMSO 50:50; pH 7.2; 10 mM HEPES buffer)
and (b) probe 2 (5 Â 10À6 M, H2O/DMSO 50:50) with increasing
KO2 analyte incubated for 10 min at rt. λexci = 310 nm; slit width
Ex, Em = 1.5.
intensity through phosphinate and 4-nitro ether deprotec-
tion. In this regard, a reaction of the probe with 1 equiv of
KO2 was performed. After filtration of the reaction mixture,
the filtrate was then subjected to a 31P NMR spectroscopic
study in which a singlet (δ 33.40) assigned to the phos-
phinate phosphorus completely disappears (Supporting
Information), indicating full phosphinate deprotection
(Figure S4). When tested with the increasing concentration
of superoxide there was a steady increase in fluorescence
intensity with the increase in concentration of superoxide
(Figure 5). By using these data we have calculated the
detection limit, which is found to be 3.0 Â 10À3 M and
6.38 Â 10À5 M for probe 1 and 2, respectively.
HEPES buffer (10 μM, pH.7.2). Analyte detection proper-
ties of probes were assessed by UVÀvis absorption and
emission spectroscopy (Supporting Information). The pre-
sence of the phosphinate and 4-nitro ether groups allowed
us to explore a secondary ROS screening in which depro-
tection gives back the parent 2-(benzothiazol-2-yl)-phenol
(HBT). The fluorescence quantum yield of HBT in DMSO
was found to be (Φ = 0.083 ( 0.005) (Fluorescein in 0.1 M
was used as a standard). Probes 1 and 2 were tested with
reactive oxygen species (ROS) m-CPBA, KO2, H2O2,
NaOCl, tBuOOH, •OH, and •OtBu. With the addition of
small portions of superoxide only, there was a dramatic
increase in fluorescence intensity involving an ∼58-fold
increase and a bathochromic shift of 85 nm relative to
probe 1 (Figure 4a). A 6-fold increase and bathochromic
shift of 101 nm was found relative to probe 2 (Figure 4b).
Superoxide gave a considerable change in emission
To obtain biological insight with these probe systems,
the cytotoxicity of probe 1 was examined with SH-SY5Y
cells. The neuroblastoma cells were treated with probe 1
in the range 1À36 μM in media for 1.0 h. Compared to
control values (CTL or Alc CTL), there was no significant
decrease in cell viability (%) induced by probe 1 in this
media. Cell viability was measured using an MTT solution
(MTT = 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide, 1.0 mg/mL) which was added to each vial
(14) DMSO is used as solvent because the probe was sparingly
soluble in water only. Importantly, the nucleophilic reaction rate is
solvent dependent. Polar aprotic solvents like DMSO are better for this
reaction: these solvents will not form strong hydrogen bonding networks
with the nucleophile, allowing for facile nucleophilic attack.
Org. Lett., Vol. XX, No. XX, XXXX
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one facile step; probes 1 and 2 act as ratiometric, fluorescent,
and selective molecular recognition devices for the detection
of superoxide in combined aqueous media. There is a bath-
ochromic shift of 85 nm and nearly a 60-fold increase
in fluorescence intensity. These probes are highly selective
for the detection of superoxide without interference of any
other ROS; the MTT assay for cell viability of probe 1
demonstrated probe non-neurotoxicity as tested using
SH-SY5Y cells. Also, this is a novel superoxide sensing
approach by ether hydrolysis giving a detection limit of
6.38Â 10À5 M.
Figure 6. Cell viability (%) of SH-SY5Y cells in response to
probe 1 determined by MTT assaying. SH-SY5Y cells were
exposed to probe 1 (1À36 μM) for 1 h; cell viabilities were
compared to control values (CTL = 100%). Data are expressed
as means ( STD of three independent measurements. CTL =
control; Alc CTL = alcohol control.
Acknowledgment. The Molecular Logic Gate Labora-
tory operated by D.G.C. acknowledges research support
from the National Research Foundation (NRF) (Grant
Nos. 2010-0013660 and 2011-0017280) and Mr. Hack Soo
Shin is acknowledged for his help in acquiring NMR
spectroscopic data. The research support staff at KAIST
facilitated the acquisition of MS data.
and then incubated with cells at 37 °C for 4.0 h. Formazan
crystals were first dissolved in dimethyl sulfoxide. UVÀvis
absorbances were then measured at 570 nm using a
microplate reader (Tecan, Maennedorf, Switzerland). This
assay supports the utility of the probe for use in biological
systems in detecting superoxide without short-term da-
mage to the neuronal systems (Figure 6).
Supporting Information Available. Methods, experi-
mental procedures, additional spectral data. This materi-
al is available free of charge via the Internet at http://
pubs.acs.org.
In conclusion, two novel fluorogenic conjugates of
2-(benzothiazol-2-yl)-phenol (HBT) were synthesized in
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX