Reactive probes for ratiometric detection of Co2+ and Cu + based on excited-state intramolecular proton transfer mechanism

Article abstract of DOI:10.1021/ol302904c

An excited-state intramolecular proton transfer (ESIPT) mechanism based two reactive probes HBTCo and HBTCu is reported for the selective detection of Co²⁺ and Cu⁺ respectively in a reducing aqueous environment. Co²⁺ and Cu⁺ mediated oxidative benzylic ether (C-O) bond cleavage offers ratiometric detection of these metal ions.

Full text of DOI:10.1021/ol302904c

ORGANIC
LETTERS
2012
Vol. 14, No. 23
6008–6011
Reactive Probes for Ratiometric Detection
of Co2þ and Cuþ Based on Excited-State
Intramolecular Proton Transfer Mechanism
Debabrata Maity, Vikash Kumar, and T. Govindaraju*
Bioorganic Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for
Advanced Scientific Research (JNCASR), Jakkur P.O., Bangalore-560064, India
tgraju@jncasr.ac.in
Received October 22, 2012
ABSTRACT
An excited-state intramolecular proton transfer (ESIPT) mechanism based two reactive probes HBTCo and HBTCu is reported for the selective
detection of Co2þ and Cuþ respectively in a reducing aqueous environment. Co2þ and Cuþ mediated oxidative benzylic ether (CꢀO) bond
cleavage offers ratiometric detection of these metal ions.
A number of chemosensors have been developed for the
optical detection of various analytes based on conven-
tional signaling mechanisms such as photoinduced elec-
tron transfer (PET), intramolecular charge transfer (ICT),
metalꢀligand charge transfer (MLCT), twisted intramol-
ecular charge transfer (TICT), fluorescence resonance
energy transfer (FRET), and excimer/exciplex formation.
In recent times, a new sensing mechanism known as excited-
state intramolecular proton transfer (ESIPT) is emerging as
a suitable optical response for designing a molecular probe
to sense biologically important species and their functions in
vitro and in vivo.1 The ESIPT chromophores exhibit inter-
esting photophysical properties including a large Stokes
shift, a desired optical response from any fluorescent probe
as it minimizes artifacts such as self-absorption and an
inner-filter effect.2 Dual channel emissive ESIPT chromo-
phores are particularly useful for developing ratiometric
probes as they become strategically more advantageous
over normal probes by minimizing the error arising from
physical or chemical fluctuations in the sample and experi-
mental conditions.3 While several anion selective probes
have been developed, metal cation detection based on mod-
ulation of ESIPT has not been much explored.4
Cobalt is an important element for all multicellular
organisms present in minute amounts as part of cobalamin
and a few metalloproteins.5 Although cobalt is mildly
toxic, unregulated exposure may cause detrimental effects
(3) (a) Xu, Z.; Yoon, J.; Spring, D. R. Chem. Commun. 2010, 46, 2563.
(b) Zhang, J. F.; Zhou, Y.; Yoon, J.; Kim, Y.; Kim, S. J.; Kim, J. S. Org.
Lett. 2010, 12, 3852. (c) Maity, D.; Govindaraju, T. Chem. Commun.
2010, 46, 4499. (d) Liu, Z.; Zhang, C.; Wang, X.; He, W.; Guo, Z. Org.
Lett. 2012, 14, 4378. (e) Maity, D.; Govindaraju, T. Chem. Commun.
2012, 48, 1039. (f) Maity D.; Karthigeyan D.; Kundu T. K.; Govindaraju
T. Sens. Actuators, B 2013, 162, 831.
(4) (a) Chu, Q. H.; Medvetz, D. A.; Pang, Y. Chem. Mater. 2007, 19,
6421. (b) Singh, N.; Kaur, N.; Mulrooney, R. C.; Callan, J. F. Tetra-
hedron Lett. 2008, 49, 6690. (c) Kim, T.; Kang, H. J.; Han, G.; Chung,
S. J.; Kim, Y. Chem.Commun. 2009, 5895. (d) Jung, H. S.; Kim, H. J.;
Vicens, J.; Kim, J. S. Tetrahedron Lett. 2009, 50, 983. (e) Xu, Y. Q.; Pang,
Y. Chem. Commun. 2010, 46, 4070. (f) Hu, R.; Feng, J.; Hu, D. H.;
Wang, S. Q.; Li, S. Y.; Li, Y.; Yang, G. Q. Angew. Chem., Int. Ed. 2010,
49, 4915. (g) Xu, Z.; Xu, L.; Zhou, J.; Xu, Y.; Zhu, W.; Qian, X. Chem.
Commun. 2012, 48, 10871.
(1) (a) Henary, M. M.; Fahrni, C. J. J. Phys. Chem. A 2002, 106, 5210.
(b) Kim, T. H.; Choi, M. S.; Sohn, B. H.; Park, S. Y.; Lyoo, W. S.; Lee,
T. S. Chem. Commun. 2008, 2364. (c) Wu, J.; Liu, W.; Ge, J.; Zhang, H.;
Wang, P. Chem. Soc. Rev. 2011, 40, 3483.
(2) (a) Taki, M.; Wolford, J. L.; O’Halloran, T. V. J. Am. Chem. Soc.
2004, 126, 712. (b) Seo, J.; Kim, S.; Park, S. Y. J. Am. Chem. Soc. 2004,
126, 11154. (c) Zhao, J.; Ji, S.; Chen, Y.; Guo, H.; Yang, P. Phys. Chem.
Chem. Phys. 2012, 14, 8803.
(5) Okamoto, S.; Eltis, L. D. Metallomics 2011, 3, 963.
r
10.1021/ol302904c
Published on Web 11/29/2012
2012 American Chemical Society

including decreased cardiac output, cardiac and thyroid
enlargements, heart disease, and elevated red blood cells
accompanied by increased cells in bone marrow, increased
blood volume and vasodilation, and flushing.6 Soft transi-
tion metal copper is the third most abundant essential trace
element in the human body.7 The alterations in the cellular
homeostasis of copper are connected to serious neurodegen-
erative diseases, including Menkes and Wilson, Alzheimer’s,
and prion diseases.7 Therefore, it is necessary to develop
a novel method for the detection of these transition metals in
biological and environmental samples in bioavailable forms.
In recent years chemists are more fascinated about develop-
ing metal-responsive fluorescent probes for their selectivity,
sensitivity, and real-time monitoring of exchangeable metal
ions in living cells.8 Designing fluorescent probes for Co and
Cu is challenging due to the fluorescence quenching nature
of paramagnetic character associated with these metal ions.
We and others have developed colorimetric and fluorometric
probes for Co2þ.9 Although there are several probes re-
Our design strategy is basedon the 2-(20-hydroxyphenyl)
benzothiazole (HBT) molecular platform as this ESIPT
chromophore shows a large Stokes shift and correspond-
ing efficient ratiometric fluorescence response. We de-
signed two probes HBTCo and HBTCu with ESIPT
chromophore HBT linked to tetradentate ligands N3O9h
11d
and N4
respectively (Scheme 1). The tetradentate
ligands N3O and N4 linked chromophores were developed
as nonfluorescent probes for reaction-based turn-on sen-
sing of Co2þ and Cuþ by Chang et al.9h and Taki et al.11d
respectively. The metal ion mediated cleaving of the
benzylic ether bond in the nonfluorescent probes generates
a fluorescent dye under physiologically reducing condi-
tions. In our probes Co2þ and Cuþ assisted benzyl ether
bond (CꢀO) cleavage releases an ESIPT active HBT
Scheme 1. Synthesis of HBTCo and HBTCu
ported for Cu2þ 10
probes to monitor intracellular copper
,
(Cuþ) are rare. Cuþ is the dominant oxidation state in a
cytosolic reducing environment. Researchers have devel-
oped a few ‘turn-on’ probes for Cuþ.11 These facts emphasize
the need for the development of novel ratiometric fluoros-
cence probes for Co2þ and Cuþ based on a common
molecular platform. Furthermore such a design principle
can be easily adopted to develop a ratiometric fluorescent
probe library for other cations as well.
ꢀ
(6) (a) Barceloux, D. G. Clin. Toxicol. 1999, 37, 201. (b) Selden, A. I.;
€
Norberg, C.; Karlson-Stiber, C.; Hellstrom-Lindberg, E. Environ. Toxicol.
Pharmacol. 2007, 23, 129.
(7) (a) Kim, B. E.; Nevitt, T.; Thiele, D. J. Nat. Chem. Biol. 2008, 4,
176. (b) Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol. 2008,
4, 168. (c) Maity, D.; Govindaraju, T. Chem.;Eur. J. 2011, 17, 1410. (d)
Nagarjun, N.; Govindaraju, T. Sens. Actuators, B 2012, 161, 304.
(8) (a) Hargrove, A. E.; Nieto, S.; Zhang, T.; Sessler, J. L.; Anslyn,
E. V. Chem. Rev. 2011, 111, 6603. (b) Maity, D.; Govindaraju, T. Eur.
J. Inorg. Chem. 2011, 5479. (c) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012,
41, 52.
(9) (a) Monteil-Rivera, F.; Dumonceau J. Anal. Bioanal. Chem. 2002,
374, 1105. (b) Montalti, M.; Prodi, L.; Zaccheroni, N. J. Mater. Chem.
2005, 15, 2810. (c) Lin, W.; Yuan, L.; Long, L.; Guo, C.; Feng, J. Adv.
Funct. Mater. 2008, 18, 2366. (d) Yao, Y.; Tian, D.; Li, H. ACS Appl.
Mater. Interfaces 2010, 2, 684. (e) Zhen, S. J.; Guo, F. L.; Chen, L. Q.; Li,
Y. F.; Zhang, Q.; Huang, C. Z. Chem. Commun. 2011, 47, 2562. (f)
Grabchev, I.; Staneva, D.; Dumas, S.; Chovelon, J.-M. J. Mol. Struct.
2011, 999, 16. (g) Maity, D.; Govindaraju, T. Inorg. Chem. 2011, 50,
11282. (h) Au-Yeung, H. Y.; New, E. J.; Chang, C. J. Chem. Commun.
2012, 48, 5268.
(10) (a) Jung, H. S.; Park, M.; Han, D. Y.; Kim, E.; Lee, C.; Ham, S.;
Kim, J. S. Org. Lett. 2009, 11, 3378. (b) Ko, K. C.; Wu, J.-S.; Kim, H. J.;
Kwon, P. S.; Kim, J. W.; Bartsch, R. A.; Lee, J. Y.; Kim, J. S. Chem.
Commun. 2011, 47, 3165. (c) Maity, D.; Manna, A. K.; Karthigeyan, D.;
Kundu, T. K.; Pati, S. K.; Govindaraju, T. Chem.;Eur. J. 2011, 17,
11152. (d) Jo, J.; Lee, H. Y.; Liu, W.; Olasz, A.; Chen, C.-H.; Lee, D.
J. Am. Chem. Soc. 2012, 134, 16000.
(11) (a) Zeng, L.; Miller, E. W.; Pralle, A.; Isacoff, E. Y.; Chang, C. J.
J. Am. Chem. Soc. 2006, 128, 10. (b) Viguier, R. F. H.; Hulme, A. N.
J. Am. Chem. Soc. 2006, 128, 11370. (c) Verma, M.; Chaudhry, A. F.;
Morgan, M. T.; Fahrni, C. J. Org. Biomol. Chem. 2010, 8, 363. (d) Taki,
M.; Iyoshi, S.; Ojida, A.; Hamachi, I.; Yamamoto, Y. J. Am. Chem. Soc.
2010, 132, 5938. (e) Chaudhry, A. F.; Verma, M.; Morgan, M. T.;
Henary, M. M.; Siegel, N.; Hales, J. M.; Perry, J. W.; Fahrni, C. J. J. Am.
Chem. Soc. 2010, 132, 737. (f) Dodani, S. C.; Leary, S. C.; Cobine, P. A.;
Winge, D. R.; Chang, C. J. J. Am. Chem. Soc. 2011, 133, 8606. (g)
Morgan, M. T.; Bagchi, P.; Fahrni, C. J. J. Am. Chem. Soc. 2011, 133,
15906. (h) Chaudhry, A. F.; Mandal, S.; Hardcastle, K. I.; Fahrni, C.
J. Chem. Sci. 2011, 2, 1016. (i) Hirayama, T.; Van de Bittner, G. C.; Gray,
L. W.; Lutsenko, S.; Chang, C. J. Proc. Natl. Acad. Sci. U.S.A. 2012, 109,
2228. (j) Cao, X.; Lin, W.; Wan, W. Chem. Commun. 2012, 48, 6247.
chromophore. The released HBT chromophore undergoes
enol to keto tautomeric transformation as a result of
ESIPT, and thus a large Stokes shift in the fluorescence
emission was achieved.
In this letter, we report the synthesis and fluorometric
properties of two ESIPT probes for the detection of
Co2þ and Cuþ under physiologically reducing conditions.
HBTCo and HBTCu were synthesized by linking HBT
with tetradentate N3O and N4 respectively under basic
conditions in excellent yields (Scheme 1). The metal ion
selective N3O and N4 ligands were synthesized following
the literature procedure.9h,12 We studied the fluorescence
properties of HBTCo and HBTCu in aqueous buffer
solution (50 mM HEPES, pH 7.2) in the presence of
2 mM glutathione (GSH) for mimicking the intracellular
(12) Lucchese, B.; Humphreys, K. J.; Lee, D.; Incarvito, C. D.;
Sommer, R. D.; Rheingold, A. L.; Karlin, K. D. Inorg. Chem. 2004,
43, 5987.
Org. Lett., Vol. 14, No. 23, 2012
6009

environment (Figure 1). Upon 350 nm excitation probes
HBTCo and HBTCu emit blue fluorescence (Emax = 380 nm)
that correspond to HBT ‘enol-form’ emission. The fluoro-
metric behavior of 20.0 μM probes was investigated in the
presence of several metal ions such as Liþ, Naþ, Kþ, Ba2þ
Mg2þ, Ca2þ, Al3þ, Mn2þ, Fe2þ, Co2þ, Ni2þ, Cuþ, Cu2þ
,
,
Zn2þ, Cd2þ, Agþ, Hg2þ, and Pb2þ after 2 h of mixing. The
examined millimolar alkali and alkaline earth metals showed
no or negligible change in the fluorescence of HBTCo and
HBTCu. While the addition of Co2þ to the HBTCo solution
led to quenching of the blue emission (Emax = 380 nm) and
subsequent appearance of a new intense emission band in the
green region (Emax = 510 nm) (Figure 1a). Other metal ions
tested quenched the blue fluorescence of HBTCo to different
extents, but there was no new emission band in the green
region. The observed large red shift (130 nm) in emission
indicates the Co2þ catalyzed oxidative cleavage of benzylic
ether (CꢀO) linkage in the presence of O2 releasing ESIPT
active phenolic HBT. The decrease in blue emission and
corresponding increase inthe green emission bandwas also
observed during sequential addition of Co2þ in a titration
experiment. A minimum of 20.0 μM Co2þ can be easily
detected by this ratiometric study employing HBTCo
(Figure S1, Supporting Information). A time dependent
study showed that the addition of Co2þ quenches HBTCo
blue fluorescence (Emax = 380 nm) rapidly with the
appearance of a new peak around 460 nm (Figure 2a).
The rapid fluorescence quenching of the probe is due to
binding of paramagnetic Co2þ. Subsequent Co2þ cata-
lyzed oxidative cleavage of the benzyl ether bond releases
HBT. The weak emission band at 460 nm is due to
an enolic rotamer,2c as the Co2þ coordination with the
phenolic (ꢀOH) moiety seems to initially stabilize the enol
rotamer. Thus a weak emission band at 460 nm may be
viewed as evidence for the existence of an enolic inter-
mediate. Then, the enol form slowly converts to a keto
tautomer upon excitation to emit bright green fluorescence
at a longer wavelength (Emax = 510 nm).
Figure 1. Fluorescence responses of (a) HBTCo (20 μM) and
(b) HBTCu (20 μM) and upon addition 1 mM of Liþ, Naþ,
Kþ, Ba2þ, Mg2þ, Ca2þ, and Al3þ and 20 μM of Mn2þ, Fe2þ
,
Co2þ, Ni2þ, Cuþ, Cu2þ, Zn2þ, Cd2þ, Agþ, Hg2þ, and Pb2þ after
2 h in aqueous solution (50 mM HEPES, pH 7.2, 2 mM GSH)
(λex = 350 nm).
Similarly, quenching ofbluefluorescence(Emax =390nm)
accompanied by a new intense green emission band at
Emax = 515 nm was observed when HBTCu was treated
with copper ions (Figure 1b). This significant transforma-
tion in fluorecence behavior of HBTCu is indistinguishable
for both oxidative states of copper, as GSH in the medium
rapidly reduces Cu2þ to Cuþ. Other control metal ions did
not show blue fluorescence quenching or a new emission
band in the green region. The ESIPT based ratiometric
emission study helped to improve the detection limit of
Cuþ down to a submicromolar concentration (Figure S2).
A time dependent study showed that the blue emis-
sion band around 390 nm decreases with the increase
in intensity of the green emission band around 515 nm
upon addition of just 1.0 μM Cuþ after a period of 1 h
(Figure 2b). In contrast to HBTCo, no emission band
around 460 nm is observed with HBTCu; instead a clear
isoemissive point is found at 460 nm. As expected Cuþ
catalyzed oxidative cleavage of the benzylic ether (CꢀO)
bond in HBTCu releases the ESIPT active HBT fluoro-
phore in the presence of O2. The phenolic HBT rapidly
transformed to the keto-form upon excitation, which is
responsible for green fluorescence emission (ESIPT) at a
longer wavelength (515 nm, Stokes shift = 125 nm).
The effect of pH on the Co2þ and Cuþ mediated oxida-
tive cleavage of the benzylic ether bond was studied to
understand the efficiency of the process (Figures S3
and S4). HBTCo and HBTCu reacted efficiently with
Co2þ and Cuþ respectively in the biologically relevant pH
range of 6.5ꢀ8.5 to release the ESIPT active HBT fluoro-
phore. Hence, these probes are very convenient for the
ratiometric detection of Co2þ and Cuþ without interference
from the pH-dependent effects. The mass peak at m/z
346.16 corresponds to the carboxylated N3O-Co complex
(calcd 346.06 for C15H15CoN3O3) and that at m/z 396.26
corresponds to the carboxylated N4-Cu complex (calcd
396.06 for C19H17CuN4O2), in agreement with the literature
data (Figures S5 and S6).9h,11d The ESI-MS data confirmed
the Co2þ and Cuþ mediated oxidative benzylic ether
(CꢀO) bond cleavage reaction in HBTCo/HBTCu and
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Org. Lett., Vol. 14, No. 23, 2012

Scheme 2. Co2þ/Cuþ Mediated Release of ESIPT Fluorophore
HBT from HBTCo and HBTCu Respectively
change in the coordination site played a major role by
rendering differential metal ion selectivity to the probes, as
CꢀO bond cleavage proceeded through the formation of
the Co/Cu complex with pentadentate moieties of N3O/N4
(Scheme 2). The reducing environment created by GSH is
very crucial as confirmed by control exeriments in the
absence of GSH, which did not lead to benzylic ether bond
cleavage (Figures S7 and S8).
Figure 2. Time dependent fluorescence responses of (a) HBTCo
(20 μM) and (b) HBTCu (20 μM) after addition of 100 μM of
Co2þ and 1 μM of Cuþ respectively in aqueous solution (50 mM
HEPES, pH 7.2, 2 mM GSH) (λex = 350 nm).
In conclusion, we developed a novel ESIPT-based mole-
cular platform (HBTCo and HBTCu) for ratiometric
fluoroscence detection of Co2þ and Cuþ under physiolo-
gically reducing conditions. The metal ion catalyzed oxi-
dative benzylic ether bond cleavage was effectively used
in the switch-on ESIPT in HBT for fluoremetric detection
of paramagnetic metal ions overcoming their inherently
associated fluorescence quenching property. These ratio-
metric probes can be efficiently used for monitoring
Co2þ or Cuþ in physiological and envirornmental samples.
Generalizing this design strategy for the development of
ESIPT-based ratiometric probes for other analytes is in
progress.
formation of Co/Cu complexes with carboxylated penta-
dentate ligands N3O and N4 respectively (Scheme 2).
We propose a tentative meachanism for the metal ion
(Co2þ/Cuþ) catalyzed oxidative clavage of the benzylic
ether (CꢀO) bond between the HBT and N3O/N4
ligand based on the CꢀN bond cleavage reported by
C. J. McKenzie et al.13 The benzylic carbon of the ligand
(N3O/N4) oxidized to a carboxylate via benzylic radical
formation in the presence of activated oxygen (Figures S9
and S10). A subsequent transformation involves the for-
mation of an oxonium ion which hydrolyzed to aldehyde
liberating ESIPT active HBT. The aldehyde further oxi-
dized to a carboxylate by the hydroperoxide of the metal
complex through a BayerꢀVilliger type reaction to form
the final N3O-Co and N4-Cu complexes (Scheme 2).14
Structurally, HBTCo and HBTCu only differ in the single
cation coordination (hydroxyl/pyridyl) site. This minor
Acknowledgment. We thank Prof. C. N. R. Rao, FRS
for encouragement, JNCASR and the DST, India for a
research grant, and CSIR for awarding SRF to D.M.
Supporting Information Available. Experimental and
synthetic and meachanistic details, characterization, and
spectroscopic data. This material is available free of charge
via the Internet at http://pubs.acs.org.
(13) Vad, M. S.; Nielsen, A.; Lennartson, A.; Bond, A. D.; McGrady,
J. E.; McKenzie, C. J. Dalton Trans. 2011, 40, 10698.
(14) Bayer, A.; Villiger, V. Ber. 1899, 32, 3625.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 23, 2012
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