A highly selective off-on red-emitting phosphorescent thiol probe with large stokes shift and long luminescent lifetime

Article abstract of DOI:10.1021/ol100999j

(Figure presented) An OFF-ON red-emitting phosphorescent thiol probe is designed by using the ³MLCT photophysics of Ru(II) complexes, i.e., with Ru(II) as the electron donor. The probe is non-luminescent because the MLCT is corrupted by electron transfer from Ru(II) to an intramolecular electron sink (2,4-dinitrobenzenesulfonyl). Thiols cleave the electron sink, and the MLCT is re-established. Phosphorescence at 598 nm was enhanced by 90-fold, with a 143 nm (5256 cm^-1) Stokes shift and a 1.1 μs luminescent lifetime.

Full text of DOI:10.1021/ol100999j

ORGANIC
LETTERS
2010
Vol. 12, No. 12
2876-2879
A Highly Selective OFF-ON Red-Emitting
Phosphorescent Thiol Probe with Large
Stokes Shift and Long Luminescent
Lifetime
Shaomin Ji,† Huimin Guo,† Xiaolin Yuan,‡ Xiaohuan Li,‡ Haidong Ding,‡,§
Peng Gao,⊥ Chunxia Zhao,⊥ Wenting Wu,† Wanhua Wu,† and Jianzhang Zhao*,†
State Key Laboratory of Fine Chemicals, School of Chemical Engineering,
158 Zhongshan Road, Dalian UniVersity of Technology, Dalian 116012,
P. R. China, Center Laboratory, Affiliated Zhongshan Hospital of Dalian UniVersity,
Dalian 116001, P. R. China, Department of Immunology, Harbin Medical UniVersity,
194 Xuefu Road, Harbin 150081, P. R. China, and National Chromatographic R & A
Center, Dalian Institute of Chemical Physics, Chinese Academy of Science,
457 Zhongshan Road, Dalian 116023, P. R. China.
zhaojzh@dlut.edu.cn
Received May 1, 2010
ABSTRACT
An OFF-ON red-emitting phosphorescent thiol probe is designed by using the 3MLCT photophysics of Ru(II) complexes, i.e., with Ru(II) as the
electron donor. The probe is non-luminescent because the MLCT is corrupted by electron transfer from Ru(II) to an intramolecular electron
sink (2,4-dinitrobenzenesulfonyl). Thiols cleave the electron sink, and the MLCT is re-established. Phosphorescence at 598 nm was enhanced
by 90-fold, with a 143 nm (5256 cm-1) Stokes shift and a 1.1 µs luminescent lifetime.
Thiols, such as glutathione and cysteine, play pivotal roles
in living organisms, and thus fluorescent molecular probes
for selective detection of thiols are of great interest.1-7
Various mechanisms have been used for design of fluorescent
thiol probes, e.g., attaching electrophilic groups to fluoro-
phores (iodoacetamides or benzyl halides),8 fluorophores with
maleimide appendents, etc.4,9-11 More recently, fluorophores
containing a -CHO group were also used.5,12-16 As a new
sensing motif, fluorophores protected by 2,4-dinitrobenze-
nesulfonyl (DNBS) have been used for thiol probes, which
† Dalian University of Technology.
‡ Affiliated Zhongshan Hospital of Dalian University.
§ Harbin Medical University.
⊥ Dalian Institute of Chemical Physics.
(4) Matsumoto, T.; Urano, Y.; Shoda, T.; Kojima, H.; Nagano, T. Org.
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10.1021/ol100999j  2010 American Chemical Society
Published on Web 05/25/2010

demonstrated perfect emission OFF-ON switch effect and
good selectivity.1,2,17,18 With DFT calculations we demon-
strated that DNBS induces a dark excited state to the probe
(S1, the transition oscillator strength for S0 f S1 is close to
zero; thus S0 f S1 is a forbidden transition and S1 f S0
relaxation is nonradiative),17 and thus the probe is nonfluo-
rescent.17,19 Cleavage of the electron sink DNBS with thiols
will re-establish the radiative S1 state of the fluorophore
(oscillator strength of S0 f S1 is close to unity, indicating
the transition is allowed and S1fS0 is probably a radiative
transition), and thus the emission is switched on.17
Previously we designed OFF-ON fluorescent thiol probes
with DNBS as intramolecular electron sink.17 However, our
probes suffer from drawbacks: (1) the pyrene moiety is not
an ideal electron donor; the nonefficient electron transfer (ET)
deteriorates the quenching efficiency or the contrast ratio,23b
and as a result, only 20- to 53-fold emission enhancement
was observed;17 (2) low emission quantum yield in aqueous
solutions, which is common for most of the fluorophores;11,17
and (3) short luminescent lifetimes (ns).17
To tackle the above limitations, herein we designed a thiol
probe by using Ru(II) poly(1,10-phenanthroline) complex as
the luminophore, which is known to show environment-non-
sensitive metal-to-ligand charge transfer (3MLCT) red emission
(at ca. 600 nm), large Stokes shift (ca. 150 nm, 5250 cm-1),
and long luminescent lifetimes (µs, 10-6 s).27,28 Thus we
envisaged the new phosphorescent thiol probe 2 (Scheme 1).
However, most of the probes are based on fluorescence,1
for which the singlet excited state is the emissive state
(usually S1, Kasha’s rule).11,17 These thiol probes show some
drawbacks: (1) small Stokes shift, e.g., probes based on
BODIPY or rhodamines;4,5 (2) short excitation-emission
wavelength, which induces undesired background fluores-
cence;20 (3) microenvironment-sensitive emission, e.g., being
effected by pH or polarity of the media;16 and (4) short
luminescent lifetimes (ns, 10-9 s, due to the spin manifold
of the S0 f S1 transition),11 and so it is difficult for these
probes to be used for lifetime-based analysis.11 Therefore,
new thiol probes with drastically different lumophores are
desired to tackle these problems.
Scheme 1. Syntheses of Complex 1 and Probe 2
Much investigation has been focused on binding sites and
the luminophores of molecular probes. However, fewer
efforts have been made to explore new sensing mecha-
nisms.16,21-23 Recently we have been interested in the study
of molecular probes with new binding sites, chromophores,
and new sensing mechanisms.17,24-26
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Probe 2 is based on tuning the MLCT photophysics by
DNBS. The typical MLCT photophysics of the Ru(II)
complex, i.e., the photoinduced ET from the Ru(II) center
(23) (a) Hong, V.; Kislukhin, A. A.; Finn, M. G. J. Am. Chem. Soc.
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Org. Lett., Vol. 12, No. 12, 2010
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(a strong electron donor) to the N∧N coordination ligand,
was perturbed by DNBS, a strong intramolecular electron
acceptor. The ET destination will be diverted from the N∧N
ligand to DNBS, and thus MLCT will be corrupted and the
emission will be switched off. Cleavage of the electron sink
(DNBS) by thiols will re-establish the MLCT of the Ru(II)
complex, with the N∧N ligand as electron acceptor, and thus
efficient ET, as well as the high luminescence quantum yield
of the Ru(II) complex phosphore in aqueous solutions.11,27,28
To the best of our knowledge, probe 2 is the first
phosphorescent probe with OFF-ON switch effect for selec-
tive thiol detection. A thiol-reactive Ru(II) complex with a
maleimide group was reported,3
but with significant back
-
ground luminescence. An elegant N∧N platinum(II) bis(acetyl-
ide) complex containing -CHO groups was reported as
cysteine probe, but the emission was decreased in presence
of thiols and only minor red-shifting was observed.7b
Moreover, the background emission of a trinuclear hetero-
bimetallic Ru(II)/Pt(II) complex thiol probe is not negligible,
and the emission enhancement is only ca. 15-fold in the
presence of thiols.29 Although a thiol probe based on
squaraine dye with emission at 600 nm was reported, the
emission enhancement is only ca. 6-fold.30 In our case, probe
2 shows emission in the deep red range, and the emission
enhancement is up to 90-fold. Furthermore, 2 shows a
significant phosphorescence response only in the presence
of thiols, thus good selectivity is observed (Figure 2).
3
the MLCT phosphorescence will be switched on.
The absorption bands of the complexes below 300 nm are
dominated by the ligand-centered absorption (1π-π, S0 f
1IL, intraligand singlet excited state) (Figure 1a). The
Figure 1. (a) UV-vis absorption of the complex 1 and probe 2
Figure 2. Response of probe 2 to different analytes. Relative
before and after addition of L-cysteine. (b) Phosphorescence
emission spectra (λex ) 455 nm) of probe 2 before and after addition
of L-cysteine. In acetonitrile/water (4:1 v/v) solution. The concen-
trations of the probe and L-cysteine are 2.0 × 10-5 mol dm-3 and
1.0 × 10-2 mol dm-3, respectively. Temperature was 30 °C.
fluorescence intensity of 20 µM probe at 598 nm (λex )455 nm)
before and after incubation in the presence of 10 mM analytes at
30 °C, pH 7.4, acetonitrile/water (4:1 v/v) solution.
The excited probe 2 shows biexponential decay kinetics,
includes a fast decay process (0.5 ns) and is followed by a
slow decay process (ca. 1.1 µs) (see Supporting Information).
absorption in the visible range of the spectra at 400-500
nm are the MLCT bands (S0 f MLCT).
11,17,27,28
1
Probe 2 is non-luminescent (Figure 1b) because of the
corrupted MLCT by the efficient photoinduced ET between
the potent electron donor (Ru center) and the strong electron
acceptor (DNBS). Phosphorescence (598 nm) was switched
on by cleavage of the DNBS with thiols; the enhancement
is as high as 90-fold (Figure 1b) versus the 20- to 53-fold
enhancement of our previous thiol probes with pyrene as
electron donor.17 The sensing mechanism was verified by
mass spectrometry (see Supporting Information).1,17 We
attribute the improved emission enhancement in the presence
of thiol to the greatly reduced background emission by the
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James, T. D.; Zhao, J. J. Am. Chem. Soc. 2009, 131, 17452.
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Org. Lett., Vol. 12, No. 12, 2010

We propose the electron transfer from Ru(II) to DNBS in 2
as a two-step cascade process, i.e., the electron first transfers
from Ru(II) to the N∧N ligand and finally to DNBS.2,17,18
The fast kinetics is probably due to the N∧N ligand f DNBS
3
ET process, and the long-lived decay is due to the MLCT
excited state. In the presence of thiols, the cleaved product,
i.e., 1, shows intensive phosphoresence at 598 nm, with a
luminescent lifetime of 1.1 µs (Supporting Information).
We have been interested in studying the sensing mecha-
nism of molecular probes with DFT calculations.17,24-26 The
HOMO and LUMO orbitals of 2 were calculated (Figure
3).31 ET from Ru(II) center to DNBS was observed, which
Figure 4. Luminescence images of NCI-H446 cells. (a) Images of
cell. (d) Images of cells incubated with probe 2 (30 µM) for 8 h at
37 °C. (g) Images of cells pretreated with N-methylmaleimide (0.5
mM) for 1 h at 37 °C and then incubated with probe 2 (30 µM) for
8 h at 37 °C. (b, e, h) Bright field images corresponding to images
a, d, g. (c) Overlay of images a and b. (f) Overlay of images d and
e. (i) Overlay of images g and h.
strong intramolecular electron acceptor DNBS to modulate the
intrinsic photophysics of the Ru(II) poly(1,10-phenanthroline)
complex, i.e., the metal-to-ligand charge transfer (MLCT) with
Ru(II) as the electron donor. The MLCT of the probe is
corrupted by the ET from Ru to DNBS, and thus the
phosphorescence is quenched. Thiols cleave the DNBS, the
MLCT process is re-established, and thus the phosphorescence
is switched on, with emission enhancement up to 90-fold. The
emission of the thiol detection with probe 2 is featured with
long emission wavelength (598 nm), large Stokes shift (143
nm, 5256 cm-1), and long luminescent lifetime (1.1 µs).
Luminescent imaging of intracellular thiols were performed.
The design rational of the phosphorescent thiol probe 2 by
tuning the MLCT of Ru(II) complexes with intramolecular
electron sink will be helpful for development of OFF-ON
phosphorescent probes, which usually show longer emission
wavelength, larger Stokes shift, and much longer luminescent
lifetimes compared to the fluorescent molecular probes.
Figure 3. Frontier molecular orbitals (MOs) of probe 2 before and
after cleavage by thiols. (a) HOMO and LUMO of probe 2. (b)
HOMO and LUMO of 1 (i.e., the cleavage product of 2 by thiols).
The energy levels of the MOs are shown. Calculation is based on
the ground state geometry by DFT at the B3LYP/6-31G(d)/
LanL2DZ level using Gaussian 09.32
leads to quenching of the phosphorescence of probe 2.17,19
Conversely, ligand-to-ligand charge transfer (LLCT) and
MLCT were found for 1, i.e., the cleavage product of 2 by
thiols (Figure 3b). The electronic transition of 1 is consistent
with the typical photophysics of the Ru(II) polypyridine
complexes.11,27,28 Thus, the phosphorescence of probe 2 will
be switched ON in the presence of thiols.
Imaging of intracellular thiols of NCI-H446 cells with probe 2
was carried out (Figure 4). The cells were incubated with 2, and
red emission was observed with 488 nm laser excitation (Figure
4d). In order to prove that probe 2 is specific to the intracellular
thiols, we used N-methylmaleimide to pretreat cells to remove the
intracellular thiols,17 then the cells were incubated with probe 2,
but no red phosphorescence was observed (Figure 4g and i). These
results confirm that 2 is specific for thiols over other analytes in
living cells.
Acknowledgment. We thank the NSFC (20634040, 20972024),
MOE (SRFDP-200801410004 and NCET-08-0077), State Key
Laboratory of Fine Chemicals (KF0802), PCSIRT(IRT0711), State
Key Laboratory of Chemo/Biosensing and Chemometrics (2008009),
the Education Department of Liaoning Province (2009T015) and
DUT (SFDUT07005/1000-893394) for financial support. We are
grateful to the Royal Society (UK) and NSFC (China) for a China-
UK Cost-Share program.
In conclusion, a highly selective OFF-ON red-emitting
phosphorescent thiol probe has been designed by using the
Supporting Information Available: General experimental
1
methods, H and 13C NMR data, and spectra of the com-
plexes. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Shuai, Z. Chem. Commun. 2009, 77.
(32) Frisch, M. J. et al. Gaussian 09, ReVision A. 1; Gaussian, Inc.:
Wallingford, CT, 2009.
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