One-and two-photon turn-on fluorescent probe for cysteine and homocysteine with large emission shift

Article abstract of DOI:10.1021/ol802979n

A novel dendritic chromophore with efficient intramolecular charge transfer (ICT) and strong two-photon absorption (TPA) was designed as a turn-on fluorescent probe for cysteine (Cys) and homocysteine (Hcy). The probe exhibited greatly enhanced fluorescen

Full text of DOI:10.1021/ol802979n

ORGANIC
LETTERS
2009
Vol. 11, No. 6
1257-1260
One- and Two-Photon Turn-on
Fluorescent Probe for Cysteine and
Homocysteine with Large Emission Shift
Xuanjun Zhang,† Xinsheng Ren,† Qing-Hua Xu,† Kian Ping Loh,*,† and
Zhi-Kuan Chen*,‡
Department of Chemistry, National UniVersity of Singapore, 3 Science DriVe 3,
Singapore 117543, Singapore. Institute of Materials Research and Engineering,
3 Research Link, Singapore 117602, Singapore
chmlohkp@nus.edu.sg; zk-chen@imre.a-star.edu.sg
Received December 28, 2008
ABSTRACT
A novel dendritic chromophore with efficient intramolecular charge transfer (ICT) and strong two-photon absorption (TPA) was designed as
a turn-on fluorescent probe for cysteine (Cys) and homocysteine (Hcy). The probe exhibited greatly enhanced fluorescence intensity as well
as a very large emission peak shift (165 nm) upon addition of Cys/Hcy due to ICT switch off. The sensing process was also monitored by
two-photon excited fluorescence (TPEF).
The detection of important biological thiols such as cysteine
(Cys) and homocysteine (Hcy) is highly relevant to a variety
of fundamental physiological processes in organisms.1 For
example, the levels of glutathione (GSH), Cys, and Hcy in
the plasmas have been linked to human diseases such as
AIDS,2 Alzheimer’s, and Parkinson’s diseases.3 Thus, the
search for sensitive and selective sensors for Cys, Hcy, and
related peptides has stimulated intense interest.4,5 Some
fluorescent probes have been developed recently. However,
most of the fluorescent probes reported only exhibited
emission intensity enhancement or decrease but with little
peak shift. A major limitation of intensity-based probes is
(4) Recent reviews: (a) Nekrassova, O.; Lawrence, N. S.; Compton, R. G.
Talanta 2003, 60, 1085. (b) Refsum, H.; Smith, A. D.; Ueland, P. M.; Nexo,
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R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.;
Strongin, R. M. J. Am. Chem. Soc. 2004, 126, 438. (c) Ros-Lis, J. V.; Garc´ıa,
B.; Jime´nez, D.; Mart´ınez-Ma´n˜ez, R.; Sanceno´n, F.; Soto, J.; Gonzalvo,
F.; Valldecabres, M. C. J. Am. Chem. Soc. 2004, 126, 4064. (d) Tanaka,
F.; Mase, N.; Barbas, C. F., III. Chem. Commun. 2004, 1762. (e) Wang,
W.; Rusin, O.; Xu, X.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.;
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† National University of Singapore.
‡ Institute of Materials Research and Engineering.
(1) (a) Wood, Z. A.; Schro¨der, E.; Harris, J. R.; Poole, L. B. Trends
Biochem. Sci. 2003, 28, 32. (b) Homocysteine in Health and Disease;
Carmel, R., Jacobsen, D. W., Eds.; Cambridge University Press: Cambridge,
U.K., 2001.
(2) Staal, F. J. T.; Ela, S. W.; Roederer, M.; Anderson, M. T.;
Herzenberg, L. A. Lancet 1992, 339, 909.
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A. C.; Sturman, S. G. Neurosci. Lett. 1990, 110, 216.
10.1021/ol802979n CCC: $40.75
Published on Web 02/23/2009
2009 American Chemical Society

selected triphenylamine as donor and high electron affinity
peripheral group -CHO as acceptor (Scheme 1) to construct
dendritic molecule P1. The electronic and vibronic coupling
between the D-π-A dipolar branches is anticipated to
increase the two-photon absorption (TPA) efficiency.10 The
efficient intramolecular charge transfer (ICT) favors the
formation of highly polar emissive state (with red-shifted
weak emission in polar medium).11,12 The selective reaction
of the aldehyde end group with N-terminal cysteines to form
thiazolidines13 (Scheme 2), for example, may alter the ICT
characteristics in the molecule, which can manifest enhanced
fluorescence. For example, after reaction with target mol-
ecules, the ICT switch off and the highly polar emissive state
disappears leading to blue-shifted and greatly enhanced
emission in polar medium.
Scheme 1. Synthesis Route of Probe P1
Scheme 2
.
Reaction Mechanism between Aldehyde and
Cysteine
that variations in sample environment and probe distribution
may be problematic for utilization in quantitative measure-
ments. On the other hand, to image the distribution of Cys/
Hcy in cellular processes, suitable turn-on fluorescence
chemosensors for Cys/Hcy should be developed. Thus, a
probe exhibiting greatly changed emission intensity (turn-
on/off) as well as large emission peak shift is ideal for
sensing but leaves a great challenge for design and synthesis.
Compared to traditional ratiometric sensing, two-photon
excited fluorescence (TPEF) microscopy offers many ad-
vantages in the biology community. Two-photon absorption
(TPA) is a process by which two photons are simultaneously
absorbed to an excited-state in a medium via a virtual state.6
TPA has the advantage of high transmission at low incident
intensity for incident light with an optical frequency below
the band gap frequency. In recent years, materials with a
large TPA cross-section have received considerable attention
because of their interesting frequency up-conversion mech-
anism and potential applications in three-dimensional fluo-
rescence imaging, optical data storage, lithographic micro-
fabrication, and photodynamic therapy.7-9 Owing to their
ability to image at an increased penetration depth in tissue
with reduced photodamage, two-photon active fluorescent
chromophores have received increasing attention.8,9 How-
ever, creation of efficient turn-on/off TPEF probes is still in
its infancy. To address these limitations, our strategy in this
work is to design a turn-on probe with both greatly enhanced
emission intensity and large peak shift, which can be
monitored by both one- and two-photon excitations. We
Six-branched molecule P1 was synthesized by successive
Sonogashira coupling (Scheme 1). The yield of compound
2 is moderate (34%) because it is difficult to limit the reaction
to the formation of only two branches. Compounds 3, 4, and
P1 were achieved in the following steps with high yields.
Two-photon absorption cross-sections were measured by the
two-photon induced fluorescence method.14 To avoid pos-
sible complications due to the excited-state excitation, we
have used femtosecond (fs) laser pulses. TPA intensity is
characterized by the TPA cross-section (δ). The TPA
spectrum of P1 is shown in Figure 1, from which we can
see it exhibits very strong TPA with δmax of 938 GM (1 GM
) 10-50 cm4 s photon-1 molecule-1) at around 800 nm. The
log-log dependence of two-photon excited emission power
on the incident power (Figure 1, inset, at 800 nm as an
example) with slope of 1.96 indicates the occurrence of
nonlinear absorption. It is important that P1 exhibit high
emission yield in toluene (Φ ) 93%) but show marked
difference in their emission yields in polar solvents (Φ )
3.8% in dichloromethane and 1.6% in DMSO). This medium-
dependent emission is due to strong ICT. A polar emissive
state with weak emission intensity and red-shifted peak
position is favored in polar solvents.
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1258
Org. Lett., Vol. 11, No. 6, 2009

In addition, the large two-photon cross-section of P1 may
afford sensing via TPEF.
As shown in Figure 2a, after the addition of Cys, the UV-vis
absorption of P1 slightly decreases and blue shifts gradually.
The color of the solution changes from yellow to nearly
colorless. This direct visualization of color change makes the
detection straightforward. The photoluminescence of the mixture
enhanced gradually (Figure 2b) with blue-shifts. After about
1 h, the P1-Cys mixture exhibits sky-blue emission under
illumination by UV light, while the control P1 solution in
DMSO shows very weak emission. This emission enhancement
in polar solvent is due to the removal of the aldehyde group
(which is responsible for ICT and the polar emissive state) and
the formation of thiazolidines. Such a structural change switches
off the ICT and renders the emission behavior of the product
less sensitive to micropolarity.
Figure 1. Two-photon cross-section spectrum of P1. Inset: variation
of fluorescence power with incident power in a flowing condition
on a log-log scale.
The mechanism of Cys binding with P1 was also monitored
by 1H NMR (Figure 3). After reaction, the aldehyde resonance
at 10.02 ppm in DMSO-d6 solution disappeared. Two new peaks
centered at 5.51 and 5.71 ppm can be assigned to the methane
protons of the thiazolidine diastereometer.5b
If the chromophore bonds with target molecules via its
aldehyde group, the electron-withdrawing effect of the
aldehyde group will be diminished and this will result in
the quenching of the dipolar emissive state, thereby forming
the basis of a probe in polar medium. We hypothesized that
the reaction of P1 with Cys and Hcy could be readily
monitored by colorimetric as well as fluorescence responses.
1
Figure 3. HNMR of P1 and after sensing of Cys in DMSO-d6.
Control experiments in the presence of other common
amino acids (with identical concentrations) reveal that P1
exhibits good selectivity to cysteine and homocysteine, as
demonstrated by both the color changes and striking emission
enhancements (Figure 4). The sensing of Cys/Hcy can also
Figure 2
.
(a) Absorption spectra of P1 in DMSO (5 × 10-6 M)
after addition of Cys (3 × 10-4 M, measured each 10 min). Inset:
photograph of before (1) and after (2) addition of Cys. (b) Emission
spectra of P1 after addition of Cys, taken every 5 min. Inset:
emission photographs of P1 (1) and P1-Cys (2) under UV shine
(photos taken 2 h after addition of Cys).
Figure 4
.
Color changes of P1 solutions (5 × 10-5 M) and different
amino acids (in DMSO, 3 × 10-3 M) under visible light (top row)
and UV illumination (bottom row): A, no analytes; B, glycine; C,
glutathione; D, N-acetylcysteine; E, b-alanine; F, L-alanine; G,
cysteine; H, homocysteine.
Org. Lett., Vol. 11, No. 6, 2009
1259

enhancement and emission peak shifts enable P1 to be used
as an effective TPEF sensor for Cys/Hcy, especially at a
practical wavelength (800 nm) for the fs-laser.
The multipolar chromophore P1 affords a powerful mul-
tisignal detection strategy. First, visual detection by eye is
possible even at a concentration of 5 × 10-6 M because of
the large extinction coefficient (ε ) 260000 M-1 cm-1) and
the fact that efficient ICT red-shifts the absorption into the
visible region (Figure 2a, inset). Second, the very weak
emission of P1 in polar solvents is enhanced greatly after
reaction with Cys, which can be readily monitored by
fluorescence spectra and also visualized under UV light. In
addition, the large TPA cross-section of P1 affords effective
TPEF sensing.
In conclusion, we have designed a dendritic fluorescent
probe exhibiting strong two-photon absorption and efficient
sensing due to ICT presence/absence before and after reaction
with Cys/Hcy. Upon binding to Cys/Hcy, the probe exhibited
both turn-on fluorescence and large peak shift (165 nm) due
to the ICT switch off. To the best of our knowledge, this is
the first report that TPEF probe exhibits both greatly
enhanced emission intensity and large peak shift. Owing to
the potential reactivity of aldehyde toward other nucleophiles,
this work should be a general guideline for the design of
novel multisignal one- and two-photon turn-on probes, based
on ICT switch on/off, with various sensing applications.
Further modification, for example, functionalization of the
branch using short poly(ethylene glycol) (PEG), would make
it water soluble and biocompatible for in vitro application.
Figure 5. Two-photon excited fluorescence spectra (ex: 800 nm)
of P1 (5 × 10-6 M) in DMSO with various amino acids (3 × 10-4
M). Inset: enlarged view of emission spectra of other amino acids
and blank.
be monitored by two-photon excited fluorescence. P1 exhibits
very weak TPEF in DMSO (peaked at about 600 nm), due
to the very low emission quantum yield in polar solvents.
After reacting with Cys/Hcy, the TPEF was enhanced by
∼30 times and the peak wavelengths blue-shifted to 435 nm
(with 165 nm shifts), which is comparable with the one-
photon emission wavelength. This is much larger than the
recent one-photon probes15 reported by Lin, Duan, and co-
workers, which exhibited large emission peak shifts after
sensing. Figure 5 showed the TPEF spectra excited with an
800 nm fs-laser. Most of the amino acid controls did not
show TPEF enhancement except for glutathione, which
displayed a 2× enhancement of the intensity along with about
50 nm blue shift (Figure 5, inset), this is insignificant
compared to the ∼30× enhancement and 165 nm blue shifts
of the emissions for Cys and Hcy. This remarkable TPEF
Acknowledgment. We are grateful to SERC (Grant No.
052 117 00029 “Molecular and Material Engineering Ap-
proaches to Organic and Polymer Electronics Devices”) for
financial support.
Supporting Information Available: Synthesis, charac-
terization, and measurement details of P1. This material is
available free of charge via the Internet at http://pubs.acs.org.
(15) (a) Lin, W.; Long, L.; Yuan, L.; Cao, Z.; Chen, B.; Tan, W. Org.
Lett. 2008, 10, 5577. (b) Duan, L.; Xu, Y.; Qian, X.; Wang, F.; Liu, J.;
Cheng, T. Tetrahedron Lett. 2008, 49, 6624.
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Org. Lett., Vol. 11, No. 6, 2009