A ratiometric fluorescent probe for cysteine and homocysteine displaying a large emission shift

Article abstract of DOI:10.1021/ol802436j

(Figure Presented) 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzaldehyde 1 was rationally designed as a novel ratiometric fluorescent probe for cysteine and homocysteine. Upon addition of cysteine or homocysteine, notably, the probe displayed a very large (125 nm) hypsochromic shift in emission due to switching off intramolecular charge transfer. This large emission wavelength shift may allow probe 1 to be employed for quantitatively detecting Cys/Hcy.

Full text of DOI:10.1021/ol802436j

ORGANIC
LETTERS
2008
Vol. 10, No. 24
5577-5580
A Ratiometric Fluorescent Probe for
Cysteine and Homocysteine Displaying
a Large Emission Shift
Weiying Lin,* Lingliang Long, Lin Yuan, Zengmei Cao, Bingbing Chen, and
Wen Tan
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, Hunan UniVersity, Changsha, Hunan 410082, P. R. China
weiyinglin@hnu.cn
Received October 22, 2008
ABSTRACT
4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzaldehyde 1 was rationally designed as a novel ratiometric fluorescent probe for cysteine and
homocysteine. Upon addition of cysteine or homocysteine, notably, the probe displayed a very large (125 nm) hypsochromic shift in emission
due to switching off intramolecular charge transfer. This large emission wavelength shift may allow probe 1 to be employed for quantitatively
detecting Cys/Hcy.
Cysteine (Cys) and homocysteine (Hcy) play crucial roles
in many physiological processes. For instance, an abnormal
level of cysteine is implicated in diseases such as liver
damage, skin lesions, slowed growth,1 and an elevated level
of homocysteine is a risk factor for Alzheimer’s and
cardiovascular diseases.2 Thus, the detection of Cys/Hcy has
been the subject of much attention. A number of fluorescent
probes for Cys/Hcy have been developed recently.3 However,
most of these fluorescent probes response to Cys/Hcy with
changes only in fluorescent intensity. A major limitation of
intensity-based probes is that variations in sample environ-
ment and probe distribution may be problematic for utiliza-
tion in quantitative measurements.4 By contrast, ratiometric
fluorescent probes allow the measurement of emission
intensities at two different wavelengths, which should provide
a built-in correction for environmental effects and can also
increase the dynamic range of fluorescence measurement.5
(1) Shahrokhian, S. Anal. Chem. 2001, 73, 5972.
(2) (a) Refsum, H.; Ueland, P. M.; Nyga˚rd, O.; Vollset, S. E. Annu.
ReV. Med. 1998, 49, 31. (b) Seshadri, S.; Beiser, A.; Selhub, J.; Jacques,
P. F.; Rosenberg, I. H.; D’Agostino, R. B.; Wilson, P. W. F. New Engl.
J. Med. 2002, 346, 476.
The aim of our work was to develop a novel fluorescent
probe which displayed a ratiometric response to Cys/Hcy.
Ideally, the newly developed ratiometric fluorescent probe
should exhibit a large emission wavelength shift (>80 nm)
(3) Selected examples of fluorescent Cys/Hcy probes: (a) Rusin, O.;
Luce, N. N. S.; Agbaria, 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.
(b) Wang, W.; Rusin, O.; Xu, X.; Kim, K. K.; Escobedo, J. O.; Fakayode,
S. O.; Fletcher, K. A.; Lowry, M.; Schowalter, C. M.; Lawrence, C. M.;
Fronczek, F. R.; Warner, I. M.; Strongin, R. M. J. Am. Chem. Soc. 2005,
127, 15949. (c) Tanaka, F.; Mase, N.; Barbas, C. F., Chem. Commun. 2004,
15, 1762. (d) Zhang, M.; Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.;
Liu, Z.; Zhang, J.; Zhang, D.; Yi, T.; Huang, C. J. Am. Chem. Soc. 2007,
129, 10322. (e) Sreejith, S.; Divya, K. P.; Ajayaghosh, A. Angew. Chem.,
Int. Ed. 2008, 47, 7883.
(4) (a) Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. J. Am.
Chem. Soc. 2008, 130, 4596. (b) Komatsu, K.; Urano, Y.; Kojima, H.;
Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447.
(5) (a) Tremblay, M. S.; Halim, M.; Sames, D. J. Am. Chem. Soc. 2007,
129, 7570. (b) Deo, S.; Godwin, H. A. J. Am. Chem. Soc. 2000, 122, 174.
(c) Mello, J. V.; Finney, N. S. Angew. Chem., Int. Ed. 2001, 40, 1536.
10.1021/ol802436j CCC: $40.75
Published on Web 11/18/2008
2008 American Chemical Society

for practical use in ratiometric measurement.6 Clearly, it is
a great challenge to acquire such a probe for Cys/Hcy.
When a dye possesses an electron rich group conjugated
to an electron deficient group, intramolecular charge transfer
(ICT) from the donor to the acceptor will proceed upon
excitation. ICT is an effective signaling mechanism employed
in design of ratiometric fluorescent probes. For a probe
functioning through ICT to undergo a spectral shift, the ICT
efficiency of the probe should be modulated upon interaction
with an analyte. One typical way to achieve this is to directly
engage the charge of an analyte with the electron donating
or withdrawing group of the probe in the binding process.
However, due to the structures of Cys/Hcy, it is challenging
to judiciously design such a ratiometric fluorescent chemosen-
sor in the context of Cys/Hcy coordination. To circumvent
this difficulty, we reasoned that, if reaction of Cys/Hcy to a
probe can result in deconjugation of the electron donor-
acceptor system, the ICT will be switched off. Thus a blue
shift in absorption and emission spectra should occur.
Based on this strategy, in this work, we developed 4-(1H-
phenanthro[9,10-d]imidazol-2-yl)benzaldehyde, compound 1
(Figure 1), as a new ratiometric fluorescent probe for Cys/
instead appear. It is worthy to note that, although the
formation of thiazolidines or thiazinanes has been used in
the Cys/Hcy probe development, it has not been employed
in the context of modulation of ICT to design ratiometric
fluorescent probes for Cys/Hcy.
Probe 1 was readily synthesized in one step by coupling
9,10-phenanthroquinone with terephthalaldehyde according
to a reported procedure (Scheme S1),8 and the reference
compound 3 (2-p-tolyl-1H-phenanthro[9,10-d] imidazole,
Figure 1) was also prepared by the same procedure (Scheme
S1). In this work, the photophysical properties of probe 1 in
the absence or presence of Cys/Hcy were studied in 10 mM
HEPES buffer, pH 7.4/DMF (v/v, 1: 3) at ambient temper-
ature. In comparison to the absorption spectrum of the
reference compound 3, that of probe 1 displayed a red-shifted
and broad peak around 376 nm (Figure S1). This 46 nm red
shift is apparently attributed to the ICT between the phenan-
throimidazole moiety and the aldehyde group, as designed.
The bathochromic shift due to ICT was also observed in the
emission spectrum. Probe 1 exhibited a strong ICT fluores-
cence emission peak at 519 nm, which is bathochromic shift
about 125 nm when compared to that of the reference
compound 3 (Figure S2).
The optical response of probe 1 to various amino acids,
glucose, and related thio-containing compounds was inves-
tigated by the UV-visible and emission spectroscopy. Probe
1 (2 × 10-5 M) was treated with 100 equiv of a series of
amino acids, glucose, reduced glutathione (GSH), or 2-mer-
captoethanol (MER) separately. As displayed in Figure 2a,
Figure 1. Design concept of ratiometric fluorescent probe 1 and
the structure of the reference compound 3.
Hcy. Upon introduction of Cys/Hcy, the probe displayed a
drastic (125 nm) hypsochromic shift in emission. Probe 1 is
composed of a phenanthroimidazole moiety and an aldehyde
group. The electron rich phenanthroimidazole moiety was
selected to act as both a fluorescent dye and electron donor7
in the ICT process, which is the key to the success of this
strategy. The aldehyde group was chosen for two reasons.
First, it was expected to function as an electron acceptor in
the ICT process, so it was conjugated to the phenanthroimi-
dazole donor through a phenyl spacer for the effective ICT.
Second, the aldehyde group has been known to react
specifically to Cys and Hcy to afford thiazolidine and
thiazine, respectively.3a-c Notably, in adducts 2a or 2b, the
thiazolidine or thiazine group is deconjugated from the
phenanthroimidazole moiety, so the ICT should be turned
off. In the emission spectra, the ICT emission band should
disappear, and a blue-shifted locally excited (LE) band should
Figure 2
.
Absorption (a) and emission (b) spectra of probe 1 (2 ×
10-5 M) with or without various amino acids, glucose, GSH, and
MER (100 equiv).
(6) Kimura, E.; Koike, T. Chem. Soc. ReV. 1998, 27, 179.
(7) Tsai, M. S.; Hsu, Y. C.; Lin, J. T.; Chen, H. Ch.; Hsu, C. P. J. Phys.
Chem. C 2007, 111, 18785.
5578
Org. Lett., Vol. 10, No. 24, 2008

reaction of Cys/Hcy with the probe resulted in decrease of
the absorption band around 376 nm and formation of a new
hypsochromic absorption band with maximum at 330 nm.
By contrast, no visible changes in absorption were observed
upon addition of other amino acids, glucose, or thio-
containing compounds, indicating that the aldehyde group
specifically reacted with the ꢀ- and γ-aminoalkylthio moi-
eties. In good agreement with the changes in the absorption
spectra, Cys/Hcy elicited a large blue shift in the emission
spectra (Figure 2b and Figure S3). However, no visible
variations were observed upon addition of other amino acids,
glucose, or thio-containing compounds, suggesting that the
probe is highly selective to Cys/Hcy. Figure 3 shows the
1 could be used for selective detection of Cys/Hcy even under
competition from other related species.
Addition of an increasing concentration of Cys (0-100
equiv) to the solution of probe 1 (2 × 10-5 M) resulted in a
gradual decrease of the absorption peak at 376 nm and a
progressive increase of a new absorption peak around 330
nm (Figure 4). The marked blue shift signals that, indeed,
the ICT is “off” in the adduct, in good agreement with the
formation of thiazolidine. Similar hypsochromic shift was
also found for Hcy (Figure S6). The high similarity between
the absorption spectrum of the reference compound 3 and
those of the probe titrated with Cys/Hcy corroborates that
the ICT is off in the adducts. Consistent with the hypso-
chromic shift in the absorption spectra, addition of Cys/Hcy
to the solution of probe 1 also caused blue shift in the
emission spectra (Figure 5). With an increasing amount of
Figure 3. Visual fluorescence emissions of probe 1 to representative
amino acids or thio-containing compounds on excitation at 365 nm
using UV lamp at rt. (From left to right: probe 1, probe 1 + Gly,
probe 1 + GSH, probe 1 + Hcy, probe 1 + Cys.)
visual fluorescence emissions of probe 1 to amino acids or
thio-containing compounds. The free probe exhibited a green
emission color, and the addition of representative amino acids
such as Gly or reduced glutathione did not change the color.
By contrast, introduction of Cys/Hcy elicited a significant
variation in emission color from green to blue. To further
test the effective applications of compound 1 as a ratiometric
fluorescent probe for Cys/Hcy, the fluorescence response of
probe 1 to Cys/Hcy in the presence of typical competing
species such as various amino acids, glucose, and related
thio-containing compounds was studied. As shown in Figures
S4-5, most of the competing species showed minimum
interference in the detection of Cys/Hcy, indicating that probe
Figure 5
.
Fluorescence spectral changes of probe 1 (2 × 10-5 M)
upon addition of increasing concentration (0-100 equiv) of Cys
(a) and Hcy (b) (λex ) 325 nm).
Cys/Hcy, the intensity of the ICT emission band of probe 1
at 519 nm gradually decreased with the concomitant growth
of a new LE band at 394 nm due to the adduct formation.
The significant hypsochromic shift, up to 125 nm, enables
the ICT and the LE emission peaks to resolve very well to
afford a remarkable I394/I519 change from 0.05 to 8.8 (an 176-
fold enhancement) for Cys and from 0.05 to 46.0 (a 920-
fold enhancement) for Hcy (Figure S3). The fluorescence
quantum yields of the adducts 2a and 2b were determined
to be 0.08 and 0.45, respectively (see the Supporting
Figure 4
.
Absorption spectral changes of probe 1 (2 × 10-5 M)
upon addition of Cys (0-100 equiv).
Org. Lett., Vol. 10, No. 24, 2008
5579

Information).9,10 The time course of the fluorescence spectra
of probe 1 in the presence of Cys (Figure S7) or Hcy (Figure
S8) indicates that there were significant spectral changes
within minutes of addition of Cys/Hcy. The reaction es-
sentially reached completion after 60 min, comparable to
previous reports.3b,c An assay time of 60 min was chosen in
the evaluation of the selectivity and sensitivity of probe 1
toward Cys/Hcy.
To confirm the formation of thiazolidine 2a and thiazinane
2b, probe 1 was treated with Cys/Hcy, and the reaction
1
products were isolated. The partial H NMR spectra of 1
and the isolated 2a are shown in Figure 6. The resonance
Figure 7. Plot of the fluorescent intensity ratios at 394 and 519
nm as a function of the Cys concentration.
tions of Cys in healthy plasma are typically in the range of
24 - 36 × 10-5 M).3a As shown in Table 1, ratiometric
Figure 6.
1H NMR spectra of (a) probe 1 and (b) the isolated
thiazolidine derivative 2a in DMSO-d6.
Table 1. Determination of the Cys Concentration in the HEPES
Buffer and the Newborn Calf Serum Solution
Cys spiked
(mol L-1
Cys recovered
recovery
(%)
a
)
(mol L-1
)
signal corresponding to the aldehyde proton at 10.1 ppm
disappeared; however, concomitantly, two new peaks at 5.55
and 6.02 ppm assigned to the methine proton of the
thiazolidine diastereometer emerged, consistent with a previ-
ous report.3a The structure of thiazolidine 2a was further
characterized by the mass spectrometry analysis (Figure S9,
Supporting Information). Furthermore, the isolated 2a and
the probe titrated with Cys have the identical absorption and
emission spectra (Figure S10, Supporting Information).
HEPES buffer
0
not detected
HEPES buffer 3.00 × 10-4 (2.96 ( 0.03) × 10-4
98.7
95.8
HEPES buffer 3.60 × 10-4 (3.45 ( 0.03) × 10-4
Serum
Serum
Serum
0
not detected
3.00 × 10-4 (2.96 ( 0.05) × 10-4
98.7
98.1
3.60 × 10-4 (3.53 ( 0.03) × 10-4
a Relative standard deviations were calculated on the basis of three
measurements.
1
Similarly the H, NMR, mass, and optical spectrometry
fluorescent probe 1 was able to determine the concentrations
of spiked Cys in the HEPES buffer and the new born calf
serum solution with good recovery, indicating that probe 1
can potentially be employed for quantitatively detecting Cys.
In conclusion, compound 1 was developed as a novel
ratiometric fluorescent Cys/Hcy probe. Importantly, upon
introduction of Cys/Hcy, the probe displayed a remarkable
blue shift (125 nm) in emission due to switching off ICT.
This large emission wavelength shift in the ICT and LE
emission bands may allow probe 1 to be employed for
quantitatively detecting Cys/Hcy.
analysis also support the formation of thiazinane 2b upon
treatment of the probe with Hcy.
As the probe showed a large emission wavelength shift
upon treatment of Cys/Hcy, the ICT and LE emission bands
are nearly completely resolved. This characteristic may allow
precise quantitative detection for Cys/Hcy. To investigate
this possibility, as an example, probe 1 (2 × 10-5 M) was
treated with various concentrations of Cys, and the fluores-
cence was recorded at 30 min.3c The fluorescent intensity
ratios at 394 and 519 nm were plotted as a function of the
Cys concentration, and a typical calibration graph of the
response to Cys under the optimum experimental conditions
was obtained as shown in Figure 7. This plot shows a good
linear relationship ranging from 0.6 to 80 × 10-5 M,
essentially inclusive the physiological levels of Cys (the total
concentra-
Acknowledgment. Funding was partially provided by the
Key Project of Chinese Ministry of Education (No. 108167),
the National Science Foundation of China (20872032), the
Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry (2007-24), and
the Hunan University research funds.
(8) Day, A. R.; Steck, E. A. J. Am. Chem. Soc. 1943, 65, 452
(9) (a) Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587. (b) Forgues,
S. F.; Lavabre, D. J. Chem. Educ. 1999, 76, 1260
.
.
Supporting Information Available: Synthesis of the
probe and some spectra of the probe. This material is
available free of charge via the Internet at http://pubs.acs.org.
(10) (a) Berlman, I. B. Handbook of fluorescence spectra of aromatic
molecules; Academic Press: New York, 1971. (b) Ajayaghosh, A.; Carol,
P.; Sreejith, S. J. Am. Chem. Soc. 2005, 127, 14962. (c) Lin, W.; Yuan, L.;
Feng, J.; Cao, X. Eur. J. Org. Chem. 2008, 2689. (d) Lin, W.; Long, L.;
Feng, J.; Wang, B.; Guo, C. Eur. J. Org. Chem. 2007, 4301
.
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Org. Lett., Vol. 10, No. 24, 2008