Rational design of a highly reactive ratiometric fluorescent probe for cyanide

Article abstract of DOI:10.1021/ol2013928

A novel highly reactive ratiometric fluorescent cyanide probe was judiciously designed based on 2-formylacrylonitrile moiety as a new cyanide reaction site. A DFT study was conducted to rationalize the extremely high reactivity nature of the ratiometric f

Full text of DOI:10.1021/ol2013928

ORGANIC
LETTERS
2011
Vol. 13, No. 14
3730–3733
Rational Design of a Highly Reactive
Ratiometric Fluorescent Probe for
Cyanide
Lin Yuan, Weiying Lin,* Yueting Yang, Jizeng Song, and Jiaoliang Wang
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, Hunan University, Changsha, Hunan 410082, People’s
Republic of China
weiyinglin@hnu.cn
Received May 23, 2011
ABSTRACT
A novel highly reactive ratiometric fluorescent cyanide probe was judiciously designed based on 2-formylacrylonitrile moiety as a new cyanide
reaction site. A DFT study was conducted to rationalize the extremely high reactivity nature of the ratiometric fluorescent cyanide probe.
Cyanide is a basic component in the preparation of a
wide variety of products ranging from plastic, fibers,
gold, dyes, chelating agents for water treatment, to
pharmaceuticals.1 However, the widespread use of
cyanide presents a major health risk to human due to its
high toxicity.1a,2 Cyanide is a potent inhibitor for some
metallo-enzymesandnon-metallo-enzymes.2,3 This leads to
diseases in the vascular, cardiac, visual, endocrine, central
nervous, and metabolic systems.3 Thus, the development of
fluorescent probes for cyanide is very important.
A diverse array of fluorescent cyanide probes have been
constructed based on reversible binding and reaction-
based approaches.3ꢀ5 However, some of them have de-
layed response time and require high equivalents (100 or
higher) of cyanide to reach a maximal fluorescent signal.
Furthermore, some of them are based on fluorescence
measurement at a single wavelength, which may be
influenced by variations in the sample environment. By
contrast, ratiometricfluorescent probesallow the measure-
ment of emission intensities at two wavelengths, which
should provide a built-in correction for environmental
effects.6 Therefore, it is of high interest to develop new
(1) (a) Acheampong, M. A.; Meulepasa, R. J. W.; Lens, P. N. L.
J. Chem. Technol. Biotechnol. 2010, 85, 590. (b) Young, C.; Tidwell, L.;
Anderson, C. Cyanide: Social, Industrial and Economic Aspects: Minerals,
Metals, and Materials Society: Warrendale, PA, 2001.
(2) Kulig, K. W. Cyanide Toxicity; U.S. Department of Health and
Human Services: Atlanta, GA, 1991.
(3) Xu, Z.; Chen, X.; Kim, H. N.; Yoon, J. Chem. Soc. Rev. 2010, 39,
127.
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Lees, A. J. Tetrahedron 2010, 66, 7465. (b) Saha, S.; Ghosh, A.; Mahato,
P.; Mishra, S.; Mishra, S. K.; Suresh, E.; Das, S.; Das, A. Org. Lett. 2010,
12, 3406.
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M.; Caltagirone, C.; Bazzicalupi, C.; Bencini, A.; Garau, A.; Isaia, F.;
Laguna, A.; Lippolis, V.; Meli, V. Chem. Commun. 2011, 47, 3805. (b)
Wang, J.; Ha, C.-S. Tetrahedron 2010, 66, 1846. (c) Jamkratoke, M.;
Ruangpornvisuti, V.; Tumcharern, G.; Tuntulani, T.; Tomapatanaget,
B. J. Org. Chem. 2009, 74, 3919. (d) Cho, D.-G.; Kim, J. H.; Sessler, J. L.
J. Am. Chem. Soc. 2008, 130, 12163. (e) Tomasulo, M.; Sortino, S.;
White, A. J. P.; Raymo, F. M. J. Org. Chem. 2006, 71, 744. (f) Ekmekci,
Z.; Yilmaz, M. D.; Akkaya, E. U. Org. Lett. 2008, 10, 461. (g) Jo, J.; Lee,
D. J. Am. Chem. Soc. 2009, 131, 16283. (h) Shiraishi, Y.; Sumiya, S.;
Hirai, T. Chem. Commun. 2011, 47, 4953. (i) Lee, K.-S.; Kim, H.-J.; Kim,
G.-H.; Shin, I.; Hong, J.-I. Org. Lett. 2008, 10, 49. (j) Kim, G.-J.; Kim,
H.-J. Tetrahedron Lett. 2010, 51, 185.
(6) For some examples, see: (a) Tsien, R. Y.; Harootunian, A. T. Cell
Calcium 1990, 11, 93. (b) Demchenko, A. P. J Fluoresc. 2010, 20, 1099.
(c) Bao, Y.; Liu, B.; Wang, H.; Tian, J.; Bai, R. Chem. Commun. 2011, 47,
3957. (d) Fu, L.; Jiang, F.-L.; Fortin, D.; Harvey, P. D.; Liu, Y. Chem.
Commun., 2011, DOI: 10.1039/c1cc10784d.
r
10.1021/ol2013928
Published on Web 06/22/2011
2011 American Chemical Society

ratiometric fluorescent probes for cyanide, in particular,
with rapid response and high sensitivity.
red in Figure 1), and the other is the acrylonitrile moiety
(as shown in green in Figure 1). Thus, we anticipated that
probe 1 should be highly reactive to CNꢀ. To the best of
our knowledge, the 2-formylacrylonitrile moiety has not
been previously exploited as a cyanide reaction site for
design of fluorescent probes.
Compound 1 was readily prepared in one step by con-
densation of coumarinyl aldehyde 4 with 3,3-diethoxypro-
pionitrile 5 in the presence of FeCl3 (Scheme 1).9 The
synthesis of control compound 2 was based on a reported
procedure.7b The control compound 3 was made in two
steps (Scheme 1). Treatment of 2-bromoacetonitrile 6 with
Ph3P 7 in ethyl acetate afforded the Wittig reagent 8, which
was then reacted with coumarinyl aldehyde 4 to give
compound 3. The products were well characterized by
1H NMR, 13C NMR, MS, and HRMS.
Herein, we describe the rational design of compound 1
(Figure 1) as a new ratiometric fluorescent probe for
cyanide based on a novel CNꢀ reaction site, 2-formylacry-
lonitrile moiety. Importantly, probe 1 displays a very fast
response (<1 min) to cyanide at room temperature, and a
maximal ratiometric fluorescent signal is achieved in the
presence of only 5 equiv of cyanide.
Figure 1. Structures of the new ratiometric fluorescent probe 1
and the control compounds 2 and 3.
Scheme 1. Synthesis of Probe 1 and the Control Compound 3
In this work, 7-diethylaminocoumarin was selected as
the fluorescent signal reporting unit in light of its high
photostability, large Stokes shift, and emission in the
visible region.7 Furthermore, diethylamino moiety is a
strong electron-donating group and thus is suitable as a
donor in an intramolecular charge transfer (ICT) system.
To effectively manipulate an ICT process, we then judi-
ciously placed an acrylaldehyde moiety on the 3-position
of the coumarin dye. This may afford compound 2
(Figure1). Weenvisionedthatthereisastrong ICT process
in the free compound 2. However, the ICT will be blocked
uponadditionof CNꢀ to theβ-carbon of the acrylaldehyde
moiety due to breaking of the conjugation. The drastic
change in ICT efficiency should elicit a ratiometric optical
response. Thus, we preliminarily tested the possibility of
compound2 asa ratiometricfluorescentprobefor cyanide.
Indeed, CNꢀ can induce a marked blue shift in the ab-
sorption spectra of compound 2 as shown in Figure S2a
in the Supporting Information. However, like some
reaction-based probes for CNꢀ, compound 2 responded to
CNꢀ relatively sluggishly. The reaction was not complete
after incubation of compound 2 (10 μM) with CNꢀ (5 equiv)
for over 1 h (Figure S2a and its inset).
Clearly, to improve the sensitivity, it is necessary to
enhance the reactivity of the probe to cyanide. We rea-
soned that this could be accomplished by increasing the
electrophilicity of the β-carbon. With this in mind, we
decided to introduce a cyano moiety, a strong electron-
withdrawing group,8 on the R-carbon to afford probe 1
(Figure 1). Another key advantage of the incorporation of
the cyano moiety is to provide a 2-formylacrylonitrile
moiety, a dual R,β-unsaturated system, to be attacked by
CNꢀ. One system is the acrylaldehyde moiety (as shown in
The absorption spectrum of probe 1 exhibits a main
absorption peak at 523 nm (Figure S1a), which is notably
red-shifted relative to that of compounds 2 (451 nm) and 3
(442 nm), indicating that indeed 2-formylacrylonitrile is a
stronger electron-withdrawing group than acrylaldehyde
or acrylonitrile, as anticipated.
The time-dependent changes inthe absorption spectra of
probe 1 (10 μM) upon reaction with CNꢀ (5 equiv) is
shown in Figure 2. Upon introduction of CNꢀ, a new peak
at 363 nm appearsimmediately accompanied with a drastic
decrease in the peak at 523 nm. Three well-defined iso-
sbestic points at 417, 323, and 272 nm, respectively, are
observed.10 Notably, the main absorption peak at 523 nm
completely disappears in less than 1 min, indicating that
probe 1 is much more reactive than compounds 2 (Figure
S2a) and 3 (Figure S2b) under the same conditions, as
judiciously designed. Importantly, only 5 equiv of cyanide
was used in the assay. By contrast, some fluorescent
cyanide probes require high equivalents (100 or higher)
of cyanide to reach reaction completion under prolonged
incubation. Thus, high reactivity is the unique feature of
probe 1 when compared to compounds 2, 3, and some
known fluorescent cyanide probes.
Toshedlight onthe highly reactivenatureofprobe 1, the
electrophilic Fukui function from density functional reac-
tivity theory (DFRT)11 was performed to estimate the
(7) (a) Dong, Y.; Li, J.; Jiang, X.; Song, F.; Cheng, Y.; Zhu, C. Org.
Lett. 2011, 13, 2252. (b) Yuan, L.; Lin, W.; Yang, Y. Chem. Commun.
2011, 47, 6275. (c) Voutsadaki, S.; Tsikalas, G. K.; Klontzas, E.;
Froudakis, G. E.; Katerinopoulos, H. E. Chem. Commun. 2010, 46,
3292.
(8) (a) Tang, X.; Liu, W.; Wu, J.; Lee, C.-S.; You, J.; Wang, P. J. Org.
Chem. 2010, 75, 7273. (b) Guo, Z.; Zhu, W.; Shen, L.; Tian, H. Angew.
Chem., Int. Ed. 2007, 46, 5549.
(9) Maeda, S.; Horikawa, N.; Obora, Y.; Ishii, Y. J. Org. Chem. 2009,
74, 9558.
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Commun. 2010, 46, 6329.
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4738. (b) Liu, S. Acta Phys. Chim. Sin. 2009, 25, 590.
Org. Lett., Vol. 13, No. 14, 2011
3731

Figure 2. Time-dependent changes in the absorption spectra of
probe 1 (10 μM) upon reaction with CNꢀ (5 equiv) in CH3CN.
The arrows indicate the change of incubation time from 0 to 60 s.
Inset: Time-dependent absorption intensity of probe 1 at 523 nm
(b in red) and 363 nm (9) in the presence of CNꢀ (5 equiv).
electrophilicity of the β-carbon in compounds 1, 2, and 3
using the B3LYP exchange functional, together with
6-31þG(d) basis sets with tight SCF convergence and
ultrafine integration grids. The electronic structures of
stationary points were analyzed by the natural bond
orbital (NBO) method. All calculations were carried out
with a suite of Gaussian 09 programs.12 The calculations
provide the electrophilicity of the β-carbon in compounds
1, 2, and 3 as 2.848978, 0.347214, and 0.284229 au, respec-
tively. Thus, the β-carbon of probe 1 is much more electro-
philic than that of compounds 2 and 3, in good agreement
with the observation that probe 1 is much more reactive
than compounds 2 and 3.
We then investigated the concentration-dependent
changes in the absorption and fluorescence spectra upon
incubation of probe 1 (10 μM) with CNꢀ (0ꢀ5 equiv) for
1 min. As displayed in Figure 3a, addition of CNꢀ induced a
significant blue shift from 523 to 363 nm, and 5 equiv of
cyanide is sufficient to drive the reaction to completion
within 1 min, re-enforcing the high reactivity feature of the
probe. Interestingly, the free probe 1 shows two character-
istic fluorescence bands at 573 and 473 nm. Obviously, the
emission band at 573 nm is attributed to the ICT band.
However, the formation of the emission band at 473 nm is
likely due to the steric hindrance between the 2-formyla-
crylonitrile moiety and the carbonyl group ofthe coumarin
dye, which forces them to be slightly nonplanar and
deconjugated, and thus the emission of the diethylamino
coumarin dye at 473 nm is present. To get insight into the
intriguing dual-emission feature of probe 1, the structures
of probe 1 and the control compounds 2 and 3 were
optimized by DFT calculations.12 As shown in Figure
S3, the optimized structure of probe 1 has a dihedral angle
about 25° between the coumarin ring and formylacryloni-
trile moiety, whereas compounds 2 and 3 are essentially
planar between the coumarin ring and the aldehyde (or
cyano) moiety with a dihedral angle <0.1°. Thus, the
calculation results are consistent with the observation that
Figure 3. Concentration-dependent changes in absorption (a)
and fluorescence (b) spectra of probe 1 (10 μM) upon gradual
addition of CNꢀ (0ꢀ5 equiv) for 1 min in CH3CN with excita-
tion at 420 nm. Inset in panel a: Changes of absorption intensity
of probe 1 at 523 nm (9) and 363 nm (2 in red) in the presence of
different equiv of CNꢀ (0ꢀ6 equiv).
compounds 2 and 3 only have an ICT emission band
(Figure S1b). However, the dual fluorescence of com-
pound 1 in polar solvents due to the TICT (twisted
intramolecular charge transfer) state cannot be excluded.13
Upon gradual addition of CNꢀ to probe 1, the intensity
of the emission band at 473 nm is slightly increased with a
minor shift and that of the emission band at 573 nm is
significantly decreased (Figure 3b). There is a ca. 11.2-fold
variation in the fluorescence ratio (I480/I573) from 0.65 in
the absence of CNꢀ to7.3 in the presenceof5 equiv of CNꢀ
(Figure 4b). The detection limit (S/N = 3) was calculated
to be 328 nM, superior or comparable to the known
fluorescent cyanide probes.
To examine the selectivity, probe 1 was incubated with
various anion species including CNꢀ, Fꢀ, Clꢀ, Brꢀ, Iꢀ,
SCNꢀ, N3ꢀ, NO3ꢀ, HCO3ꢀ, CO32ꢀ, SO32ꢀ, SO42ꢀ, and
H2PO4ꢀ. Among various anion species tested, only CNꢀ
responded to 1 with a large blue shift (Figure 4a) and a
marked color change from pink to colorless (the inset in
Figure 4a). Consistent with the results of the absorption
spectra, the fluorescence ratio data (I480/I573) reveal that
probe 1 is highly selective to CNꢀ over various anion
species tested (Figure 4b). We further examined the col-
orimetric and fluorescent ratio response of the probe
toward CNꢀ in the presence of other potentially com-
peting species. The other species only displayed minimum
(12) Frisch, M. J. et al. GAUSSIAN 09, revision A.01; Gaussian, Inc.:
Pittsburgh, PA, 2009.
(13) Valeur, B. Molecular Fluorescence: Principles and Applications;
Wiley-VCH: Weinheim, Germany, 2001; pp 62ꢀ66.
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Org. Lett., Vol. 13, No. 14, 2011

DFT calculations12 for compound 1, the electrophilicity
(2.848978 au) of the β-carbon is larger than that (0.200041
au) of the carbon in carbonyl functional group, indi-
cating that β-carbon is more susceptible to attack by CNꢀ.
Thus, the results of 1H NMR, mass spectroscopic, absorp-
tion spectra, and calculation studies are in good agreement
with the proposed interaction mechanism as shown in
Figure S5.
Figure 4. (a) Absorption spectra of probe 1 (10 μM) in the
presence of 5 equiv of various anion species (CNꢀ, Fꢀ, Clꢀ, Brꢀ,
Iꢀ, SCNꢀ, N3ꢀ, NO3ꢀ, HCO3ꢀ, CO32ꢀ, SO32ꢀ, SO42ꢀ, and
H2PO4ꢀ as n-Bu4N salts in CH3CN). Inset: Color of probe 1 in
the absence or presence of various anion species; (b) emission
ratio (I480/I573) of probe 1 in the absence or presence of various
species (5 equiv) in CH3CN with excitation at 420 nm.
Figure 5. 1H NMR spectral changes for probe 1 upon addition
of CNꢀ (5 equiv). (a) Probe 1 only; (b) probe 1 and CNꢀ (5
equiv); [probe 1] = 6 mM in CD3CN at room temperature.
In summary, we have rationally designed and synthe-
sized new ratiometric fluorescent cyanide probe 1 based on
the novel CNꢀ reaction site, 2-formylacrylonitrile moiety.
Ratiometric probe 1 displays a large emission ratio re-
sponse (ca. 11.2-fold variation) to CNꢀ. Importantly,
ratiometric probe 1 is highly reactive to CNꢀ with a very
fast response (<1 min) to cyanide at room temperature,
and a maximal fluorescent signal is achieved in the pre-
sence of only 5 equiv of cyanide. In addition, the unique
feature of probe 1, extremely high reactivity, is rationalized
by DFT calculations. We expect that the 2-formylacrylo-
nitrile moiety as a novel CNꢀ reaction site will be useful for
development of other highly reactive fluorescent cyanide
probes.
interference (Figure S4). This indicates that probe 1 is
potentially useful for sensing CNꢀ in the presence of other
related species.
A likely interaction mechanism of probe 1 with CNꢀ is
shown in Figure S5. To shed light on the proposed
mechanism, 1H NMR, mass spectroscopy, absorption
spectroscopy, and DFT calculations were conducted. Ad-
dition of CNꢀ to the probe results in disappearance of the
resonance signal of the double-bond proton Hb at around
δ = 8.91 ppm (Figure 5), suggesting the formation of the
1-CN adduct. Furthermore, a major peak at m/z 321.9,
corresponding to [1-CN-adduct-H]ꢀ, is observed in the
ESI-MS spectrum (negative ion mode) (Figure S6) upon
treatment of probe 1 with CNꢀ. This implies that a 1:1
adduct between probe 1 and CNꢀ is formed. However, the
possibility of the interaction of probe 1 with CNꢀ to form a
cyanohydrin adduct can be excluded. The maximal ab-
sorption wavelength of the cyanohydrin adduct should be
comparable to compound 3 as they have the similar core
structure (Figure S7). However, in fact, the 1-CN adduct
displayed a large blue shift (78 nm) when compared with
compound 3 (Figure S8). Furthermore, according to the
Acknowledgment. This research was supported by
NSFC (20872032, 20972044), NCET (08-0175), the Doc-
toral Fund of Chinese Ministry of Education (No.
20100161110008).
Supporting Information Available. Experimental pro-
cedures, characterization data, and some spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Org. Lett., Vol. 13, No. 14, 2011
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