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Colorimetric and fluorometric probe for the highly selective and sensitive detection of cyanide based on coumarinyloxime
Sensors and Actuators B 188 (2013) 1043–1047
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Colorimetric and fluorometric probe for the highly selective and sensitive detection of cyanide based on coumarinyloxime Sang-Yun Na, Jong-Young Kim, Hae-Jo Kim ∗ Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
a r t i c l e
i n f o
Article history: Received 20 June 2013 Received in revised form 24 July 2013 Accepted 25 July 2013 Available online 6 August 2013
a b s t r a c t We report a coumarinyloxime-based fluorescent probe (1) for cyanide. The probe has shown a highly selective and sensitive response to the cyanide anion over other various anions with a submicromolar limit of fluorimetric detection (LOD = 0.49 M). When CN− was added to 1, probe 1 showed dramatic fluorescence and color changes, which were easily observable with the naked eye in aqueous solvent. © 2013 Elsevier B.V. All rights reserved.
Keywords: Cyanide probe Fluorescent probe Optical sensor Reaction-based chemodosimeter
1. Introduction Cyanide anions (CN− ) are extremely toxic to living organisms, and a trace amount of intake of the cyanide anions can therefore result in death [1]. Nevertheless, cyanides are industrially useful materials and are still widely employed for various uses such as synthetic resins, medicines, pesticides, fertilizers, and in gold extraction. The World Health Organization (WHO) sets the maximum permissive level of cyanides in drinking water at 1.9 M [2]. Therefore, the selective and sensitive detection of cyanide is very important for environmental protection, food analysis and even anti-terrorism [3]. Over the past ten years, a variety of reversible chemosensors has been studied based on the characteristic properties of the cyanide anion, such as its strong nucleophilicity to carbon atoms and high binding affinity to metal ions [4]. Recently, reactionbased chemodosimeters were developed by utilizing the strong nucleophilic character of cyanides to the carbocations [5]. Although many chemodosimters have been reported as probes for cyanide, challenges remain for the sensitive and optical detection of cyanide, especially in aqueous environments. Herein, we report an easily accessible oxime-based probe for the highly sensitive and optical detection of cyanides in the aqueous environment. For sensitive detection, we introduced a coumarin scaffold as the signaling
∗ Corresponding author. Tel.: +82 31 330 4703; fax: +82 31 330 4566. E-mail address: [email protected] (H.-J. Kim). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.098
fluorophore and an oxime functional group as the reaction unit of cyanide. 2. Materials and methods 2.1. General All reagents and solvent were purchased from commercial sources and used without further purification, unless otherwise stated. All reactions were carried out on a magnetic stirrer and monitored using thin-layer chromatography (TLC). Compounds were separated by flash chromatography on silica gel 60 (230–400 mesh). Absorption and fluorescence spectra were obtained using an Agilent 8453 spectrophotometer and a JASCO FP-6500 fluorescence spectrometer, respectively. NMR measurement was performed with a 200 MHz (1 H) and 50 MHz (13 C) spectrometer. The solvent for NMR measurements was dimethyl sulfoxide (DMSO-d6 ). All peaks were recorded as ı in ppm relative to the signals of residual nondeuterated solvent peak. The following abbreviations were used to explain the multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. 2.2. Preparation of 1 A mixture solution of 7-(diethylamino)-2-oxo-2H-chromene3-carbaldehyde (122.5 mg, 0.50 mmol) [6] and hydroxylamine (24.8 mg, 0.75 mmol) in ethanol (EtOH, 10 mL) and dichloromethane (DCM, 10 mL) was stirred at room temperature (rt) for 1 h. After evaporation of all solvents, the reaction mixture was extracted with ethyl acetate (EtOAc, 3× 20 mL). The organic
Et2N
S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047
O
O
NH2OH (aq) O
Et2N
O
O N
CH2Cl2/EtOH
H
0.6
1
OH
H
Scheme 1. Synthesis of 1.
layer was concentrated under reduced pressure. Chromatographic purification of the crude product on silica gel using DCM and methanol (10:1, v/v) as an eluent, gave the desired product as a yellow solid (65 mg) in 50% yield. 1 H NMR (DMSO-d , 200 MHz): ı 11.30 (s, 1H), 8.16(s, 1H), 7.99 6 (s, 1H), 7.44 (d, 3 J = 9.0 Hz, 1H), 6.74 (dd, 3 J = 9.0 Hz, 4 J = 2.4 Hz, 1H), 6.56 (d, 3 J = 2.4 Hz, 1H), 3.45 (q, 3 J = 7.0 Hz, 4H), 1.13 (t, 3 J = 7.0 Hz, 6H). 13 C NMR (DMSO, 50 MHz): ı 160.7, 156.7, 151.5, 143.3, 138.8, 130.8, 112.0, 110.0, 108.3, 96.9, 44.6, 12.8 (12 carbon peaks). HRMS (FAB− ): m/z obsd 260.1163 ([M−H]− , calcd 260.1161 for C14 H16 N2 O3 ). 2.3. Preparation of 1–CN Sodium cyanide (0.1 M NaCN in D2 O, 1 equiv. excess) was added to a solution of 1 (10 mol, 20 mM) in 0.5 mL of DMSO-d6 . After shaking several times, the reaction mixture was monitored in 1 H NMR spectroscopy and we found the reaction was complete within 4 h and then measured its mass spectra. 1 H NMR (D O/DMSO-d , 25:1, v/v, 200 MHz): ı 7.71 (s, 1H), 6.93 2 6 3 (d, J = 8.2 Hz, 1H), 6.11–6.06 (m, 2H), 4.73 (s, 1H), 3.23 (q, 3 J = 7.0 Hz, 4H), 1.04 (t, 3 J = 7.0 Hz, 6H). LRMS (FAB− , glycerol): m/z obsd 286 ([M−H]− , cald 286.1197 for C15 H16 N3 O3 ). 2.4. General UV–vis and fluorescence measurements A stock solution (10 mM) of 1 in DMSO was prepared and used by diluting with a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). For UV–vis measurement, a sample solution was prepared by mixing an appropriate amount of stock solution of 1 (10 mM in DMSO) with an appropriate amount of anion and finally diluted with a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) to afford the desired concentration of 1 and the anion. Fluorescence measurement was carried out similarly with a slit width of 3 nm × 3 nm.
0.4
Abs
1044
02 0.2
0 300
400
500
600
λ/nm Fig. 1. Time-dependent UV–vis spectra of 1 (20 M) upon addition of cyanide ions (60 mM) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). Inset: its kinetics.
the addition of CN− (60 mM) to 1 (20 M), the UV–vis spectra of 1 showed a typical bathochromic shift with a dramatic ratiometric change (Fig. 1). The maximum absorbance at 426 nm was rapidly decreasing while a new maximum band at 492 nm was increasing with a clear isosbestic point at 463 nm. The clear isosbestic point in this ratiometric UV–vis response indicates that the transformation of 1 into the 1–CN conjugate was achieved without the formation of other byproducts. The reaction of 1 with CN− was complete within 4 h under the experimental conditions. The initial rate analysis of 1 in the presence of cyanide anions gave a mild rate constant with the second order rate constant of k2 = 4.5 × 10−3 M−1 s−1 at 25 ◦ C (Fig. S2). To further evaluate the selectivity of 1 for cyanide against other anions, we measured the fluorescence intensity of 1 (20 M, ex 463 nm) in the presence of various anions (F− , Cl− , Br− , I− , HSO4 − , AcO− , N3 − , ClO4 − , NO3 − ) including CN− (60 mM) in a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) (Fig. 2A). The maximum fluorescence intensity of 1 at 493 nm (F493 ) was dramatically decreased by CN− (60 mM), whereas other anions did not induce any detectable fluorescence changes except for F− , where the basicity of F− anion
2.5. Reaction stoichiometry The reaction stoichiometry of 1 with CN− was determined by using Job’s plot [7]. For the Job’s plot, a series of solution with a varying mole fraction of cyanide anions were prepared by maintaining the total concentration of 1 and cyanide to be constant (20 M). The fluorescence emission was measured for each sample by exciting at 463 nm and spectra were measured from 400 to 700 nm. The fluorescence intensity at 493 nm was monitored for each solution and the fluorescence intensities of the mixture of 1 and CN− were plotted against X1 (mole fraction of CN− ). 3. Results and discussion We designed a coumarinyloxime-based fluorescent probe (1) for cyanide anions, where coumarin was employed as a signaling unit and oxime as a reaction unit. The reaction of 7-(diethylamino)2-oxo-2H-chromene-3-carbaldehyde with hydroxylamine at rt in the DCM/EtOH produced the desired probe (1) very efficiently (Scheme 1). Time-dependent UV–vis spectral changes of 1 with cyanide were monitored in a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). Upon
Fig. 2. Fluorescence spectra of 1 (20 M, ex /em = 463/493 nm) with various anions (60 mM) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4).
S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047
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Fig. 3. Naked-eye visible (up) and fluorescence (down, ex 365 nm) images of 1 (20 M) upon the addition of various anions (60 mM) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4).
gave a slight interference to the nucleophilic addition reaction of cyanide. The addition of CN− to the mixtures of 1 and the other anions induced responses as significant as that of the 1–CN solution (Fig. 2B). This competitive assay has also shown a consistent result for the selectivity of 1 toward cyanide. The prominent UV–vis and fluorescence changes of 1–CN conjugate were also observable with the naked eye. Upon the addition of CN− to the solution of 1 (20 M) in the DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4), the initial light green color of 1 disappeared to afford a colorless solution. The resulting complex (1–CN) exhibited a dramatic fluorescence change from strong green fluorescence to no fluorescence, while other anions did not elicit any photophysical changes under a portable UV spectroscope (Fig. 3). The reaction stoichiometry between 1 and CN− was determined in a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) using Job’s plot. The maximum fluorescence intensities (F493 ) were measured for each molar solution of 1 and CN− and plotted against the mole fraction of CN− . The Job’s plot revealed a 1:1 reaction ratio between 1 and CN− (Fig. S3). The mass spectral analysis also showed corroborative
Et2 N
O
O
CN N
Ha
Et2N
evidence for the one-to-one formation of 1–CN conjugate at m/z obsd 286 ([M−H]− , cald 286.1197 for C15 H16 N3 O3 − ) (Fig. S7). In order to gain insight into the reaction mechanism, we investigated the 1 H NMR spectral changes of 1 caused by the incremental addition of − to 1 and compared the spectra of 1–CN with the spectrum of 1 itself. Upon the addition of 0.5 equiv. CN− to 1, a new set of 1 H NMR peaks started to appear and the reaction was almost complete with 1.0 equiv. CN− . No further reaction was observed even in the presence of 3.0 equiv. CN− (Fig. 4). As cyanide anions were added to probe 1, the imine proton (Ha ) of 1 was dramatically (ı = −3.3 ppm) shifted to a highly upfield region (8.0–4.7 ppm) with a concomitant disappearance of the OH proton. The aromatic protons of 1, however, were slightly shifted (ı < −1.0 ppm) toward the upfield aromatic regions. These 1 H NMR spectral changes, together with the bathochromic shift of 1–CN conjugate in its UV–vis spectra, indicate that cyanide was added to an electrophilic site of the oxime carbon rather than that of the aromatic carbon of 1. The nucleophilic addition of cyanide to the oxime carbon site of 1 would generate an anionic intermediate, which subsequently underwent a proton transfer from acidic oxime
O
O
H+ N
OH
OH
H CN
Et2N
transf er
O
O H N H b CN
O
Hb
D C B
A
Ha
OH
10.0
Fig. 4.
1
ppm (f1)
5.0
H NMR spectra of 1 (20 mM) in the absence (A) and presence of 0.5 equiv. (B) and 1.0 equiv. (C), and 3.0 equiv. (D) of cyanides in DMSO-d6 /D2 O (25:1, v/v).
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S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047
100
77
y = -0.1364x + 76.409 R² = 0.9981
F493
F 493
80
[2]
75
[3]
60 73
40
0
4
8
12
16
[CN]/μM 20 0
[4] 0
1000
2000
3000
equiv. of CN
_
4000
5000
6000
Fig. 5. Linear plot of 1 (20 M, ex /em 463/493 nm) upon the addition of different amounts of CN− (0, 2, 6, 10 or 16 M) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). The limit of detection of CN− was determined as 0.49 M.
OH to the amine anion to afford the 1–CN conjugate. The resulting hydroxylamine (1–CN) would play the role of a fluorescence quencher through the photoinduced-electron transfer mechanism from nitrogen to coumarin and then might induce the dramatic On–Off transition of the fluorescence of 1 upon the addition of CN− . The detection limit of CN− was measured in the DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) using a fluorescence titration experiment. The standard deviation of the emission intensities of 1 without CN− was obtained as = 0.022 (n = 3). The fluorescence intensities at 493 nm were measured by the incremental addition of CN− to 1 (20 M), the slope of which gave m = −0.0391. From the fluorescence experiment of 1, we found that the limit of detection (LOD) of CN− was 0.49 M at 3/m [8], which was almost 4 times more sensitive than the level of cyanide in drinking water permitted by WHO (Fig. 5) [2]. The probe (1) showed a stable pH profile at pH 3–11, where 1 displayed a strong green fluorescence but 1–CN did not exhibit any fluorescence (Fig. S4). We expect that the characteristic onoff fluorescence property of 1 in the broad range of pHs could be utilized for the selective and sensitive detection of cyanides in the aqueous enviroment.
[5]
4. Conclusion In summary, we report a coumarinyloxime-based fluorescent probe (1) for cyanide. The fluorescent probe has shown a highly selective and sensitive response to the cyanide anion over other various anions with a submicromolar limit of fluorimetric detection (LOD = 0.49 M). When CN− was added to 1, the probe (1) showed a dramatic On–Off fluorescence change as well as a distinct color change, which was observable with the naked eye in aqueous solvent. Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF 2011-0028456). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.07.098.
[6]
[7] [8]
(b) Agency for Toxic Substances and Disease Registry, Toxicological Profile for Cyanide, Department of Health and Human Services, Atlanta, 2006. Guidelines for Drinking-water Quality, World Health Organization, Geneva, 1996. (a) C.A. Young, L.G. Tidwell, C.G. Anderson, Cyanide: Social Industrial and Economic Aspects, The Minerals, Metals and Materials Society Press, Warrendale, 2001; (b) R.A. Greenfield, B.R. Brown, J.B. Hutchins, J.J. Iandolo, R. Jackson, L.N. Slater, M.S. Brone, Microbiological, biological, and chemical weapons of warfare and terrorism, American Journal of the Medical Sciences 323 (2002) 326. (a) R. Badugu, J.R. Lakowicz, C.D. Geddes, Enhanced fluorescence cyanide detection at physiologically lethal levels: reduced ICT-based signal transduction, Journal of the American Chemical Society 127 (2005) 3635; (b) S.-Y. Chung, S.-W. Nam, J. Lim, S. Park, J. Yoon, A highly selective cyanide sensing in water via fluorescence change and its application to in vivo imaging, Chemical Communications 20 (2009) 2866; (c) J.H. Lee, A.R. Jeong, I.-S. Shin, H.-J. Kim, J.-I. Hong, Fluorescence turn-on sensor for cyanide based on a cobalt(II)–coumarinylsalen complex, Organic Letters 12 (2010) 764; (d) Z.C. Xu, X.Q. Chen, H.N. Kim, J.Y. Yoon, Sensors for the optical detection of cyanide ion, Chemical Society Reviews 39 (2010) 127; (e) Y.M. Kim, H.S. Huh, M.H. Lee, I.L. Lenov, H.Y. Zhao, F.P. Gabbai, Turn-on fluorescence sensing of cyanide ions in aqueous solution at parts-per-billion concentrations, Chemistry: A European Journal 17 (2011) 2057. (a) Y.M. Chung, B. Raman, D.S. Kim, K.H. Ahn, Fluorescence modulation in anion sensing by introducing intramolecular H-bonding interactions in host–guest adducts, Chemical Communications 2 (2006) 186; (b) Y.K. Yang, J.S. Tae, Acridinium salt based fluorescent and colorimetric chemosensor for the detection of cyanide in water, Organic Letters 8 (2006) 5721; (c) K.-S. Lee, H.-J. Kim, G.H. Kim, I. Shin, J.-I. Hong, Fluorescent chemodosimeter for selective detection of cyanide in water, Organic Letters 10 (2008) 49; (d) Y. Sun, G.F. Wang, W. Guo, Colorimetric detection of cyanide with nitrophenyl benzamide derivatives, Tetrahedron 65 (2009) 3480; (e) J. Jo, D. Lee, Turn-on fluorescence detection of cyanide in water: activation of latent fluorophores through remote hydrogen bonds that mimic peptide -turn motif, Journal of the American Chemical Society 131 (2009) 16283; (f) S.J. Hong, J. Yoo, S.H. Kim, J.S. Kim, J. Yoon, C.H. Lee, ␣-Vinyl substituted calix[4] pyrrole as a selective ratiometric sensor for cyanide anion, Chemical Communications 2 (2009) 189; (g) G.-J. Kim, H.-J. Kim, Doubly activated coumarin as a colorimetric and fluorescent chemodosimeter for cyanide, Tetrahedron Letters 51 (2010) 185; (h) G.-J. Kim, H.-J. Kim, Coumarinyl aldehyde as a Michael acceptor type of colorimetric and fluorescent probe for cyanide in water, Tetrahedron Letters 51 (2010) 2914; (i) S. Park, H.-J. Kim, Highly activated Michael acceptor by an intramolecular hydrogen bond as a fluorescence turn-on probe for cyanide, Chemical Communications 46 (2010) 9197; (j) Y. Shiraishi, S. Sumiya, T. Hirai, Highly sensitive cyanide anion detection with a coumarin–spiropyran conjugate as a fluorescent receptor, Chemical Communications 47 (2011) 4953; (k) H.J. Kim, K.C. Ko, J.H. Lee, J.Y. Lee, J.S. Kim, KCN sensor: unique chromogenic and ‘turn-on’ fluorescent chemodosimeter: rapid response and high selectivity, Chemical Communications 47 (2011) 2886; (l) S. Park, H.-J. Kim, Highly selective chemodosimeter for cyanide based on a doubly activated Michael acceptor type of coumarin thiazole fluorophore, Sensors & Actuators B 161 (2012) 317; (m) S. Park, H.-J. Kim, Reaction-based chemosensor for the reversible detection of cyanide and cadmium ions, Sensors & Actuators B 168 (2012) 376; (n) H. Lee, H.-J. Kim, Highly selective sensing of cyanide by a benzochromenebased ratiometric fluorescence probe, Tetrahedron Letters 53 (2012) 5455; (o) S. Park, K.-H. Hong, J.-I. Hong, Azo dye-based latent colorimetric chemodosimeter for the selective detection of cyanides in aqueous buffer, Sensors & Actuators B 174 (2012) 140; (p) K.-H. Hong, H.-J. Kim, Azo dye-based colorimetric cheomodosimeter for cyanide in aqueous solution, Supramolecular Chemistry 25 (2013) 24. (a) D.-N. Lee, D.Y. Kim, S.H. Ghil, H.-J. Kim, Coumarin–benzothiazoline conjugate as a fluorescence turn-on probe for reactive oxygen species and its cellular expression, Bulletin of the Korean Chemical Society 32 (2011) 3109; (b) G.-J. Kim, K. Lee, H. Kwon, H.-J. Kim, Ratiometric fluorescence imaging of cellular glutathione, Organic Letters 13 (2011) 2799. P. Job, Formation and stability of inorganic complexes in solution, Annales de Chimie 9 (1928) 113. D. MacDougall, W.B. Crummett, Guidelines for data acquisition and data quality evaluation in environmental chemistry, Analytical Chemistry 52 (1980) 2242.
Biographies References [1] (a) K.W. Kulig, Cyanide Toxicity, U.S. Department of Health and Human Services, Atlanta, 1991;
Sang-Yun Na is a master course student at the Hankuk University of Foreign Studies, Korea. His research interest is to develop highly selective fluorescence sensors for biological thiols and toxic chemicals.
S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047 Jong-Young Kim is an undergraduate course student at the Hankuk University of Foreign Studies, Korea. He joined Dr. H.-J. Kim’s lab for his junior research program at HUFS. His research interest is to develop highly selective fluorescence sensors for toxic chemicals. Hae-Jo Kim received his B.S. degree in chemistry education, and M.S. and Ph.D. degrees in chemistry from Seoul National University, Korea with Professor Jong-In
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Hong (2002). After completing his postdoctoral research at University of Toronto, Canada with Professor Jik Chin (2005), he joined Kyonggi University, Korea as a faculty member (2006). He is currently associate professor and chairperson of the Department of Chemistry at Hankuk University of Foreign Studies, Korea. His research focuses on fluorescent molecular probes for tumor imaging and therapy.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Colorimetric and fluorometric probe for the highly selective and sensitive detection of cyanide based on coumarinyloxime Sang-Yun Na, Jong-Young Kim, Hae-Jo Kim ∗ Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
a r t i c l e
i n f o
Article history: Received 20 June 2013 Received in revised form 24 July 2013 Accepted 25 July 2013 Available online 6 August 2013
a b s t r a c t We report a coumarinyloxime-based fluorescent probe (1) for cyanide. The probe has shown a highly selective and sensitive response to the cyanide anion over other various anions with a submicromolar limit of fluorimetric detection (LOD = 0.49 M). When CN− was added to 1, probe 1 showed dramatic fluorescence and color changes, which were easily observable with the naked eye in aqueous solvent. © 2013 Elsevier B.V. All rights reserved.
Keywords: Cyanide probe Fluorescent probe Optical sensor Reaction-based chemodosimeter
1. Introduction Cyanide anions (CN− ) are extremely toxic to living organisms, and a trace amount of intake of the cyanide anions can therefore result in death [1]. Nevertheless, cyanides are industrially useful materials and are still widely employed for various uses such as synthetic resins, medicines, pesticides, fertilizers, and in gold extraction. The World Health Organization (WHO) sets the maximum permissive level of cyanides in drinking water at 1.9 M [2]. Therefore, the selective and sensitive detection of cyanide is very important for environmental protection, food analysis and even anti-terrorism [3]. Over the past ten years, a variety of reversible chemosensors has been studied based on the characteristic properties of the cyanide anion, such as its strong nucleophilicity to carbon atoms and high binding affinity to metal ions [4]. Recently, reactionbased chemodosimeters were developed by utilizing the strong nucleophilic character of cyanides to the carbocations [5]. Although many chemodosimters have been reported as probes for cyanide, challenges remain for the sensitive and optical detection of cyanide, especially in aqueous environments. Herein, we report an easily accessible oxime-based probe for the highly sensitive and optical detection of cyanides in the aqueous environment. For sensitive detection, we introduced a coumarin scaffold as the signaling
∗ Corresponding author. Tel.: +82 31 330 4703; fax: +82 31 330 4566. E-mail address: [email protected] (H.-J. Kim). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.098
fluorophore and an oxime functional group as the reaction unit of cyanide. 2. Materials and methods 2.1. General All reagents and solvent were purchased from commercial sources and used without further purification, unless otherwise stated. All reactions were carried out on a magnetic stirrer and monitored using thin-layer chromatography (TLC). Compounds were separated by flash chromatography on silica gel 60 (230–400 mesh). Absorption and fluorescence spectra were obtained using an Agilent 8453 spectrophotometer and a JASCO FP-6500 fluorescence spectrometer, respectively. NMR measurement was performed with a 200 MHz (1 H) and 50 MHz (13 C) spectrometer. The solvent for NMR measurements was dimethyl sulfoxide (DMSO-d6 ). All peaks were recorded as ı in ppm relative to the signals of residual nondeuterated solvent peak. The following abbreviations were used to explain the multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. 2.2. Preparation of 1 A mixture solution of 7-(diethylamino)-2-oxo-2H-chromene3-carbaldehyde (122.5 mg, 0.50 mmol) [6] and hydroxylamine (24.8 mg, 0.75 mmol) in ethanol (EtOH, 10 mL) and dichloromethane (DCM, 10 mL) was stirred at room temperature (rt) for 1 h. After evaporation of all solvents, the reaction mixture was extracted with ethyl acetate (EtOAc, 3× 20 mL). The organic
Et2N
S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047
O
O
NH2OH (aq) O
Et2N
O
O N
CH2Cl2/EtOH
H
0.6
1
OH
H
Scheme 1. Synthesis of 1.
layer was concentrated under reduced pressure. Chromatographic purification of the crude product on silica gel using DCM and methanol (10:1, v/v) as an eluent, gave the desired product as a yellow solid (65 mg) in 50% yield. 1 H NMR (DMSO-d , 200 MHz): ı 11.30 (s, 1H), 8.16(s, 1H), 7.99 6 (s, 1H), 7.44 (d, 3 J = 9.0 Hz, 1H), 6.74 (dd, 3 J = 9.0 Hz, 4 J = 2.4 Hz, 1H), 6.56 (d, 3 J = 2.4 Hz, 1H), 3.45 (q, 3 J = 7.0 Hz, 4H), 1.13 (t, 3 J = 7.0 Hz, 6H). 13 C NMR (DMSO, 50 MHz): ı 160.7, 156.7, 151.5, 143.3, 138.8, 130.8, 112.0, 110.0, 108.3, 96.9, 44.6, 12.8 (12 carbon peaks). HRMS (FAB− ): m/z obsd 260.1163 ([M−H]− , calcd 260.1161 for C14 H16 N2 O3 ). 2.3. Preparation of 1–CN Sodium cyanide (0.1 M NaCN in D2 O, 1 equiv. excess) was added to a solution of 1 (10 mol, 20 mM) in 0.5 mL of DMSO-d6 . After shaking several times, the reaction mixture was monitored in 1 H NMR spectroscopy and we found the reaction was complete within 4 h and then measured its mass spectra. 1 H NMR (D O/DMSO-d , 25:1, v/v, 200 MHz): ı 7.71 (s, 1H), 6.93 2 6 3 (d, J = 8.2 Hz, 1H), 6.11–6.06 (m, 2H), 4.73 (s, 1H), 3.23 (q, 3 J = 7.0 Hz, 4H), 1.04 (t, 3 J = 7.0 Hz, 6H). LRMS (FAB− , glycerol): m/z obsd 286 ([M−H]− , cald 286.1197 for C15 H16 N3 O3 ). 2.4. General UV–vis and fluorescence measurements A stock solution (10 mM) of 1 in DMSO was prepared and used by diluting with a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). For UV–vis measurement, a sample solution was prepared by mixing an appropriate amount of stock solution of 1 (10 mM in DMSO) with an appropriate amount of anion and finally diluted with a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) to afford the desired concentration of 1 and the anion. Fluorescence measurement was carried out similarly with a slit width of 3 nm × 3 nm.
0.4
Abs
1044
02 0.2
0 300
400
500
600
λ/nm Fig. 1. Time-dependent UV–vis spectra of 1 (20 M) upon addition of cyanide ions (60 mM) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). Inset: its kinetics.
the addition of CN− (60 mM) to 1 (20 M), the UV–vis spectra of 1 showed a typical bathochromic shift with a dramatic ratiometric change (Fig. 1). The maximum absorbance at 426 nm was rapidly decreasing while a new maximum band at 492 nm was increasing with a clear isosbestic point at 463 nm. The clear isosbestic point in this ratiometric UV–vis response indicates that the transformation of 1 into the 1–CN conjugate was achieved without the formation of other byproducts. The reaction of 1 with CN− was complete within 4 h under the experimental conditions. The initial rate analysis of 1 in the presence of cyanide anions gave a mild rate constant with the second order rate constant of k2 = 4.5 × 10−3 M−1 s−1 at 25 ◦ C (Fig. S2). To further evaluate the selectivity of 1 for cyanide against other anions, we measured the fluorescence intensity of 1 (20 M, ex 463 nm) in the presence of various anions (F− , Cl− , Br− , I− , HSO4 − , AcO− , N3 − , ClO4 − , NO3 − ) including CN− (60 mM) in a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) (Fig. 2A). The maximum fluorescence intensity of 1 at 493 nm (F493 ) was dramatically decreased by CN− (60 mM), whereas other anions did not induce any detectable fluorescence changes except for F− , where the basicity of F− anion
2.5. Reaction stoichiometry The reaction stoichiometry of 1 with CN− was determined by using Job’s plot [7]. For the Job’s plot, a series of solution with a varying mole fraction of cyanide anions were prepared by maintaining the total concentration of 1 and cyanide to be constant (20 M). The fluorescence emission was measured for each sample by exciting at 463 nm and spectra were measured from 400 to 700 nm. The fluorescence intensity at 493 nm was monitored for each solution and the fluorescence intensities of the mixture of 1 and CN− were plotted against X1 (mole fraction of CN− ). 3. Results and discussion We designed a coumarinyloxime-based fluorescent probe (1) for cyanide anions, where coumarin was employed as a signaling unit and oxime as a reaction unit. The reaction of 7-(diethylamino)2-oxo-2H-chromene-3-carbaldehyde with hydroxylamine at rt in the DCM/EtOH produced the desired probe (1) very efficiently (Scheme 1). Time-dependent UV–vis spectral changes of 1 with cyanide were monitored in a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). Upon
Fig. 2. Fluorescence spectra of 1 (20 M, ex /em = 463/493 nm) with various anions (60 mM) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4).
S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047
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Fig. 3. Naked-eye visible (up) and fluorescence (down, ex 365 nm) images of 1 (20 M) upon the addition of various anions (60 mM) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4).
gave a slight interference to the nucleophilic addition reaction of cyanide. The addition of CN− to the mixtures of 1 and the other anions induced responses as significant as that of the 1–CN solution (Fig. 2B). This competitive assay has also shown a consistent result for the selectivity of 1 toward cyanide. The prominent UV–vis and fluorescence changes of 1–CN conjugate were also observable with the naked eye. Upon the addition of CN− to the solution of 1 (20 M) in the DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4), the initial light green color of 1 disappeared to afford a colorless solution. The resulting complex (1–CN) exhibited a dramatic fluorescence change from strong green fluorescence to no fluorescence, while other anions did not elicit any photophysical changes under a portable UV spectroscope (Fig. 3). The reaction stoichiometry between 1 and CN− was determined in a DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) using Job’s plot. The maximum fluorescence intensities (F493 ) were measured for each molar solution of 1 and CN− and plotted against the mole fraction of CN− . The Job’s plot revealed a 1:1 reaction ratio between 1 and CN− (Fig. S3). The mass spectral analysis also showed corroborative
Et2 N
O
O
CN N
Ha
Et2N
evidence for the one-to-one formation of 1–CN conjugate at m/z obsd 286 ([M−H]− , cald 286.1197 for C15 H16 N3 O3 − ) (Fig. S7). In order to gain insight into the reaction mechanism, we investigated the 1 H NMR spectral changes of 1 caused by the incremental addition of − to 1 and compared the spectra of 1–CN with the spectrum of 1 itself. Upon the addition of 0.5 equiv. CN− to 1, a new set of 1 H NMR peaks started to appear and the reaction was almost complete with 1.0 equiv. CN− . No further reaction was observed even in the presence of 3.0 equiv. CN− (Fig. 4). As cyanide anions were added to probe 1, the imine proton (Ha ) of 1 was dramatically (ı = −3.3 ppm) shifted to a highly upfield region (8.0–4.7 ppm) with a concomitant disappearance of the OH proton. The aromatic protons of 1, however, were slightly shifted (ı < −1.0 ppm) toward the upfield aromatic regions. These 1 H NMR spectral changes, together with the bathochromic shift of 1–CN conjugate in its UV–vis spectra, indicate that cyanide was added to an electrophilic site of the oxime carbon rather than that of the aromatic carbon of 1. The nucleophilic addition of cyanide to the oxime carbon site of 1 would generate an anionic intermediate, which subsequently underwent a proton transfer from acidic oxime
O
O
H+ N
OH
OH
H CN
Et2N
transf er
O
O H N H b CN
O
Hb
D C B
A
Ha
OH
10.0
Fig. 4.
1
ppm (f1)
5.0
H NMR spectra of 1 (20 mM) in the absence (A) and presence of 0.5 equiv. (B) and 1.0 equiv. (C), and 3.0 equiv. (D) of cyanides in DMSO-d6 /D2 O (25:1, v/v).
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100
77
y = -0.1364x + 76.409 R² = 0.9981
F493
F 493
80
[2]
75
[3]
60 73
40
0
4
8
12
16
[CN]/μM 20 0
[4] 0
1000
2000
3000
equiv. of CN
_
4000
5000
6000
Fig. 5. Linear plot of 1 (20 M, ex /em 463/493 nm) upon the addition of different amounts of CN− (0, 2, 6, 10 or 16 M) in DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4). The limit of detection of CN− was determined as 0.49 M.
OH to the amine anion to afford the 1–CN conjugate. The resulting hydroxylamine (1–CN) would play the role of a fluorescence quencher through the photoinduced-electron transfer mechanism from nitrogen to coumarin and then might induce the dramatic On–Off transition of the fluorescence of 1 upon the addition of CN− . The detection limit of CN− was measured in the DMSO/HEPES buffer (9:1, 0.10 M, pH 7.4) using a fluorescence titration experiment. The standard deviation of the emission intensities of 1 without CN− was obtained as = 0.022 (n = 3). The fluorescence intensities at 493 nm were measured by the incremental addition of CN− to 1 (20 M), the slope of which gave m = −0.0391. From the fluorescence experiment of 1, we found that the limit of detection (LOD) of CN− was 0.49 M at 3/m [8], which was almost 4 times more sensitive than the level of cyanide in drinking water permitted by WHO (Fig. 5) [2]. The probe (1) showed a stable pH profile at pH 3–11, where 1 displayed a strong green fluorescence but 1–CN did not exhibit any fluorescence (Fig. S4). We expect that the characteristic onoff fluorescence property of 1 in the broad range of pHs could be utilized for the selective and sensitive detection of cyanides in the aqueous enviroment.
[5]
4. Conclusion In summary, we report a coumarinyloxime-based fluorescent probe (1) for cyanide. The fluorescent probe has shown a highly selective and sensitive response to the cyanide anion over other various anions with a submicromolar limit of fluorimetric detection (LOD = 0.49 M). When CN− was added to 1, the probe (1) showed a dramatic On–Off fluorescence change as well as a distinct color change, which was observable with the naked eye in aqueous solvent. Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF 2011-0028456). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.07.098.
[6]
[7] [8]
(b) Agency for Toxic Substances and Disease Registry, Toxicological Profile for Cyanide, Department of Health and Human Services, Atlanta, 2006. Guidelines for Drinking-water Quality, World Health Organization, Geneva, 1996. (a) C.A. Young, L.G. Tidwell, C.G. Anderson, Cyanide: Social Industrial and Economic Aspects, The Minerals, Metals and Materials Society Press, Warrendale, 2001; (b) R.A. Greenfield, B.R. Brown, J.B. Hutchins, J.J. Iandolo, R. Jackson, L.N. Slater, M.S. Brone, Microbiological, biological, and chemical weapons of warfare and terrorism, American Journal of the Medical Sciences 323 (2002) 326. (a) R. Badugu, J.R. Lakowicz, C.D. Geddes, Enhanced fluorescence cyanide detection at physiologically lethal levels: reduced ICT-based signal transduction, Journal of the American Chemical Society 127 (2005) 3635; (b) S.-Y. Chung, S.-W. Nam, J. Lim, S. Park, J. Yoon, A highly selective cyanide sensing in water via fluorescence change and its application to in vivo imaging, Chemical Communications 20 (2009) 2866; (c) J.H. Lee, A.R. Jeong, I.-S. Shin, H.-J. Kim, J.-I. Hong, Fluorescence turn-on sensor for cyanide based on a cobalt(II)–coumarinylsalen complex, Organic Letters 12 (2010) 764; (d) Z.C. Xu, X.Q. Chen, H.N. Kim, J.Y. Yoon, Sensors for the optical detection of cyanide ion, Chemical Society Reviews 39 (2010) 127; (e) Y.M. Kim, H.S. Huh, M.H. Lee, I.L. Lenov, H.Y. Zhao, F.P. Gabbai, Turn-on fluorescence sensing of cyanide ions in aqueous solution at parts-per-billion concentrations, Chemistry: A European Journal 17 (2011) 2057. (a) Y.M. Chung, B. Raman, D.S. Kim, K.H. Ahn, Fluorescence modulation in anion sensing by introducing intramolecular H-bonding interactions in host–guest adducts, Chemical Communications 2 (2006) 186; (b) Y.K. Yang, J.S. Tae, Acridinium salt based fluorescent and colorimetric chemosensor for the detection of cyanide in water, Organic Letters 8 (2006) 5721; (c) K.-S. Lee, H.-J. Kim, G.H. Kim, I. Shin, J.-I. Hong, Fluorescent chemodosimeter for selective detection of cyanide in water, Organic Letters 10 (2008) 49; (d) Y. Sun, G.F. Wang, W. Guo, Colorimetric detection of cyanide with nitrophenyl benzamide derivatives, Tetrahedron 65 (2009) 3480; (e) J. Jo, D. Lee, Turn-on fluorescence detection of cyanide in water: activation of latent fluorophores through remote hydrogen bonds that mimic peptide -turn motif, Journal of the American Chemical Society 131 (2009) 16283; (f) S.J. Hong, J. Yoo, S.H. Kim, J.S. Kim, J. Yoon, C.H. Lee, ␣-Vinyl substituted calix[4] pyrrole as a selective ratiometric sensor for cyanide anion, Chemical Communications 2 (2009) 189; (g) G.-J. Kim, H.-J. Kim, Doubly activated coumarin as a colorimetric and fluorescent chemodosimeter for cyanide, Tetrahedron Letters 51 (2010) 185; (h) G.-J. Kim, H.-J. Kim, Coumarinyl aldehyde as a Michael acceptor type of colorimetric and fluorescent probe for cyanide in water, Tetrahedron Letters 51 (2010) 2914; (i) S. Park, H.-J. Kim, Highly activated Michael acceptor by an intramolecular hydrogen bond as a fluorescence turn-on probe for cyanide, Chemical Communications 46 (2010) 9197; (j) Y. Shiraishi, S. Sumiya, T. Hirai, Highly sensitive cyanide anion detection with a coumarin–spiropyran conjugate as a fluorescent receptor, Chemical Communications 47 (2011) 4953; (k) H.J. Kim, K.C. Ko, J.H. Lee, J.Y. Lee, J.S. Kim, KCN sensor: unique chromogenic and ‘turn-on’ fluorescent chemodosimeter: rapid response and high selectivity, Chemical Communications 47 (2011) 2886; (l) S. Park, H.-J. Kim, Highly selective chemodosimeter for cyanide based on a doubly activated Michael acceptor type of coumarin thiazole fluorophore, Sensors & Actuators B 161 (2012) 317; (m) S. Park, H.-J. Kim, Reaction-based chemosensor for the reversible detection of cyanide and cadmium ions, Sensors & Actuators B 168 (2012) 376; (n) H. Lee, H.-J. Kim, Highly selective sensing of cyanide by a benzochromenebased ratiometric fluorescence probe, Tetrahedron Letters 53 (2012) 5455; (o) S. Park, K.-H. Hong, J.-I. Hong, Azo dye-based latent colorimetric chemodosimeter for the selective detection of cyanides in aqueous buffer, Sensors & Actuators B 174 (2012) 140; (p) K.-H. Hong, H.-J. Kim, Azo dye-based colorimetric cheomodosimeter for cyanide in aqueous solution, Supramolecular Chemistry 25 (2013) 24. (a) D.-N. Lee, D.Y. Kim, S.H. Ghil, H.-J. Kim, Coumarin–benzothiazoline conjugate as a fluorescence turn-on probe for reactive oxygen species and its cellular expression, Bulletin of the Korean Chemical Society 32 (2011) 3109; (b) G.-J. Kim, K. Lee, H. Kwon, H.-J. Kim, Ratiometric fluorescence imaging of cellular glutathione, Organic Letters 13 (2011) 2799. P. Job, Formation and stability of inorganic complexes in solution, Annales de Chimie 9 (1928) 113. D. MacDougall, W.B. Crummett, Guidelines for data acquisition and data quality evaluation in environmental chemistry, Analytical Chemistry 52 (1980) 2242.
Biographies References [1] (a) K.W. Kulig, Cyanide Toxicity, U.S. Department of Health and Human Services, Atlanta, 1991;
Sang-Yun Na is a master course student at the Hankuk University of Foreign Studies, Korea. His research interest is to develop highly selective fluorescence sensors for biological thiols and toxic chemicals.
S.-Y. Na et al. / Sensors and Actuators B 188 (2013) 1043–1047 Jong-Young Kim is an undergraduate course student at the Hankuk University of Foreign Studies, Korea. He joined Dr. H.-J. Kim’s lab for his junior research program at HUFS. His research interest is to develop highly selective fluorescence sensors for toxic chemicals. Hae-Jo Kim received his B.S. degree in chemistry education, and M.S. and Ph.D. degrees in chemistry from Seoul National University, Korea with Professor Jong-In
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Hong (2002). After completing his postdoctoral research at University of Toronto, Canada with Professor Jik Chin (2005), he joined Kyonggi University, Korea as a faculty member (2006). He is currently associate professor and chairperson of the Department of Chemistry at Hankuk University of Foreign Studies, Korea. His research focuses on fluorescent molecular probes for tumor imaging and therapy.