Novel coumarin-based fluorescent probe for selective detection of Cu(II)

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 18–22

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Novel coumarin-based fluorescent probe for selective detection of Cu(II) Ahmadreza Bekhradnia ⇑, Elham Domehri, Masome Khosravi Pharmaceutical Sciences Research Center, Department of Medicinal Chemistry, Mazandaran University of Medical Sciences, Sari, Iran

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A new fluorescent chemosensor for

Cu2+ has been reported.  We designed and synthesized a new carboxamide coumarin sensor through microwave irradiation.  The association constant between 3 and Cu2+ was found to be 3.01  104 M1.

a r t i c l e

i n f o

Article history: Received 6 February 2015 Received in revised form 24 June 2015 Accepted 6 July 2015 Available online 7 July 2015 Keywords: Cation recognition Fluorescent chemosensor Coumarin derivatives

O O2N

O O2N

N H O

O

N(Me)2

Fluorescence OFF

r.t

O

O

Cu

N(Me)2

Fluorescence ON

a b s t r a c t We report an efficient and convenient method for preparing nitro-3-carboxamide coumarin derivatives, proposed as novel fluorescent chemosensor, through microwave irradiation. This compound can be used as fluorescent probe for Cu2+ with selectivity over other metal ions in aqueous solution. The fluorescence of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide(3) is the highest in the presence of Cu2+, with stronger excitation at k = 320 nm than for the other cations tested. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Coumarin derivatives are an important group of fluorescent organic heterocycles with photochemical, photophysical, and biological properties that have led to variety of applications as pharmaceuticals [1,2], fluorescent whiteners [3], fluorescent indicators [4], and laser dyes [5]. Coumarin derivatives can be used as fluorescent probes for metal ions because of their highly variable size, hydrophobicity, and chelation 6. Selective detection of heavy metal ions such as copper [7a] is important because of its toxic and carcinogenic effects on humans and wildlife. For example, the average diet provides substantial amounts of copper, and the recommended intake is 0.9 mg/day [7b,c]. However, exposure to higher levels of copper can cause rheumatoid arthritis, gastrointestinal disturbances, and Wilson’s disease. Copper is released into ⇑ Corresponding author at: Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden. E-mail addresses: [email protected], [email protected] (A. Bekhradnia). http://dx.doi.org/10.1016/j.saa.2015.07.029 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

N

Cu(II)

the bloodstream and is deposited in the kidneys, cornea, and particularly, the brain [7c]. Therefore, detection and elimination of this metal is important. In this regard, many analytical techniques are available for the determination of heavy metals. Among them, substantial attention has been paid to fluorometry because of its high sensitivity. Fluorescence can vary with polarity and viscosity because it is sensitive to the local environment [6]. Coumarin is a suitable fluorophore because of its desirable photophysical properties such as its large Stokes shift and visible excitation and emission wavelengths [8]. There are several methods for synthesizing substituted coumarins, including the Pechmann and Duisberg [9], Perkin and Henry [10], Brafola et al. [11], Claisen et al. [12], Shringer [13], and Wittig reactions [14]. Because of the formation of chromone by-products and variable yields, the synthesis of coumarins through the Kostanecki–Robinson reaction is considered an inferior procedure. Other approaches to coumarin preparation have additional challenges. Most of these procedures must be carried under severe conditions such as high temperatures, require long reactions times, and afford low yields [15]. To overcome these

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challenges, increasing the rate of chemical reactions in organic syntheses through microwave activation has attracted substantial interest in recent years [16]. We wish to report microwaveassisted procedure for the preparation of nitro-coumarin amide derivative. Also, the effectiveness of UV–vis analysis and fluorescence properties of the synthesized compound and its reactivity has been investigated toward several heavy metal cations. 2. Experimental section 2.1. Chemicals and instruments Melting point was measured in open capillary tubes with an Electrothermal-9200 melting point apparatus. 1H and 13C-NMR spectra were recorded using a Bruker (400 MHz) Avance (III) spectrometer. Chemical shifts (d) were reported in ppm downfield from the internal standard tetramethylsilane (TMS). The Uv–vis spectra were obtained using a Perkin-Elmer lambda-EZ 201 and a Jasco FP-200 spectrofluorometer was used to record fluorescence emission spectra. Data were recorded on-line and analyzed by Excel software on a PC computer. Fluorescence intensity measurements were performed at room temperature. Infrared (IR) spectra in cm1 were recorded on FT-IR Perkin-Elmer spectrometer. All microwave irradiation reactions were carried out on a Milestone Micro-SYNTH apparatus. Internal temperatures were measured with fiber-optic sensor in conjunction with Milestone immersion well. 2.2. Synthesis of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2Hchromene-3-carboxamide(3) In the present study, we prepared coumarin ester derivative through the condensation of nitro-2-hydroxy benzaldehyde and malonic ester under solvent-free conditions and microwave irradiation. The produced ester was hydrolyzed to afford the related carboxylic acid. Subsequently, 3-carboxamide coumarin was produced through reaction with N,N0 -dimethylethylenediamine, and its fluorescence spectra was studied in the presence of trace amounts of cations, including Na+, K+, Li+, Ca2+, Ag+, Cu2+, Pb2+, Hg2+, Co2+, Cr3+, Mn2+, Fe2+, Ni2+, Cd2+, and Zn2+. Compound 1 was prepared by the reaction of salicylaldehyde and diethyl malonate in the presence of piperidine and glacial acetic acid under microwave irradiation for the time indicated in Tables 1 (Scheme 1). Compound 2 was prepared by heating 1 in alcohol-based solution. Subsequently, dicyclohexylcarbodiimide (DCC), N,N0 -dimethylethylenediamine, and a catalytic amount of 4-(dimethylamino) pyridine (DMAP) were added to a stirred suspension of 2 in chloroform. The white N,N0 -dicyclohexylurea (DCU) precipitate was then filtered from the yellow solution, and finally, the respective carboxamide 3 was obtained after solvent removal and further purification. This fluorophore was solved in (HEPES:DMSO) 9:1, v/v and evaluated the cation chelating by fluorescence spectroscopy. The selected physical and spectral data for synthetic compounds are as follows: (1): mp: 180–182 °C. 1H NMR (400 MHz, CDCl3) d: 1.4 Table 1 Microwave settings for 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3carboxamide (1). Step

Time

Temperature (T1)

Temperature (T1)

Max power

1 2 3 4

14 min 15 min 5 min 5 min

Ramp to 105 °C 105 °C Ramp to 130 °C 130 °C

85 85 100 100

800 W 650 W 650 W 650 W

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(Me, t, 3H, J = 7.2), 4.5 (CH2, q, 2H, J = 7.2), 7.6–8.3 (m-Ar, 3H), 8.6 (@CH, s, 1H). IR (KBr): 1693 (ester C@O), 1778 (lacton C@O) cm1. (2): m.p. 198–201 °C. 1H NMR (400 MHz, CDCl3) d: 7.5–8.0 (m-Ar, 3H), 9.2 (@CH, s, 1H), 12.5 (OH, s, 1H). IR (KBr): 3438 (OH), 1967 (acid C@O), 1776.68 (lacton C@O) cm1. (3); m.p. 209–211 °C. 1H NMR (400 MHz, CDCl3) d: 2.5 (N(Me)2, s, 6H), 3.7–4.0 (HNCH2, m, 2H, JCH2-CH2 = 7.3), 2.7–2.8 (Me)2NCH2, m, 2H, JCH2-CH2 = 7.3), 8.0–8.4 (m-Ar, 3H), 8.5 (@CH, s, 1H), 6.9 (NH, 1H). IR (KBr): 3419 (amide NH), 1648 (amide NH), 1604 (amide C@O) cm1. 13C NMR (400 MHz, CDCl3) d: 35.3, 43.7, 44.7, 56.5, 114.1, 116.9, 117.8, 120.2, 127.7, 133.1, 138.2, 154.81, 162.3, 162.4. FAB-MS calcd for C14H15N3O5 [M+H]+ 306.26, found 306.00. Elemental analysis data is the following: C, 54.90%; H, 4.97%; N, 13.82%; O: 26.31 Anal. Calcd.: C, 55.08%; H, 4.92%; N, 13.77%; O: 26.23. 3. Results and discussion Figs. 1 and 2 show changes in the absorption and fluorescence spectra of aqueous solution of 3, upon the addition of various cation salts. The absorption intensity of 3 decreased with the addition of various heavy metal ions, while addition of Hg2+ produced a higher hypochromic effect at k = 335 nm than with the other ions. All the cations studied led to hypochromic effects in absorbance in the UV–vis spectra of 3 (Fig. 1). By UV–vis analysis, it was difficult to identify specific cations over other metal ions because of the ambiguous absorption spectra [17]. Therefore, to investigate the selectivity of 3 for Cu2+ in the presence of other metal ions, the fluorescence response 3 toward various metal ion solutions (0.3 mM) was studied. To obtain reliable results, the excitation spectra for compounds 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carbo xamide, 3, and 3-Cu(II) were obtained and given in Fig. 3. The excitation spectrum would represent the relative emission of the 3 and 3-Cu(II) in aqueous solution (HEPES:DMSO) 9:1, v/v). Since the excitation spectrum of a fluorophore could be superimposable on its absorption spectrum, the excitation spectra of 3 and 3-Cu(II) were recorded at 320 nm with an emission at 420 nm. Fig. 2 shows the fluorescence spectra of 3, before and after the addition of Cu2+ to a mixture of various cations, with excitation at 320 nm. The fluorescence intensity of 3 increased when Cu2+ was added, with the highest fluorescence intensity observed when Cu(NO3) solution was added (Figs. 2 and 4). In summary, the fluorescence intensity was enhanced with excitation at 320 nm for Cu2+ over the other metal cations when 3 was used as a probe. To investigate the binding modes between sensor 3 and its respective cation, we carried out ab initio calculations using HF/6-31G⁄ [18]. The optimized structure of the 3 complex is shown in Fig. 5, indicating 1:1 stoichiometry between the sensor and Cu2+. In this structure, Cu2+ binds with an oxygen atom of a carbonyl group, a nitrogen atom of an amide, a nitrogen atom of an amine, and two oxygen atoms of a nitrate. To find the mole percent of the sensor to moles of the metal, UV/vis titration experiments were performed using 0.6 lmol (2 mL 0.3 mM solution) of 3 in solution (HEPES:DMSO) 9:1 with varying concentrations of metal nitrate salts (0–4.5  106 mol). Fig. 6 shows the relationship between absorbance and a mole ratio of 1:1, consistent with the theoretical results. The association constant (Ka) of compound 3 with Cu2+ was determined using the Benesi–Hildebrand equation [19] as follows:

1 1 1 þ ¼ F  F min K a ðF max F min Þ½Cu2þ  ðF max F min Þ F and Fmin represent the fluorescent intensity of the ligand 3 at moderate concentration and absence of Cu+2, respectively. Fmax is the

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O

O

O Piperidine, AcOH

OC2H5

NO2

H

NO2

OC2H5

+ OH

OC2H5

Microwave(600W-850W)

O

O

O

1

NaOH, EtOH, HCl (pH=2) O NO2

N H O

O

DCC , DMAP

NO2

OH

N,N-dimethyl ethylene diamine

N(Me)2

O

O

3

O

2

O NO2

O Cu

N H O

O

N(Me)2

2+

NO2

(HEPES:DMSO )

Fluorescence OFF

N O

O

N(Me)2 Cu2+

Fluorescence ON

Scheme 1. Synthetic pathways to 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide (3).

Fig. 1. Absorption spectra of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide before and after addition of heavy metal ions.

saturated fluorescent intensity of compound 3 in the presence of excess amount of Cu2+. According to Benesi–Hildebrand equation, the association constant between 3 and Cu2+ was calculated from the fluorescence titration result and was found to be 3.01  104 M1.

Moreover, the detection limit was calculated based on the fluorescence titration [20]. To determine the S/N ratio, the emission intensity of the complex (3-Cu2+) was measured and the standard deviation of blank measurements was calculated. The detection limit was then calculated according to 3  db/m. where db was

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Fig. 4. Flourescence spectra of 3 solutions (0.3 mM) with addition of metal solutions in aqueous solution (HEPES:DMSO) 9:1, v/v) (0.1 mM) with an excitation at 320 nm, excitation and emission slit widths at 3.0 nm, and room temperature.

Fig. 2. Fluorescence spectra of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2Hchromene-3-carboxamide upon the addition of Cu2+ in the presence of various mixture of cations with an excitation at 320 nm, excitation and emission slit widths at 3.0 nm, and room temperature. Fig. 5. Optimized structure of 3-Cu(II) by ab initio calculation (HF/6-31G⁄). All hydrogen atoms are omitted for clarity.

Fig. 3. Excitation spectra of 3 and complex 3-Cu(II) (1:1) in aqueous solution (HEPES:DMSO) 9:1, v/v) with an emission at 420 nm, excitation and emission slit widths at 3.0 nm, and room temperature.

Fig. 6. Molar ratio of complex 3-Cu(II) by using UV Spectrum with addition of varying concentrations of the metal nitrate salts (0–4.5  106 mole) to 2 ml of 0.3 mM solution of 3 (0.6 lmoles) in solution (HEPES:DMSO) 9:1.

4. Fluorescence and absorption spectra the standard deviation of blank solutions, and m was the slope between intensity vs. sample concentration. The detection limit for Cu2+ was calculated to 2.7  106 mol/L. For the biological application of the ligand, the sensing should perform in physiological pH (6.8–8.2). Therefore, all of experiments were done in the aqueous solution (HEPES:DMSO) 9:1, v/v).

Stock solutions of 3 (0.3 mM) was prepared in aqueous solution (HEPES:DMSO) 9:1 v/v. Stock solutions of metal nitrate salts (0.1 mM Na+, K+, Li+, Ca2+, Ag+, Cu2+, Pb2+, Hg2+, Co2+, Cr3+, Mn2+, Fe2+, Ni2+, Cd2+, or Zn2+) was prepared in water. The prepared metal solutions (4.5  106 mol or 45 mL 0.1 mM solution) were mixed

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with solution of 3 (6.0  107 mol or 2 mL 0.3 mM solution), and the absorption and fluorescence intensities of the free ligand and complexed form of the probe compound were measured. The UV–vis absorbance and fluorescence spectra of receptor varied with the concentration of metal ions. Fluorescence quantum yield was determined by comparing the emission and absorption intensities of the probe with those of a fluorescence standard, fluorescein in 0.1 N NaOH. For all fluorescence measurements, the excitation wavelength was 320 nm, while for excitation spectra, the emission wavelength was 400 nm, with excitation and emissions slit widths of 3.0 nm. Quantum yield of 12.1% was calculated for the 3-Cu(II) complex. Nevertheless, low quantum yields were obtained for combined receptor and other cations as well as free receptor and no worth to report.

5. Conclusion In conclusion, we synthesized new coumarin carboxamide 3 through microwave irradiation, which showed no significant fluorescence. The receptor can be used for complexation of heavy toxic metals and exhibit enhanced fluorescence in the presence of Cu2+ with selectivity over other metal ions in aqueous solution. The highest fluorescence intensity for 3 was observed in the presence of Cu2+ in preference to a variety of other common heavy and toxic metal ions in aqueous solution, with excitation at 320 nm.

Acknowledgments The authors gratefully acknowledge the financial support for this work received from the Mazandaran University of Medical Sciences ‘‘Professor’s Projects and Sabbatical Funds’’. The authors wish to express their special thanks to Professor Per-Ola Norrby from University of Gothenburg for his valuable assistance and contribution.

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.saa.2015.07.029. References [1] (a) M.E. Marshall, K. Kervin, C. Benefield, A. Umerani, S. Albainy Jenei, Q. Zhao, M.B. Khazaeli, J. Cancer. Res. 120 (1994) 3–10; (b) T. Tanaka, T. Kumamoto, T. Ishikawa, J. Tet. Lett. 41 (2000) 10229–10233. [2] (a) S.N. Azizi, P. Shakeri, M.J. Chaichi, A.R. Bekhradnia, et al., Spectrochim. Acta A 122 (2014) 482–488; (b) S.N. Azizi, M.J. Chaichi, P. Shakeri, A.R. Bekhradnia, J. Fluoresc. 23 (2013) 227–235; (b) S.N. Azizi, M.J. Chaichi, P. Shakeri, A.R. Bekhradnia, J. Lumin. 144 (2013) 34–40. [3] K. Aslam, M.K. Khosa, N. Jahan, S. Nosheen, Pak. J. Pharm. Sci. 23 (2010) 449– 454. [4] K. Haupt, A.G. Mayes, K. Mosbach, J. Anal. Chem. 70 (1998) 3936–3939. [5] B.B. Raju, T.S. Varadarajan, Laser Chem. 16 (1995) 109–120. [6] B.D. Wagner, Molecules 14 (2009) 210–237. [7] (a) L. Yang, W. Zhu, M. Fang, Q. Zhang, C. Li, Spectrochim. Acta A 109 (2013) 186–192; (b) P.G. Georgopoulos, A. Roy, M.J. Yonone-Lioy, R.E. Opiekun, P.J. Lioy, J. Toxicol. Environ. Health, B 4 (2001) 341; c G.W. Rafter, Med Hypotheses 22 (1987) 245–249. [8] Y. Xu, X. Dai, B.-X. Zhao, Spectrochim. Acta A 138 (2015) 164–168. [9] V.H. Pechmann, C. Duisberg, J. Chem. Ber. 17 (1884) 929–979. [10] W.H. Perkin, W.S. Henry, J. Chem. Soc. 28 (1875) 10–15. [11] G. Brafola, F. Fringuelli, O. Piermatti, F. Pizzo, J. Heterocycles 43 (1996) 1257– 1266. [12] N. Cairns, L.M. Harwood, D.P. Astles, J. Chem. Soc. Perkin Trans. (1994) 3101– 3107. [13] R.L. Shringer, J. Org. React. 1 (1942) 15–18. [14] I. Yavari, R. Hekmat shoar, A. Zonouzi, J. Tet. Lett. 39 (1998) 2391–2392. [15] (a) A. De La Hoz, A. Moreno, E. Vazquez, J. Synlett. (1999) 608–610; (b) N. Britto, V.G. Gore, R.S. Mali, A.C. Ranade, J. Synth. Commun. 19 (1989) 1899–1910. [16] A.R. Bekhradnia, S. Arshadi, Synth. React. Inorg. Metal-Org. Nano-Met. Chem. 42 (2012) 705–710. [17] M.Z. Kassaee, A.R. Bekhradnia, J. Biosci. Bioeng. 2003 (95) (2003) 526–529. [18] S. Arshadi, A.R. Bekhradnia, S. Ahmadi, A.R. Karami, S. Pourbeyram, Chin. J. Chem. 29 (2011) 1347–1352. [19] L.Y. Wang, D.C. Ye, D.R. Cao, Spectrochim. Acta A 90 (2012) 40–44. [20] G.-Q. Wang, J.-C. Qin, Ch.-R. Li, Zh.-y. Yang, Spectrochim. Acta A 150 (2015) 21– 25.