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Recognition of Mg2+ by a new fluorescent “turn-on” chemosensor based on pyridyl-hydrazono-coumarin
Author’s Accepted Manuscript Recognition of Mg2+ by a new fluorescent “turnon” chemosensor based on pyridyl-hydrazonocoumarin Jessica Orrego-Hernández, Nelson Nuñez-Dallos, Jaime Portilla www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(16)30088-1 http://dx.doi.org/10.1016/j.talanta.2016.02.020 TAL16338
To appear in: Talanta Received date: 16 December 2015 Revised date: 8 February 2016 Accepted date: 9 February 2016 Cite this article as: Jessica Orrego-Hernández, Nelson Nuñez-Dallos and Jaime Portilla, Recognition of Mg2+ by a new fluorescent “turn-on” chemosensor based on pyridyl-hydrazono-coumarin, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.02.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Recognition of Mg2+ by a new fluorescent “turn-on” chemosensor based on pyridyl-hydrazono-coumarin
Jessica Orrego-Hernández, Nelson Nuñez-Dallos, Jaime Portilla*
Bioorganic Compounds Research Group, Department of Chemistry, Universidad de los Andes, Carrera 1 No. 18A 10, Bogotá, Colombia
*E-mail: [email protected]; Tel: +57 1 3394949. Ext. 2080
ABSTRACT A new fluoroionophore PyHC bearing 2-pyridylhydrazone and 7-hydroxycoumarin moieties for selective detection of Mg2+ was synthesized and characterized. This chemosensor exhibited “turn-on” fluorescence behavior and was sensitive to Mg2+ concentrations as low as 105 nmol L-1 in ethanol-water solution. Detailed spectroscopic studies revealed the binding mode of a 1:1 complex between PyHC and Mg2+ that leads to a fluorescence enhancement.
KEYWORDS: Chemosensor, Fluorescence, Mg2+, Pyridyl-hydrazono-coumarin, Schiff base
2
1. INTRODUCTION In biological systems, magnesium ion plays a crucial role as a cofactor in DNA synthesis and protein phosphorylation [1]. In human body, it is essential for many cardiovascular and neurological processes [2–4]. Abnormal concentrations of magnesium in the cytosol and subcellular regions have relation with diseases such as diabetes, hypertension, epilepsy and Alzheimer [3–6]. For these reasons, a simple and efficient analytical techniques for intracellular detection and imaging of magnesium are of great interest [7]. Here, fluorescence-based chemosensors offer a number of advantages such as simple handling, and real-time response allowing dynamic measurements. Currently, considerable efforts are spent searching for new suitable fluorescent probes matching all desired properties such as high sensitivity, selectivity, aqueous solubility, easy preparation, and low-cost synthesis [8–11]. Among the chemosensors reported for Mg2+ detection are β-diketone [12], β-ketoacid [13], crown ether [14], polymer-based ligands [15], and arylimines [16,17]. Also, commercial probes, for example mag-fura-2 and Magnesium Green showing high water solubility and low toxicity [18]. Nevertheless, the principal unresolved challenge for in vivo and in vitro monitoring of magnesium is to obtain sufficient affinity and sensitivity of the chemosensor towards magnesium under intracellular conditions, in particular in the presence of calcium. The intracellular concentration of Mg2+ is of the order 0.1-6 mmol L-1 whereas the concentration of calcium may vary in a much wider range (from 0.1 µmol L-1 to 1 mmol L-1) [8]. Therefore, the goal of the current study is to design a highly selective
3 and sensitive fluorescent chemosensor that can specifically recognize Mg2+ without the interference of other metal ions, especially Ca2+. The proposed basic principle of a “turn-on” fluorescent chemosensor for metal ions is that fluorescence is quenched in the free fluoroionophore as a result of rapid non-radiative processes, including photo-induced electron transfer (PET), intramolecular charge transfer (ICT), excited-state intra or intermolecular proton transfer (ESIPT), and isomerization. The binding of the chemosensor with metal ions interrupts the aforementioned processes and increases its structural rigidity, generating a chelation-enhanced fluorescence (CHEF) effect [19–24]. Nowadays, one of the most widely used strategy for developing tailor-made selective fluorescent chemosensors for sensing of metal ions include the synthesis of Schiff bases and coumarin derivatives [25–27]. Schiff base ligands have the C=N donor system that can coordinate with various metal ions and form stable complexes. In addition, Schiff bases are easy and inexpensive to prepare [28–30]. Coumarins as fluorophores offer many advantages, including high fluorescence quantum yield, large Stokes shift, excellent light stability, and low toxicity [31–36]. Herein, we report the synthesis of the Schiff base 7-hydroxy-4-methyl-8-((2-(pyridin-2yl)hydrazono)methyl)-2H-chromen-2-one (PyHC) (Scheme 1). This new compound acts as a “turn-on” fluorescent chemosensor for the selective detection of Mg2+ with a high sensitivity in ethanol-water solution. To the best of our knowledge, PyHC is one of the most sensitive chemosensors for Mg2+ recognition. We have developed PyHC as an integrated chemosensor with the 7-hydroxy-4methylcoumarin unit as an emitting fluorophore, which has suitable photophysical properties [31-36]. The design also includes that the cation binds by the 2-(pyridin-2-
4 yl)hydrazono group, as well as by the phenolic hydroxyl group of the fluorophore unit (Scheme 1). In addition, the hidrazono group has the C=N donor system that can quench the fluorescence of the fluorophore by PET process and C=N isomerization [19-24]. Thus, the coordination of PyHC to Mg2+ probably disrupts these processes and increases its structural rigidity producing a fluorescence enhancement. The binding mode of the PyHCMg2+ complex was studied by spectroscopic methods involving absorption, fluorescence, ESI mass, and NMR spectra. 2. EXPERIMENTAL 2.1. General information All reagents and solvents were purchased from commercial sources and used as received. All solvents used in this work were analytic grade. The reactions were monitored by TLC (DCM:MeOH 10:1) and were visualized by UV lamp (254 nm or 365 nm). NMR data were recorded on a Bruker Avance 400 (400.13 MHz for 1H; 100.61 MHz for 13C). 1H and 13C NMR chemical shifts are reported in parts per million (ppm) relative to TMS, with the residual solvent peak used as an internal reference. High resolution mass spectra (HRMS) were obtained on an Agilent Technologies Q-TOF 6520 spectrometer via an electrospray ionization (ESI). Infrared spectra (FT-IR) were recorded on a Thermo Nicolet Nexus 470 ESP FT-IR Spectrometer. Thermogravimetric analysis (TG) was performed on a NETZSCH STA 409 PC/PG instrument in a nitrogen atmosphere with a continuous flow of 60 mL/min and a heating rate of 10 K/min, from 30°C to 600°C. Melting points were determined in capillary tubes on a Stuart SMP10 melting point apparatus and are uncorrected. pH measurements were made with a Fisher Scientific accumet research AR50 pH meter at 298 K. 2.2. Synthesis and characterization
5 The chemosensor PyHC was synthesized according to the Scheme 1. The synthetic procedures of 2-hydrazinylpyridine and 8-formyl-7-hydroxy-4-methylcoumarin were adapted and slightly modified from literature [37–40]. Detailed synthetic route and characterizations of products are shown in the Supplementary data. Synthesis of 7-hydroxy-4-methyl-8-((2-(pyridin-2-yl)hydrazono)methyl)-2H-chromen2-one (PyHC). A solution of 2-hydrazinylpyridine (53.3 mg, 0.49 mmol) in EtOH (5 mL) was added dropwise to a solution of 8-formyl-7-hydroxy-4-methylcoumarin (100.8 mg, 0.49 mmol) in hot ethanol (4 mL), then the reaction mixture was refluxed for 3 h. The compound PyHC precipitated out as yellow solid. The precipitate was isolated by vacuum filtration, washed with cold ethanol, and dried under vacuum (0,132g, 92 %). mp 290-291 °C with decomposition (from ethanol). Rf = 0.43 (DCM:MeOH 10:1). IR νmax/cm-1 (KBr): 3438 (OH), 3204 (NH), 1738 (C=N), 1632 (C=O ester), 1597 (C-O ester). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.38 (s, 3H), 6.20 (s, 1H), 6.83 (d, J = 7.2 Hz, 2H), 6.92 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.69 (t, J = 7.1 Hz, 1H), 8.18 (s, 1H), 8.67 (s, 1H), 11.29 (s, 1H), 12.33 (s, 1H).
13
C NMR (100 MHz, DMSO-d6): δ (ppm) 18.3 (CH3), 106.3
(CH), 106.5 (C), 110.6 (CH), 111.9 (C), 113.1 (CH), 115.8 (CH), 126.3 (CH), 135.4 (CH), 138.2 (CH), 148.3 (CH), 151.5 (C), 153.8 (C), 154.9 (C), 159.4 (C), 159.9 (C). HRMS m/z (ESI) calculated for [C16H13N3O3+H]+: 296.1030; found 296.1031 [M+H]+. 2.3. UV-vis absorption and fluorescence studies The electronic absorption spectra were measured on Varian Cary 100 Conc (Agilent Technologies) spectrophotometer in a quartz cuvette having a path length of 1 cm. The fluorescence emission spectra were recorded by using a CARY Eclipse (Agilent Technologies) fluorescence spectrophotometer in a quartz cell (1 cm path length). UV-vis
6 and fluorescence measurements were performed at room temperature (20 ºC). For fluorescence measurements, both the excitation and emission slit widths were 5 nm. The 0.2 mmol L-1 stock solutions of the chemosensor PyHC were prepared in dichloromethane, acetonitrile, dimethyl sulfoxide and absolute ethanol. The salts used in stock solutions of metal ions were NaCl, KNO3, Mg(NO3)2.6H2O, CaCl2, Ba(NO3)2, Cr(NO3)3.9H2O, Fe(NO3)3.9H2O, Co(NO3)2.6H2O, Ni(NO3)2.6H2O, Cu(NO3)2.2.5H2O, Zn(NO3)2.6H2O, CdCl2, Hg(NO3)2.H2O, Al(NO3)3.9H2O, and Pb(NO3)2. Inorganic salts were dissolved in distilled water to afford 1 mmol L-1 aqueous solution. Aliquots of stock solution of PyHC was diluted to 5 mL to make the final concentration of 10 μmol L-1. In the selectivity and competition experiments of PyHC towards Mg2+ and other metal ions, the fluorescence emission spectra were recorded at λex = 340 nm from 10 μmol L-1 of the chemosensor in a 99:1 (v/v) ethanol-water solution (pH 7.14) and in the presence of 1.0 equiv. of various metal ions. The sensing studies were performed at pH 7.14 because it is close to the physiological pH (about 7-7.4). The fluorescence intensities were measured at λem = 449 nm. For the Job’s plot experiment of PyHC and Mg2+ the total concentration of PyHC and Mg2+ were kept as 10 μmol L-1. Fluorescence response in pictures was excitation at 365 nm using a UV lamp. 2.4. Determination of the relative quantum yields The relative quantum yields were obtained by using anthracene (φF = 0.28 in ethanol at 340 nm) as reference and calculated according to the following equation [41–46].
where x and st indicate the sample and standard solution, respectively, φ is the quantum yield, F is the integrated area of the emission, A is the absorbance at the excitation wavelength, L is the length of the absorption cell, and η is the index of refraction of the solvents. 2.5. Determination of the binding constant
7 The binding constant Ka was obtained according to the Benesi-Hildebrand equation for a 1:1 complex [31, 47-48].
wherein I0 is the fluorescence intensity of the free ligand, I are the observed fluorescence intensities upon addition of different equivalents of Mg2+, Ic is the fluorescence intensity of the ligand-metal complex, and [Mg2+] shows the concentration of magnesium ions. The binding constant is given by the ratio intercept/slope from the plot 1/(I-I0) versus 1/[Mg2+]. 2.6. Determination of the detection limit The limit of detection (LOD) of PyHC for Mg2+ was obtained by 3Sb/k, where Sb is the standard deviation of the blank measurements (by 10 times, Sb = 0.8527), and k is the slope from the plot fluorescence intensity I versus [Mg2+] [49,50]. (Fig. 4b, k = 2.43 x 107 L mol1
).
3. RESULTS AND DISCUSSION 3.1. Synthesis of chemosensor PyHC The chemosensor PyHC was synthesized with a high yield (92 %) from the condensation
reaction
between
8-formyl-7-hydroxy-4-methylcoumarin
and
2-
hydrazinylpyridine in absolute ethanol at reflux. The molecular structure of PyHC was verified unambiguously by FT-IR, 1H and
13
C NMR spectroscopy, and high resolution
mass spectra (HRMS) through electrospray ionization (ESI) (Fig. S6-S11). The thermogravimetric analysis (TG) showed that the chemosensor PyHC exhibits high thermal stability. This compound was found to be stable up to 293 °C (Fig. S12). 3.2. Photophysical properties of PyHC
8 The absorption and emission spectra of PyHC were investigated in dichloromethane (DCM), acetonitrile (ACN), dimethyl sulfoxide (DMSO) and absolute ethanol (EtOH) (Fig. S13-14 and Table S1). In the absorption spectra of PyHC, the absorption band with maximum between 325 and 345 nm is assigned to a π-π* transition. The fluorescence spectra displayed a maximum emission band in the range 450-460 nm. The chemosensor PyHC showed the highest relative quantum yield in DMSO (φF = 0.042), while it presented weaker fluorescence in EtOH, ACN and DCM (φF of 0.0021, 0.0036 and 0.0004, respectively). Ethanol was selected as solvent in further studies due to the fact that in the design of a “turn-on” fluorescent chemosensor for metal ions is important to consider that the free ligand exhibits weak fluorescence in the solvent of study. Furthermore, it was taken into account that ethanol is a green solvent, highly miscibility in water, and easy handling. 3.3. Fluorescence and UV-vis response of PyHC to Mg2+ Fluorescence spectra of PyHC (10 μmol L-1) were recorded at λex of 340 nm in ethanol -water solution (99:1 v/v) (pH 7.14) to observe its fluorescence intensity response towards Mg2+ and other metal ions (Na+, K+, Ca2+, Ba2+, Cr3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Al3+, and Pb2+) dissolved in distilled water (Fig. 1 and Fig. S15-16). An enhancement of the fluorescence quantum yield up to 17-fold was observed in presence of Mg2+ (φF = 0.0360) without a significant alteration of the maximum emission band (λem = 449 nm), while it was observed weak fluorescence with the other metal ions. These results suggest that PyHC has high selectivity for Mg2+. Furthermore, this chemosensor showed only a small enhancement of the fluorescence intensity in the presence of Ca2+ cations. The absorption spectra of PyHC with one equivalent of Mg2+ showed a new band at 390 nm, which indicates the coordination interaction between PyHC and Mg2+ (Fig. S17-18). This complexation produces a chelation-enhanced fluorescence effect.
9 In order to analyze in more detail the selectivity of PyHC, fluorescence spectra of one equivalent the chemosensor were measured with one equivalent of Mg2+ and in the presence of one equivalent of other metal ions (Fig. S19-20). In these competition experiments, it was found that alkali and alkaline earth metal ions had relatively low interference levels on the selectivity for Mg2+, while the transition metal ions quenched the fluorescence of the PyHC-Mg2+ complex. Thus, PyHC could be used as a fluorescent probe for the selective detection of Mg2+ among biologically abundant ions like Na+, K+, and Ca2+. The transition metal ions had strong interference on the selectivity of PyHC towards Mg2+ as a result of their strong coordination character, which was confirmed by the changes in the intensity and bathochromic shifts of the absorption bands of PyHC, as well as the appearance of new bands in the UV-vis spectra (Fig. S17 and S21). Job’s plot analysis of the fluorescence spectra showed a maximum at 0.5 mole fraction of Mg2+, indicating the formation of a 1:1 complex between PyHC and Mg2+ (Fig. 2). Based on a 1:1 stoichiometry, the association constant of PyHC with Mg2+ was calculated to be 5.57 x 104 L mol-1 at 20 ºC (R2 = 0.9975) from the Benesi–Hildebrand plot [31, 4748], which demonstrates the good binding ability of the chemosensor towards Mg2+ (Fig. 3). The chemosensor was found to bind Mg2+ reversibly as tested by reacting with EDTA. The addition of EDTA to a solution of PyHC-Mg2+ complex resulted in quenched of the fluorescence intensity (Fig. S22-23). The fluorescence quenching is a result of the high affinity of EDTA towards Mg2+ (Ka = 6.61 x 108 L mol-1 at 20 ºC) [51], which resulted in the decomplexation of the PyHC-Mg2+ complex. Reversibility of the fluorescence signal is an important feature of magnesium-responsive probes, confirming that the observed increase in fluorescence signal is due to Mg2+ and not the result of an artifact. Fluorescence intensity of PyHC with different concentrations of Mg2+ showed linear range from 0.1–1.7
10 μmol L-1 (Fig. 4). The detection limit (LOD) was calculated as 105 nmol L-1 (R2 = 0.9976). This result indicates that PyHC is one of the most sensitive chemosensors for Mg2+ compared to some recently reported Mg2+ fluoroionophores [9-11, 19].
3.4. ESI mass spectral analysis and 1H NMR titration The electrospray ionization mass spectrum (HRMS-ESI) was obtained for the in situ complex of PyHC with Mg(NO3)2.6H2O in MeOH (Fig. 5). The ion peak at m/z = 318.0786 corresponds to [PyHC–H++Mg2+]+ (Calcd. 318.0729), and the isotopic peak pattern supports the presence of magnesium [52-53]. To investigate the binding mode, a 1H NMR titration was also conducted (Fig. 6). During the titration, the concentration of PyHC was kept constant, and the [Mg2+]/[PyHC] ratio was increased. Upon addition of magnesium to the solution of PyHC, the phenolic Ha and amine Hb signals become broadened with observable chemical shift changes after the coordination with Mg2+. This shifting indicates the involvement of the phenolic hydroxyl group (-OH) and the nitrogen atom of the imine group (-N=C-H) in the binding with Mg2+. Moreover, the protons of water are shifted downfield (from 3.33 to 3.53 ppm) at higher concentration of Mg2+ suggesting the coordination of water molecules with the metal center. 3.5. Proposed sensing mechanism of PyHC towards Mg2+ The sensing mechanism and binding mode of PyHC with Mg2+ were proposed based on the above experimental results (Scheme 2). In ethanol-water solution (99:1 v/v), PyHC exhibited weak fluorescence probably because of the combination of non-radiative processes, including photo-induced electron transfer (PET) from the lone pair electrons of the imine moiety (-C=N-) to the coumarin fluorophore and isomerization of the C=N bond
11 in excited state [19, 22-24]. Upon addition of Mg2+, a fluorescence enhancement was observed as a result of the coordination PyHC-Mg2+, which results in the increase of its structural rigidity and the inhibition of the PET and C=N isomerization processes.
4. CONCLUSIONS In summary, we have developed a highly sensitive “turn-on” fluoroionophore for the recognition of Mg2+. This new chemosensor was synthesized efficiently by a simple Schiff base
condensation
between
2-hydrazinylpyridine
and
8-formyl-7-hydroxy-4-
methylcoumarin. Spectroscopic studies involving absorption, fluorescence, ESI mass and NMR spectra revealed the formation of a 1:1 complex between the chemosensor PyHC and Mg2+, which leads to a fluorescence enhancement. These results also suggest that cation binds to the chemosensor by the 2-(pyridin-2-yl)hydrazono group, as well as by the phenolic hydroxyl group of the coumarin unit. The chemosensor showed good binding ability towards Mg2+ with an association constant of 5.57 x 104 L mol-1, a relatively low interference from Ca2+ and a low detection limit of 105 nmol L-1 (R2 = 0.9976) in ethanolwater solution. ACKNOWLEDGEMENTS We thank the Department of Chemistry and Vicerrectoría de Investigaciones at Universidad de los Andes for financial support. Our gratitude to COLCIENCIAS for the financial support and for the doctoral scholarship conferred to N. Nuñez-Dallos (Conv. 617). SUPPLEMENTARY DATA
12 Supplementary information available: experimental procedures, spectroscopic data of compounds, and photophysical properties. See http://.
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19
SCHEME AND FIGURE CAPTIONS Scheme 1. Synthesis of the chemosensor PyHC. Fig. 1. (a) Fluorescence spectra of PyHC (10 μmol L-1) in ethanol absolute-water solution (99:1 v/v) in the presence of one equivalent of various metal ions with excitation at λex = 340 nm. (b) Fluorescence intensity response of PyHC towards Mg2+ and other metal ions. The emission intensity was measured at λem = 449 nm. Fig. 2. Job’s plot of PyHC and Mg2+ in ethanol absolute-water solution (99:1 v/v), showing 1:1 stoichiometry. The total concentration was 10 μmol L-1 (λex = 340 nm, λem = 449 nm). Fig. 3. Benesi–Hildebrand plot of PyHC (10 μmol L-1) with Mg2+. Fig. 4. (a) Fluorescence spectra of PyHC (10 μmol L-1) in ethanol absolute-water solution (99:1 v/v) upon addition of increasing concentration of Mg2+ (0.1–1.7 μmol L-1, λex = 340 nm). (b) Fluorescence intensity of PyHC at 449 nm with different concentrations of Mg2+. Fig. 5. HRMS (ESI+) spectrum for the 1:1 complex of PyHC with Mg2+ in MeOH along with isotopic peak pattern observed (left) and calculated (right). Fig. 6. Expansions in 1H NMR spectra of PyHC in DMSO-d6 in the presence of increasing equivalents of Mg2+ as Mg(NO3)2.6H2O. Scheme 2. Proposed sensing mechanism and binding mode of PyHC with Mg2+.
20
Scheme 1
21
Fluorescence Intensity (a.u)
(a) 200 Mg2+ 150 100 Other cations PyHC 50 0 350
400
450 500 Wavelength (nm)
550
600
Fluorescence Intensity (a.u)
(b) 200 150 100 50 0
Figure 1
PyHC Na+ K+ Mg2+ Ca2+ Fe3+ Co2+ Cu2+ Zn2+
22
120
I-I0
80
40
0 0.0
0.2
0.4 2+
0.6 2+
[Mg ]/([Mg ]+[PyHC])
Figure 2
0.8
1.0
23
Figure 3
24
Fluorescence Intensity (a.u)
(a) 50 40 30
Mg2+
20 10 0
400
450
500
550
Wavelength (nm)
Figure 4
600
650
25
Figure 5
26
1.0 eq.
0.5 eq.
Ha 0.0 eq.
Figure 6
Hb H 2O
DMSO-d6
CH3
27
Scheme 2
Graphical Abstract
28 Highlights A fluorescent chemosensor for Mg2+ was synthesized using pyridyl-hydrazonocoumarin moieties. The chemosensor showed high sensitivity towards Mg2+ and the detection limit was as low as 105 nmol L-1. Spectroscopic studies revealed the formation of a 1:1 complex between the chemosensor and Mg2+.
PII: DOI: Reference:
S0039-9140(16)30088-1 http://dx.doi.org/10.1016/j.talanta.2016.02.020 TAL16338
To appear in: Talanta Received date: 16 December 2015 Revised date: 8 February 2016 Accepted date: 9 February 2016 Cite this article as: Jessica Orrego-Hernández, Nelson Nuñez-Dallos and Jaime Portilla, Recognition of Mg2+ by a new fluorescent “turn-on” chemosensor based on pyridyl-hydrazono-coumarin, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.02.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Recognition of Mg2+ by a new fluorescent “turn-on” chemosensor based on pyridyl-hydrazono-coumarin
Jessica Orrego-Hernández, Nelson Nuñez-Dallos, Jaime Portilla*
Bioorganic Compounds Research Group, Department of Chemistry, Universidad de los Andes, Carrera 1 No. 18A 10, Bogotá, Colombia
*E-mail: [email protected]; Tel: +57 1 3394949. Ext. 2080
ABSTRACT A new fluoroionophore PyHC bearing 2-pyridylhydrazone and 7-hydroxycoumarin moieties for selective detection of Mg2+ was synthesized and characterized. This chemosensor exhibited “turn-on” fluorescence behavior and was sensitive to Mg2+ concentrations as low as 105 nmol L-1 in ethanol-water solution. Detailed spectroscopic studies revealed the binding mode of a 1:1 complex between PyHC and Mg2+ that leads to a fluorescence enhancement.
KEYWORDS: Chemosensor, Fluorescence, Mg2+, Pyridyl-hydrazono-coumarin, Schiff base
2
1. INTRODUCTION In biological systems, magnesium ion plays a crucial role as a cofactor in DNA synthesis and protein phosphorylation [1]. In human body, it is essential for many cardiovascular and neurological processes [2–4]. Abnormal concentrations of magnesium in the cytosol and subcellular regions have relation with diseases such as diabetes, hypertension, epilepsy and Alzheimer [3–6]. For these reasons, a simple and efficient analytical techniques for intracellular detection and imaging of magnesium are of great interest [7]. Here, fluorescence-based chemosensors offer a number of advantages such as simple handling, and real-time response allowing dynamic measurements. Currently, considerable efforts are spent searching for new suitable fluorescent probes matching all desired properties such as high sensitivity, selectivity, aqueous solubility, easy preparation, and low-cost synthesis [8–11]. Among the chemosensors reported for Mg2+ detection are β-diketone [12], β-ketoacid [13], crown ether [14], polymer-based ligands [15], and arylimines [16,17]. Also, commercial probes, for example mag-fura-2 and Magnesium Green showing high water solubility and low toxicity [18]. Nevertheless, the principal unresolved challenge for in vivo and in vitro monitoring of magnesium is to obtain sufficient affinity and sensitivity of the chemosensor towards magnesium under intracellular conditions, in particular in the presence of calcium. The intracellular concentration of Mg2+ is of the order 0.1-6 mmol L-1 whereas the concentration of calcium may vary in a much wider range (from 0.1 µmol L-1 to 1 mmol L-1) [8]. Therefore, the goal of the current study is to design a highly selective
3 and sensitive fluorescent chemosensor that can specifically recognize Mg2+ without the interference of other metal ions, especially Ca2+. The proposed basic principle of a “turn-on” fluorescent chemosensor for metal ions is that fluorescence is quenched in the free fluoroionophore as a result of rapid non-radiative processes, including photo-induced electron transfer (PET), intramolecular charge transfer (ICT), excited-state intra or intermolecular proton transfer (ESIPT), and isomerization. The binding of the chemosensor with metal ions interrupts the aforementioned processes and increases its structural rigidity, generating a chelation-enhanced fluorescence (CHEF) effect [19–24]. Nowadays, one of the most widely used strategy for developing tailor-made selective fluorescent chemosensors for sensing of metal ions include the synthesis of Schiff bases and coumarin derivatives [25–27]. Schiff base ligands have the C=N donor system that can coordinate with various metal ions and form stable complexes. In addition, Schiff bases are easy and inexpensive to prepare [28–30]. Coumarins as fluorophores offer many advantages, including high fluorescence quantum yield, large Stokes shift, excellent light stability, and low toxicity [31–36]. Herein, we report the synthesis of the Schiff base 7-hydroxy-4-methyl-8-((2-(pyridin-2yl)hydrazono)methyl)-2H-chromen-2-one (PyHC) (Scheme 1). This new compound acts as a “turn-on” fluorescent chemosensor for the selective detection of Mg2+ with a high sensitivity in ethanol-water solution. To the best of our knowledge, PyHC is one of the most sensitive chemosensors for Mg2+ recognition. We have developed PyHC as an integrated chemosensor with the 7-hydroxy-4methylcoumarin unit as an emitting fluorophore, which has suitable photophysical properties [31-36]. The design also includes that the cation binds by the 2-(pyridin-2-
4 yl)hydrazono group, as well as by the phenolic hydroxyl group of the fluorophore unit (Scheme 1). In addition, the hidrazono group has the C=N donor system that can quench the fluorescence of the fluorophore by PET process and C=N isomerization [19-24]. Thus, the coordination of PyHC to Mg2+ probably disrupts these processes and increases its structural rigidity producing a fluorescence enhancement. The binding mode of the PyHCMg2+ complex was studied by spectroscopic methods involving absorption, fluorescence, ESI mass, and NMR spectra. 2. EXPERIMENTAL 2.1. General information All reagents and solvents were purchased from commercial sources and used as received. All solvents used in this work were analytic grade. The reactions were monitored by TLC (DCM:MeOH 10:1) and were visualized by UV lamp (254 nm or 365 nm). NMR data were recorded on a Bruker Avance 400 (400.13 MHz for 1H; 100.61 MHz for 13C). 1H and 13C NMR chemical shifts are reported in parts per million (ppm) relative to TMS, with the residual solvent peak used as an internal reference. High resolution mass spectra (HRMS) were obtained on an Agilent Technologies Q-TOF 6520 spectrometer via an electrospray ionization (ESI). Infrared spectra (FT-IR) were recorded on a Thermo Nicolet Nexus 470 ESP FT-IR Spectrometer. Thermogravimetric analysis (TG) was performed on a NETZSCH STA 409 PC/PG instrument in a nitrogen atmosphere with a continuous flow of 60 mL/min and a heating rate of 10 K/min, from 30°C to 600°C. Melting points were determined in capillary tubes on a Stuart SMP10 melting point apparatus and are uncorrected. pH measurements were made with a Fisher Scientific accumet research AR50 pH meter at 298 K. 2.2. Synthesis and characterization
5 The chemosensor PyHC was synthesized according to the Scheme 1. The synthetic procedures of 2-hydrazinylpyridine and 8-formyl-7-hydroxy-4-methylcoumarin were adapted and slightly modified from literature [37–40]. Detailed synthetic route and characterizations of products are shown in the Supplementary data. Synthesis of 7-hydroxy-4-methyl-8-((2-(pyridin-2-yl)hydrazono)methyl)-2H-chromen2-one (PyHC). A solution of 2-hydrazinylpyridine (53.3 mg, 0.49 mmol) in EtOH (5 mL) was added dropwise to a solution of 8-formyl-7-hydroxy-4-methylcoumarin (100.8 mg, 0.49 mmol) in hot ethanol (4 mL), then the reaction mixture was refluxed for 3 h. The compound PyHC precipitated out as yellow solid. The precipitate was isolated by vacuum filtration, washed with cold ethanol, and dried under vacuum (0,132g, 92 %). mp 290-291 °C with decomposition (from ethanol). Rf = 0.43 (DCM:MeOH 10:1). IR νmax/cm-1 (KBr): 3438 (OH), 3204 (NH), 1738 (C=N), 1632 (C=O ester), 1597 (C-O ester). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.38 (s, 3H), 6.20 (s, 1H), 6.83 (d, J = 7.2 Hz, 2H), 6.92 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.69 (t, J = 7.1 Hz, 1H), 8.18 (s, 1H), 8.67 (s, 1H), 11.29 (s, 1H), 12.33 (s, 1H).
13
C NMR (100 MHz, DMSO-d6): δ (ppm) 18.3 (CH3), 106.3
(CH), 106.5 (C), 110.6 (CH), 111.9 (C), 113.1 (CH), 115.8 (CH), 126.3 (CH), 135.4 (CH), 138.2 (CH), 148.3 (CH), 151.5 (C), 153.8 (C), 154.9 (C), 159.4 (C), 159.9 (C). HRMS m/z (ESI) calculated for [C16H13N3O3+H]+: 296.1030; found 296.1031 [M+H]+. 2.3. UV-vis absorption and fluorescence studies The electronic absorption spectra were measured on Varian Cary 100 Conc (Agilent Technologies) spectrophotometer in a quartz cuvette having a path length of 1 cm. The fluorescence emission spectra were recorded by using a CARY Eclipse (Agilent Technologies) fluorescence spectrophotometer in a quartz cell (1 cm path length). UV-vis
6 and fluorescence measurements were performed at room temperature (20 ºC). For fluorescence measurements, both the excitation and emission slit widths were 5 nm. The 0.2 mmol L-1 stock solutions of the chemosensor PyHC were prepared in dichloromethane, acetonitrile, dimethyl sulfoxide and absolute ethanol. The salts used in stock solutions of metal ions were NaCl, KNO3, Mg(NO3)2.6H2O, CaCl2, Ba(NO3)2, Cr(NO3)3.9H2O, Fe(NO3)3.9H2O, Co(NO3)2.6H2O, Ni(NO3)2.6H2O, Cu(NO3)2.2.5H2O, Zn(NO3)2.6H2O, CdCl2, Hg(NO3)2.H2O, Al(NO3)3.9H2O, and Pb(NO3)2. Inorganic salts were dissolved in distilled water to afford 1 mmol L-1 aqueous solution. Aliquots of stock solution of PyHC was diluted to 5 mL to make the final concentration of 10 μmol L-1. In the selectivity and competition experiments of PyHC towards Mg2+ and other metal ions, the fluorescence emission spectra were recorded at λex = 340 nm from 10 μmol L-1 of the chemosensor in a 99:1 (v/v) ethanol-water solution (pH 7.14) and in the presence of 1.0 equiv. of various metal ions. The sensing studies were performed at pH 7.14 because it is close to the physiological pH (about 7-7.4). The fluorescence intensities were measured at λem = 449 nm. For the Job’s plot experiment of PyHC and Mg2+ the total concentration of PyHC and Mg2+ were kept as 10 μmol L-1. Fluorescence response in pictures was excitation at 365 nm using a UV lamp. 2.4. Determination of the relative quantum yields The relative quantum yields were obtained by using anthracene (φF = 0.28 in ethanol at 340 nm) as reference and calculated according to the following equation [41–46].
where x and st indicate the sample and standard solution, respectively, φ is the quantum yield, F is the integrated area of the emission, A is the absorbance at the excitation wavelength, L is the length of the absorption cell, and η is the index of refraction of the solvents. 2.5. Determination of the binding constant
7 The binding constant Ka was obtained according to the Benesi-Hildebrand equation for a 1:1 complex [31, 47-48].
wherein I0 is the fluorescence intensity of the free ligand, I are the observed fluorescence intensities upon addition of different equivalents of Mg2+, Ic is the fluorescence intensity of the ligand-metal complex, and [Mg2+] shows the concentration of magnesium ions. The binding constant is given by the ratio intercept/slope from the plot 1/(I-I0) versus 1/[Mg2+]. 2.6. Determination of the detection limit The limit of detection (LOD) of PyHC for Mg2+ was obtained by 3Sb/k, where Sb is the standard deviation of the blank measurements (by 10 times, Sb = 0.8527), and k is the slope from the plot fluorescence intensity I versus [Mg2+] [49,50]. (Fig. 4b, k = 2.43 x 107 L mol1
).
3. RESULTS AND DISCUSSION 3.1. Synthesis of chemosensor PyHC The chemosensor PyHC was synthesized with a high yield (92 %) from the condensation
reaction
between
8-formyl-7-hydroxy-4-methylcoumarin
and
2-
hydrazinylpyridine in absolute ethanol at reflux. The molecular structure of PyHC was verified unambiguously by FT-IR, 1H and
13
C NMR spectroscopy, and high resolution
mass spectra (HRMS) through electrospray ionization (ESI) (Fig. S6-S11). The thermogravimetric analysis (TG) showed that the chemosensor PyHC exhibits high thermal stability. This compound was found to be stable up to 293 °C (Fig. S12). 3.2. Photophysical properties of PyHC
8 The absorption and emission spectra of PyHC were investigated in dichloromethane (DCM), acetonitrile (ACN), dimethyl sulfoxide (DMSO) and absolute ethanol (EtOH) (Fig. S13-14 and Table S1). In the absorption spectra of PyHC, the absorption band with maximum between 325 and 345 nm is assigned to a π-π* transition. The fluorescence spectra displayed a maximum emission band in the range 450-460 nm. The chemosensor PyHC showed the highest relative quantum yield in DMSO (φF = 0.042), while it presented weaker fluorescence in EtOH, ACN and DCM (φF of 0.0021, 0.0036 and 0.0004, respectively). Ethanol was selected as solvent in further studies due to the fact that in the design of a “turn-on” fluorescent chemosensor for metal ions is important to consider that the free ligand exhibits weak fluorescence in the solvent of study. Furthermore, it was taken into account that ethanol is a green solvent, highly miscibility in water, and easy handling. 3.3. Fluorescence and UV-vis response of PyHC to Mg2+ Fluorescence spectra of PyHC (10 μmol L-1) were recorded at λex of 340 nm in ethanol -water solution (99:1 v/v) (pH 7.14) to observe its fluorescence intensity response towards Mg2+ and other metal ions (Na+, K+, Ca2+, Ba2+, Cr3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Al3+, and Pb2+) dissolved in distilled water (Fig. 1 and Fig. S15-16). An enhancement of the fluorescence quantum yield up to 17-fold was observed in presence of Mg2+ (φF = 0.0360) without a significant alteration of the maximum emission band (λem = 449 nm), while it was observed weak fluorescence with the other metal ions. These results suggest that PyHC has high selectivity for Mg2+. Furthermore, this chemosensor showed only a small enhancement of the fluorescence intensity in the presence of Ca2+ cations. The absorption spectra of PyHC with one equivalent of Mg2+ showed a new band at 390 nm, which indicates the coordination interaction between PyHC and Mg2+ (Fig. S17-18). This complexation produces a chelation-enhanced fluorescence effect.
9 In order to analyze in more detail the selectivity of PyHC, fluorescence spectra of one equivalent the chemosensor were measured with one equivalent of Mg2+ and in the presence of one equivalent of other metal ions (Fig. S19-20). In these competition experiments, it was found that alkali and alkaline earth metal ions had relatively low interference levels on the selectivity for Mg2+, while the transition metal ions quenched the fluorescence of the PyHC-Mg2+ complex. Thus, PyHC could be used as a fluorescent probe for the selective detection of Mg2+ among biologically abundant ions like Na+, K+, and Ca2+. The transition metal ions had strong interference on the selectivity of PyHC towards Mg2+ as a result of their strong coordination character, which was confirmed by the changes in the intensity and bathochromic shifts of the absorption bands of PyHC, as well as the appearance of new bands in the UV-vis spectra (Fig. S17 and S21). Job’s plot analysis of the fluorescence spectra showed a maximum at 0.5 mole fraction of Mg2+, indicating the formation of a 1:1 complex between PyHC and Mg2+ (Fig. 2). Based on a 1:1 stoichiometry, the association constant of PyHC with Mg2+ was calculated to be 5.57 x 104 L mol-1 at 20 ºC (R2 = 0.9975) from the Benesi–Hildebrand plot [31, 4748], which demonstrates the good binding ability of the chemosensor towards Mg2+ (Fig. 3). The chemosensor was found to bind Mg2+ reversibly as tested by reacting with EDTA. The addition of EDTA to a solution of PyHC-Mg2+ complex resulted in quenched of the fluorescence intensity (Fig. S22-23). The fluorescence quenching is a result of the high affinity of EDTA towards Mg2+ (Ka = 6.61 x 108 L mol-1 at 20 ºC) [51], which resulted in the decomplexation of the PyHC-Mg2+ complex. Reversibility of the fluorescence signal is an important feature of magnesium-responsive probes, confirming that the observed increase in fluorescence signal is due to Mg2+ and not the result of an artifact. Fluorescence intensity of PyHC with different concentrations of Mg2+ showed linear range from 0.1–1.7
10 μmol L-1 (Fig. 4). The detection limit (LOD) was calculated as 105 nmol L-1 (R2 = 0.9976). This result indicates that PyHC is one of the most sensitive chemosensors for Mg2+ compared to some recently reported Mg2+ fluoroionophores [9-11, 19].
3.4. ESI mass spectral analysis and 1H NMR titration The electrospray ionization mass spectrum (HRMS-ESI) was obtained for the in situ complex of PyHC with Mg(NO3)2.6H2O in MeOH (Fig. 5). The ion peak at m/z = 318.0786 corresponds to [PyHC–H++Mg2+]+ (Calcd. 318.0729), and the isotopic peak pattern supports the presence of magnesium [52-53]. To investigate the binding mode, a 1H NMR titration was also conducted (Fig. 6). During the titration, the concentration of PyHC was kept constant, and the [Mg2+]/[PyHC] ratio was increased. Upon addition of magnesium to the solution of PyHC, the phenolic Ha and amine Hb signals become broadened with observable chemical shift changes after the coordination with Mg2+. This shifting indicates the involvement of the phenolic hydroxyl group (-OH) and the nitrogen atom of the imine group (-N=C-H) in the binding with Mg2+. Moreover, the protons of water are shifted downfield (from 3.33 to 3.53 ppm) at higher concentration of Mg2+ suggesting the coordination of water molecules with the metal center. 3.5. Proposed sensing mechanism of PyHC towards Mg2+ The sensing mechanism and binding mode of PyHC with Mg2+ were proposed based on the above experimental results (Scheme 2). In ethanol-water solution (99:1 v/v), PyHC exhibited weak fluorescence probably because of the combination of non-radiative processes, including photo-induced electron transfer (PET) from the lone pair electrons of the imine moiety (-C=N-) to the coumarin fluorophore and isomerization of the C=N bond
11 in excited state [19, 22-24]. Upon addition of Mg2+, a fluorescence enhancement was observed as a result of the coordination PyHC-Mg2+, which results in the increase of its structural rigidity and the inhibition of the PET and C=N isomerization processes.
4. CONCLUSIONS In summary, we have developed a highly sensitive “turn-on” fluoroionophore for the recognition of Mg2+. This new chemosensor was synthesized efficiently by a simple Schiff base
condensation
between
2-hydrazinylpyridine
and
8-formyl-7-hydroxy-4-
methylcoumarin. Spectroscopic studies involving absorption, fluorescence, ESI mass and NMR spectra revealed the formation of a 1:1 complex between the chemosensor PyHC and Mg2+, which leads to a fluorescence enhancement. These results also suggest that cation binds to the chemosensor by the 2-(pyridin-2-yl)hydrazono group, as well as by the phenolic hydroxyl group of the coumarin unit. The chemosensor showed good binding ability towards Mg2+ with an association constant of 5.57 x 104 L mol-1, a relatively low interference from Ca2+ and a low detection limit of 105 nmol L-1 (R2 = 0.9976) in ethanolwater solution. ACKNOWLEDGEMENTS We thank the Department of Chemistry and Vicerrectoría de Investigaciones at Universidad de los Andes for financial support. Our gratitude to COLCIENCIAS for the financial support and for the doctoral scholarship conferred to N. Nuñez-Dallos (Conv. 617). SUPPLEMENTARY DATA
12 Supplementary information available: experimental procedures, spectroscopic data of compounds, and photophysical properties. See http://.
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19
SCHEME AND FIGURE CAPTIONS Scheme 1. Synthesis of the chemosensor PyHC. Fig. 1. (a) Fluorescence spectra of PyHC (10 μmol L-1) in ethanol absolute-water solution (99:1 v/v) in the presence of one equivalent of various metal ions with excitation at λex = 340 nm. (b) Fluorescence intensity response of PyHC towards Mg2+ and other metal ions. The emission intensity was measured at λem = 449 nm. Fig. 2. Job’s plot of PyHC and Mg2+ in ethanol absolute-water solution (99:1 v/v), showing 1:1 stoichiometry. The total concentration was 10 μmol L-1 (λex = 340 nm, λem = 449 nm). Fig. 3. Benesi–Hildebrand plot of PyHC (10 μmol L-1) with Mg2+. Fig. 4. (a) Fluorescence spectra of PyHC (10 μmol L-1) in ethanol absolute-water solution (99:1 v/v) upon addition of increasing concentration of Mg2+ (0.1–1.7 μmol L-1, λex = 340 nm). (b) Fluorescence intensity of PyHC at 449 nm with different concentrations of Mg2+. Fig. 5. HRMS (ESI+) spectrum for the 1:1 complex of PyHC with Mg2+ in MeOH along with isotopic peak pattern observed (left) and calculated (right). Fig. 6. Expansions in 1H NMR spectra of PyHC in DMSO-d6 in the presence of increasing equivalents of Mg2+ as Mg(NO3)2.6H2O. Scheme 2. Proposed sensing mechanism and binding mode of PyHC with Mg2+.
20
Scheme 1
21
Fluorescence Intensity (a.u)
(a) 200 Mg2+ 150 100 Other cations PyHC 50 0 350
400
450 500 Wavelength (nm)
550
600
Fluorescence Intensity (a.u)
(b) 200 150 100 50 0
Figure 1
PyHC Na+ K+ Mg2+ Ca2+ Fe3+ Co2+ Cu2+ Zn2+
22
120
I-I0
80
40
0 0.0
0.2
0.4 2+
0.6 2+
[Mg ]/([Mg ]+[PyHC])
Figure 2
0.8
1.0
23
Figure 3
24
Fluorescence Intensity (a.u)
(a) 50 40 30
Mg2+
20 10 0
400
450
500
550
Wavelength (nm)
Figure 4
600
650
25
Figure 5
26
1.0 eq.
0.5 eq.
Ha 0.0 eq.
Figure 6
Hb H 2O
DMSO-d6
CH3
27
Scheme 2
Graphical Abstract
28 Highlights A fluorescent chemosensor for Mg2+ was synthesized using pyridyl-hydrazonocoumarin moieties. The chemosensor showed high sensitivity towards Mg2+ and the detection limit was as low as 105 nmol L-1. Spectroscopic studies revealed the formation of a 1:1 complex between the chemosensor and Mg2+.