Antipyrine Based Arsenate Selective Fluorescent Probe for Living Cell

Article pubs.acs.org/ac

Antipyrine Based Arsenate Selective Fluorescent Probe for Living Cell Imaging Sisir Lohar,† Animesh Sahana,† Arnab Banerjee,† Avishek Banik,‡ Subhra Kanti Mukhopadhyay,‡ Jesús Sanmartín Matalobos,*,§ and Debasis Das*,† †

Department of Chemistry, The University of Burdwan, Burdwan, West Bengal, India Department of Microbiology, The University of Burdwan, Burdwan, West Bengal, India § Departamento de Química Inorgánica, Facultade de Química, Universidad de Santiago de Compostela, Avda. Das Ciencias s/n, 15782, Santiago de Compostela, Spain ‡

S Supporting Information *

ABSTRACT: Condensation of salicylaldehyde and 4-aminoantipyrine has yielded a new fluorescent probe (APSAL) capable of detecting intracellular arsenate at the micromolar level for the first time. The structure of the probe has been established by different spectroscopic techniques and confirmed from X-ray crystallography. Common anions, viz., F−, Cl−, Br−, I−, N3−, NCO−, NO2−, NO3−, SCN−, CN−, CH3COO−, SO42‑, ClO4−, and HPO42‑ do not interfere. The binding constant of APSAL for H2AsO4− has been determined using the Benesi− Hildebrand equation as 8.9 × 103 M−1. Fluorescence quantum yield of APSAL (0.016) increases more than 12 times upon binding arsenate ion.

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lower value of maximum contaminant level (MCL) for As has stimulated us to develop new methods for monitoring As. Current methodologies for As detection either generate or use toxic chemicals or require sophisticated equipment and a long analysis time.7 Fluorescence detection may offer a promising approach for fast and simple tracking of As species for environmental monitoring. Indeed, fluorescence sensors have widely been used for the determination of cations, anions, amino acids, and many more.8−24 However, H2AsO4− selective fluorescent sensors have not been reported yet. Although Harrop and co-workers have recently reported one fluorescence methodology for As(III) detection but the arsenic species they detected does not exist in environmental samples.25 Herein, we report for the first time an H2AsO4− selective fluorescent sensor, (4E)-4-(2-hydroxybenzylideneamino)-1,2-dihydro-2,3dimethyl-1-phenylpyrazol-5-one (APSAL). APSAL has been synthesized by condensation of salicylaldehyde with 4-aminoantipyrine. It has unique selectivity for H2AsO4− over other common anions, viz., F−, Cl−, Br−, I−, N3−, NCO−, NO2−, NO3−, SCN−, CN−, CH3COO−, SO42‑, ClO4−, and HPO42‑. The probe has successfully been used for intracellular H2AsO4− detection in contaminated living cells.

rsenic (As), a metalloid, occurs as the 20th most abundant element in earth’s crust and as a component of more than 245 minerals. U.S. EPA1 and International Association for Research on Cancer2 have classified As as a Group A and Category 1 human carcinogen, respectively. Globally, agriculture, the single largest user of freshwater, is a major cause of degradation of surface and groundwater through erosion and chemical runoff. Pyrite oxidation and oxyhydroxide reduction are two main theories behind the release of arsenic into groundwater. Arsenic can exist both as inorganic and organic forms in soils, plants, animals, and humans. Contaminated groundwater is the main source of inorganic arsenic exposure to the human population. Inorganic arsenic, viz., arsenite and arsenate, is being considered carcinogenic3 and more toxic than monomethylarsonic acid (MMA) and dimethylarsonic acid (DMA). Other organic forms, viz., arsenobetaine (AsBet), arsenocholine (AsCol), and arsenosugars, are considered either virtually or completely nontoxic. Arsenite and arsenate have resemblance with phosphite and phosphate ions and probably is the cause of their toxicity. Arsenite and arsenate hinder the conversion of ATP to ADP by permanently replacing the phosphate groups.4 Arsenic poisoning may occur due to its uptake at higher doses or chronic contact in occupational or environmental exposure or accidental intoxication and is associated with cancer, gastrointestinal, respiratory diseases, and neurotoxic symptoms.5 The World Health Organization (WHO) has recommended the upper limit of arsenic in drinking water as 0.01 ppm.6 This © 2013 American Chemical Society

Received: October 27, 2012 Accepted: January 8, 2013 Published: January 8, 2013 1778

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EXPERIMENTAL SECTION Materials and Methods. Salicylaldehyde and 4-aminoantipyrine have been purchased from Sigma Aldrich and used as received. All other chemicals and solvents are of analytical grade and used without further purification. Water having a resistivity of 18.2 MΩ cm was obtained from a Milli-Q Millipore water purification system (Bedford, MA) and used throughout all the experiments. Arsenate (Caution!) stock solution has been prepared using NaH2AsO4. The sources of NO3−, NO2−, SCN−, NCO−, CN−, SO42‑, HPO42‑, CH3COO−, ClO4−, F−, Cl−, Br−, I−, and N3− ions are either their sodium, potassium, or ammonium salts. Absorption and fluorescence spectra have been recorded on a Shimadzu Multi Spec 1501 absorption spectrophotometer and Hitachi 4500 fluorescence spectrometer, respectively. Mass spectra have been recorded on a QTOF Micro YA 263 mass spectrometer in the ESI positive mode. IR spectra are recorded on a PerkinElmer FT-IR spectrophotometer (model RX-1). The fluorescence imaging system is comprised of an inverted fluorescence microscope (Leica DM 1000 LED), digital compact camera (Leica DFC 420C), and an image processor (Leica Application Suite v3.3.0). The microscope is equipped with a 50 W mercury arc lamp. Diffraction data for APSAL are collected at 100 K using graphite-monochromated Mo−Kα radiation (λ = 0.7107 Å) from a fine focus sealed tube. Some significant crystal parameters and refinement data are summarized in Table 1.

of six programs known as per name of the creator, Sheldrick, SHELXL97.26−28 Preparation and Imaging of Cells. Candida albicans cells (IMTECH no. 3018) are grown in yeast extract glucose broth medium (pH 6.0, incubation temperature 37 °C, 24 h) are centrifuged at 3000 rpm for 3 min and then washed with normal saline. Then cells are treated with an aqueous solution of H2AsO4− (1 mg mL−1) for 45 min. After incubation, the cells are washed again with normal saline and observed under a fluorescence microscope equipped with a UV filter after adding APSAL. Cells incubated only with H2AsO4− or APSAL have been used as a control. Similarly, fluorescence images of freshly collected pollen grains of Allamandapuberula (Aapocynaceae) are also taken along with its respective control.

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RESULTS AND DISCUSSION Synthesis of (4E)-4-(2-Hydroxybenzylideneamino)1,2-dihydro-2,3-dimethyl-1-phenylpyrazol-5-one Scheme 1

Table 1. Crystal Parameters of APSAL identification code empirical formula formula weight temperature wavelength crystal system space group unit cell dimensions

volume Z density (calculated) absorption coefficient F(000) crystal size Θ range for data collection index ranges

reflections collected independent reflections absorption correction refinement method largest diff. peak and hole

APSAL C18H17N3O2 307.35 100 K 0.7107 Å monoclinic P21/n a = 7.4101(5) Å b = 7.4586(4) Å c = 27.2469(19) Å β = 95.595(4)0 1498.73 (17) Å3 4 1.362 Mg/m3 0.09 mm−1 648 0.44 × 0.26 × 0.23 mm3 2.8−22.7° h = −9−9 k = 0−9 l = 0−33 45 064 2939, [R(int) = 0.057] colorless full-matrix least-squares on F2 0.25 and −0.26 e Å−3

Figure 1. X-ray crystal structure of APSAL.

Figure 2. Effect of pH on the emission intensity of APSAL (5 μM) and APSAL + H2AsO4− (1:5 molar ratio).

Data are processed and corrected for Lorentz and polarization effects. Multiscan absorption corrections have been performed using the Siemens Area Detector Absorption Correction (SADABS) program. Structure is solved by standard direct methods using Semi-Invariants Representation (SIR 2004) and then refined by full matrix least-squares on F2 using a package

(APSAL) (Scheme 1). Scheme 1 shows the synthesis of APSAL. 4-Aminoantipyrine (250 mg, 1.23 mmol) is dissolved in 20 mL of dry methanol. To this solution, 15 mL of methanol solution of salicylaldehyde (150 mg, 1.23 mmol) is added and 1779

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Figure 3. Relative emission intensity of APSAL (10 μM) in the presence of different anions (500 μM) in HEPES buffer (0.1 M, methanol/water = 1/4, v/v, pH 7.4): λex = 350 nm, λem = 498 nm.

Figure 6. Relative emission intensities of [APSAL−H2AsO4−] adduct in the presence of various anions in HEPES buffered (0.1 M) solution (methanol/water = 1/4, v/v, pH 7.4); black bar, APSAL (10.0 μM); red bar, APSAL (10.0 μM) with 50 equiv of H2AsO4−; blue bar, 10.0 μM of APSAL + 50 equiv of H2AsO4− + 100 equiv of anions stated, F− (2), Cl− (3), Br− (4), N3−(5), NCO− (6), NO2− (7), NO3− (8), SCN− (9), CN− (10), CH3COO− (11), SO42‑ (12), ClO4− (13), HPO42‑ (14), I− (15), λex = 350 nm, λem = 498 nm.

of CIF file and CCDC no. are found in the Supporting Information).Yield, 92%; mp, 139 ± 1 °C. The ESI-TOF (+) mass spectral peaks at m/z 308.11 and 330.09 are assigned to [APSAL + H]+ and [APSAL + Na]+, respectively (Figure S1 in the Supporting Information). 1H NMR (CDCl3, 500 MHz, Figure S2 in the Supporting Information): δ, ppm: 2.41 (3H, s), 3.17 (3H, s), 6.9 (1H, t, J = 7), 6.9 (1H, d, J = 8), 7.2 (1H, m, J = 1.5), 7.3 (2H, t, J = 7.5), 7.3 (2H, d, J = 7.5), 7.5 (2H, t, J = 8), 9.8 (1H, s) and 13.3 (1H, s). FT-IR (KBr disc, Figure S3 in the Supporting Information) ν (cm−1): (OH); 1594 (C N), 1645 (CO). Elemental analysis data as calculated for C18H17N3O2(%): C, 70.34 ; H, 5.58; N, 13.67. Found (%): C, 69.65; H, 5.88; N, 13.5. Single Crystal X-ray Structure Analysis of APSAL. Single crystal X-ray diffraction studies on a colorless prismatic crystal obtained after slow evaporation of an ethanol solution of APSAL consist of crystallographically independent molecules that comprise one antipyrene unit connected by a −CN− group to an o-hydroxy benzene unit. Table S1 (Supporting Information) shows the bond distances and angles for free APSAL that fall within the expected ranges. APSAL crystallizes with configuration E and possesses a unique binding site ON. The planar conformation is stabilized through both an extended conjugated system and an intramolecular H bond O1−H···N1 (Figure 1). Fluorescence Studies. Performance of a fluorescence probe bearing proton sensitive donor sites strongly depends on the pH of the medium, and hence optimization of pH on the efficiency of the sensor is essential. APSAL shows very weak emission intensity at the pH range 2.0−11.0 in methanol/water = 1/4, v/v. However, in the presence of H2AsO4−, the emission intensity increases gradually with increasing pH (Figure 2). So, we have chosen pH 7.4 to perform all the experiments as it is close to the physiological pH. However, on the basis of the pKa values of H3AsO4, viz. pKa1 = 2.19, pKa2 = 6.94 and pKa3 = 11.5, it is evident that both

Figure 4. Fluorescence spectral changes of APSAL (10 μM) in HEPES buffered (0.1 M) solution (methanol/water = 1:4, v/v, pH 7.4) upon gradual addition of H2AsO4− ([H2AsO4−] = 0−500 μM, λex = 350 nm, λem = 498 nm).

Figure 5. Determination of binding constant of APSAL with H2AsO4−.

refluxed for 6 h. Slow evaporation of the solvent has yielded a colorless crystal of APSAL. The crystal structure of APSAL is presented in Figure 1 (Crystallographic information in the form 1780

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Figure 7. (a) Fluorescence image of Candida albicanscells treated only with H2AsO4−; (b) pollen grains of Allamandapuberula (Aapocynaceae) treated only with H2AsO4−; (c) Candida albicanscells treated with both H2AsO4−and APSAL; and (d) pollen grains of Allamandapuberula (Aapocynaceae) treated with H2AsO4− and APSAL.

forms viz. HAsO42‑ and H2AsO4− exists in significant percentage (74% and 26%, respectively) at pH 7.4. Figure 3 shows the fluorescence spectra of APSAL in the presence of different anions in HEPES buffered (0.1 M) solution (methanol/water =1/4, v/v, pH 7.4). Emission intensity of APSAL has increased significantly in the presence of H2AsO4−, while other common anions either play no role or quench its emission intensity. APSAL, when excited at 350 nm emits weakly at 498 nm (quantum yield, 0.016) along with another weak emission band at 396 nm. Figure 4 shows the gradual increase of emission intensity of APSAL at 498 nm upon concominant addition of H2AsO4−, while the fluorescence quantum yield increases to 0.196. The binding constant of APSAL for H2AsO4− has been determined using the Benesi−Hildebrand equation29 (Figure 5) and found as 8.9 × 103 M−1. Furthermore, APSAL can detect as low as 3 × 10−6 M H2AsO4− (Figure S4 in the Supporting Information) in HEPES buffered (0.1 M) solution (methanol/water = 1/4, v/v, pH 7.4). UV−Visible Studies. The absorption spectrum (Figure S5 in the Supporting Information) of APSAL in HEPES buffered (0.1 M) solution (methanol/water = 1:4, v/v, pH 7.4) shows a peak at 346 nm along with two shoulders at 316 and 300 nm. Upon gradual addition of H2AsO4− (0−500 μM), the absorbance at 346 nm increases sharply along with a small increase of the absorbance of two other shoulders. Selectivity. The selectivity of APSAL for H2AsO4− over other common anions is examined in HEPES buffered (0.1 M)

solution (methanol/water = 1/4, v/v, pH 7.4). Figure 6 shows the interference of common anions on the emission intensity of the [APSAL−H2AsO4−] system. An increase in fluorescence intensity of the [APSAL−H2AsO4−] system upon addition of a foreign ion is designated as positive interference, whereas the reverse phenomenon is termed as negative interference. No significant interference from common anions is observed. It is noteworthy that at pH 7.4, phosphate exists both as HPO42‑ and H2PO4− in significant percentage (61% and 39%, respectively). Therefore, the sensor is selective against both of them. The mass spectrum of the adduct between APSAL and H2AsO4− (Figure S6 in the Supporting Information) shows 1:1 stoichiometry. In free APSAL, phenolic −OH and carbonyl oxygen are anti to each other along with a hydrogen bond between −OH and imine nitrogen. In the presence of H2AsO4−, strong a H-bond forms between APSAL and H2AsO4−, resulting in significant enhancement of fluorescence intensity. Cell Imaging Studies. Figure 7 indicates that APSAL can easily permeate to the living cells tested and is harmless (cells remain alive for a considerable time after exposure to 10 μM APSAL). APSAL can efficiently detect intracellular H2AsO4− ion in living cells. Density Functional Theoretical (DFT) Studies. Molecular level interactions between APSAL and NaH2AsO4 have been studied using density functional theory (DFT) with the B3LYP/6-311G basis set.30 Figure 8 shows that the charge density at the highest occupied molecular orbital (HOMO) of 1781

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Figure 8. HOMO−LUMO energy gap of APSAL and its NaH2AsO4 adduct.

APSAL, dimethyl units of the antipyrene part do have some charge density in addition to that observed for HOMO. The energy gap between HOMO and LUMO of APSAL is 3.3764 eV. In the case of the [APSAL (NaH2AsO4) (H2O)] adduct, most of the charge density of the HOMO resides on the pyrazole-salicylaldehyde part along with a small charge density on one oxygen of NaH2AsO4. However, in LUMO, it is on the salicylaldehyde-pyrazole part. Here, the HOMO−LUMO energy gap is 3.2379 eV. Thus H-bonding interaction between APSAL and NaH2AsO4 (Figure 9) is nicely supported by DFT studies. In the optimized structure of the adduct (Figure 9), the H(39)−O(42) and H(45)−O(4) distances are 1.586 94 Å and 1.523 02 Å, respectively, within the range of the H-bonding distance. One water molecule in the adduct is also H-bonded as the bond distances, viz., H(49)−O(43) and H(48)−O(41) are 2.309 87 Å and 1.841 20 Å, respectively.

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CONCLUSION Chronic arsenic toxicity (arsenicosis) due to drinking of arsenic contaminated groundwater is a major environmental health hazard throughout the world. Therefore, detection of arsenic species is very important. Herein we report a fluorescence probe which has a unique selectivity for AsO43‑ over other common anions, viz., F−, Cl−, Br−, I−, N3−, NCO−, NO2−, NO3−, SCN−, CN−, CH3COO−, SO42‑, ClO4−, and HPO42‑. The probe has been successfully used for intracellular arsenate

Figure 9. Stereoscopic view of the optimized structure of H-bonded [APSAL−NaH2AsO4] adduct.

free APSAL resides on the salicylaldehyde unit, with no charge density on the methyl group and a very little charge density on the carbonyl oxygen of the antipyrene unit. On the other hand, in the lowest unoccupied molecular orbital (LUMO) of 1782

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(17) Banerjee, A.; Sahana, A.; Guha, S.; Lohar, S.; Hauli, I.; Mukhopadhyay, S. K.; Matalobos, J. S.; Das, D. Inorg. Chem. 2012, 51, 5699. (18) Banerjee, A.; Sahana, A.; Das, S.; Lohar, S.; Guha, S.; Sarkar, B.; Mukhopadhyay, S. K.; Mukherjee, A. K.; Das, D. Analyst 2012, 137, 2166. (19) Banerjee, A.; Karak, D.; Sahana, A.; Guha, S.; Lohar, S.; Das, D. J. Hazard. Mater. 2011, 186, 738. (20) Sahana, A.; Banerjee, A.; Das, S.; Lohar, S.; Karak, D.; Sarkar, B.; Mukhopadhyay, S. K.; Mukherjee, A. K.; Das, D. Org. Biomol. Chem. 2011, 9, 5523. (21) Das, S.; Guha, S.; Banerjee, A.; Lohar, S.; Sahana, A.; Das, D. Org. Biomol. Chem. 2011, 9, 7097. (22) Guha, S.; Lohar, S.; Banerjee, A.; Sahana, A.; Chaterjee, A.; Mukherjee, S. K.; Matalobos, J. S.; Das, D. Talanta 2012, 91, 18. (23) Sahana, A.; Banerjee, A.; Lohar, S.; Guha, S.; Das, S.; Mukhopadhyay, S. K.; Das, D. Analyst 2012, 137, 3910. (24) Karak, D.; Lohar, S.; Banerjee, A.; Sahana, A.; Hauli, I.; Mukhopadhyay, S. K.; Matalobos, J. S.; Das, D. RSC Advances 2012, 2, 12447. (25) Ezeh, V. C.; Harrop, T. C. Inorg. Chem. 2012, 51, 1213. (26) (a) SADABS-Bruker AXS area detector scaling and absorption correction, version 2008/1, University of Göttingen: Göttingen, Germany, 2008. (b) Blesing, R. H. Acta Crystallogr. 1995, A51, 33. (27) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 2005, 38, 381. (28) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112. (29) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703. (30) Gaussian 03, Rev.C.02; Gaussian Inc.: Wallingford, CT, 2004.

detection in contaminated living cells. Although the detection limit is in the micromolar range, which is higher than the maximum contaminant level (MCL) guided by the WHO, but it is a challenging task. We are continuously striving for new fluorescent probes having a detection limit below that of the WHO recommended tolerance value.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Debasis Das: e-mail, [email protected]; phone, +91-3422533913; fax, +91-342-2530452. Notes

The authors declare no competing financial interest. The CCDC number of APSAL is 868494.

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ACKNOWLEDGMENTS

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REFERENCES

S. Lohar and A. Sahana are grateful to CSIR, New Delhi, for the fellowship. Financial assistance from UGC-DAE-CSR-Kolkata is gratefully acknowledged. Authors gratefully acknowledge the Indian Institute of Chemical Biology (IICB, Kolkata) for extending their mass spectral facilities. We sincerely thank the University Science Instrumentation Center (USIC), Burdwan University, for providing the fluorescence microscope facility.

(1) U.S. EPA (U.S. Environmental Protection Agency) IRIS (Integrated Risk Information System). Online database maintained in Toxicology Data Network (TOXNET) by the National Library of Medicine, Bethesda, MD, 1997. (2) IARC (International Agency for Research on Cancer). International Agency for Research on Cancer, Toxicological Abbreviations, Arsenic and Arsenic Compounds, IARC Summary & Evaluation. 1987, Vol. 23, p 100. (3) Sabbioni, E.; Fischbach, M.; Pozzi, G.; Pietra, R.; Gallirono, M.; Piette, J. Carcinogenesis 1991, 12, 1287. (4) Jain, C. K.; Ali, I. Water Res. 2000, 34, 4304. (5) Yoshida, T.; Yamamuchi, H.; Fan Sun, G. Toxicol. Appl. Pharmacol. 2004, 198, 243. (6) WHO. Guidelines for Drinking-Water Quality, 4th ed.; World Health Organization: Geneva, Switzerland, 2011. (7) Melamed, D. Anal. Chim. Acta 2005, 1, 532. (8) Kim, S. Y.; Hong, J. I. Tetrahedron Lett. 2009, 50, 2822. (9) Sahana, A.; Banerjee, A.; Das, S.; Lohar, S.; Karak, D.; Sarkar, B.; Mukhopadhyay, S. K.; Mukherjee, A. K.; Das, D. Org. Biomol. Chem. 2011, 9, 5523. (10) Wu, J. S.; Hwang, I. C.; Kim, K. S.; Kim, J. S. Org. Lett. 2007, 9, 5. (11) Lee, M. H.; Giap, T. V.; Kim, S. H.; Lee, Y. H.; Kang, C.; Kim, J. S. Chem. Commun. 2010, 46, 1407. (12) (a) Lim, M. H.; Lippard, S. J. Acc. Chem. Res. 2007, 40, 41. (b) Miller, E. W.; Chang, C. J. Curr. Opin. Chem. Biol. 2007, 11, 620. (13) Yuan, L.; Lin, W.; Yang, Y. Chem. Commun. 2011, 47, 6275. (14) Hariharan, M.; Karunakaran, S. C.; Ramaiah, D. Org. Lett. 2007, 9, 417. (15) Wu, J.-S.; Zhou, J.-H.; Wang, P.-F.; Zhang, X.-H.; Wu, S.-K. Org. Lett. 2005, 7, 2133. (16) Sahana, A.; Banerjee, A.; Guha, S.; Lohar, S.; Chattopadhyay, A.; Mukhopadhyay, S. K.; Das, D. Analyst 2012, 137, 1544. 1783

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