Naked-eye detection of biologically important anions by a new chromogenic azo-azomethine sensor

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 133 (2014) 31–37

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Naked-eye detection of biologically important anions by a new chromogenic azo-azomethine sensor Khatereh Rezaeian, Hamid Khanmohammadi ⇑ Department of Chemistry, Faculty of Science, Arak University, Arak 38156-8-8349, 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

 New chromogenic azo-azomethine

A new chromogenic azo-azomethine sensor has been synthesized. The anion recognition studies exhibited that the receptor acts as a sensor for biologically important anions such as F, AcO and H2PO4 over other anions. Interestingly, the sensory system not only let for the naked eye detection without any spectroscopic instrumentation but also helped to discriminate between anions.

sensor has been synthesized.  The affinity of the sensor toward biologically important anions was investigated.  The sensory system is able for naked eye detection and discrimination between anions.

a r t i c l e

i n f o

Article history: Received 17 February 2014 Received in revised form 5 May 2014 Accepted 14 May 2014 Available online 28 May 2014 Keywords: Anion recognition Azo-azomethine Chromogenic Sensor UV–Vis spectroscopy

a b s t r a c t A new chromogenic azo-azomethine sensor, containing active phenolic sites, has been designed and synthesized via condensation reaction of N,N,N0 ,N0 -tetrakis(2-aminoethyl)-2,2-dimethyl propane-1,3diamine with 1-(3-formyl-4-hydroxyphenylazo)-4-nitrobenzene. The anion recognition ability of the synthesized receptor was evaluated using UV–Vis spectroscopy and 1H NMR technique. The anion recognition studies exhibited that the receptor acts as a sensor for biologically important anions such as F, AcO and H2PO4 over other anions. The binding stoichiometry between sensor and anions was found to be 1:2. 1H NMR experiment revealed that sensor recognizes anions via H-bonds and subsequent deprotonation to elicit a vivid color change. Interestingly, the sensory system not only let for the naked eye detection without any spectroscopic instrumentation but also helped to discriminate between anions. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Paramount attention has been laid over last few decades on development of molecular sensors for biologically relevant anions ⇑ Corresponding author. Tel.: +98 8614173400. E-mail address: [email protected] (H. Khanmohammadi). http://dx.doi.org/10.1016/j.saa.2014.05.049 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

[1–3]. Among various anions, fluoride, acetate and phosphate have been intensively researched owing to their imperative roles in a broad range of chemical, biological and environmental processes [4,5]. For instance, fluoride ions show beneficial effects in human health. On one hand, fluoride plays a crucial role in protecting dental health and treating osteoporosis; on the other hand, excessive fluoride intake may cause collagen breakdown, bone disorders,

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thyroid activity, depression and immune system disruption [6–8]. In addition, carboxylates are of immense interest due to their specific biochemical functions in the enzymes and antibodies and are also the vital components of numerous metabolic processes [9,10]. Also, phosphate and its derivatives are significantly involved in transduction, energy storage and gene construction in biological systems [11,12]. Thus, the impetus for development of synthetic receptors proficient for sensing these biologically important anions is compelling. Recently, substantial efforts have been devoted to fabrication of colorimetric anion sensors, since naked eye detection without requiring spectroscopic instrumentation is low cost and highly desirable for applications in immediate screen processes [13–16]. Most of the theses receptors employ common recognition moieties for anion sensing include amide, urea and thiourea, amidourea, phenol, pyrrole, imidazolium and indole [17,18]. In theses subunits, either hydrogen bonding or complete deprotonation of active NAH or OAH protons plays pivotal role in recognition phenomenon. In the last few decades, many colorimetric sensors for anion recognition have been reported [19–22]. However, most of those which interact with fluoride ion do so along with other anions like acetate or dihydrogenphosphate [23,24]. Although, some of the reported receptors are chromogenic sensors, their color change

upon the addition of the fluoride ions is quite similar to those upon the addition of acetate and dihydrogenphosphate [25]. Consequently, the discrimination of fluoride from other anions is hampered. The mentioned obstacle prompted us to develop a new chromogenic azo-azomethine sensor for facile detection and discrimination of bioactive anions. Herein, our research interest manifests design and synthesis of a new azo-azomethine based colorimetric sensor, 1, (Scheme 1), equipped with four nitroazobenzene groups as the chromogenic signaling subunits and phenolic OH groups as binding sites. The affinity of 1 toward anions was investigated by means of UV–Vis and 1H NMR spectroscopy as well as by the naked eye. Experimental Materials All of the reagents and solvents involved in synthesis were of analytically grade and used as received without further purification. All chemicals were purchased from Sigma–Aldrich and Merck. N,N,N0 ,N0 -tetrakis(2-aminoethyl)-2,2-dimethyl propane-1,3-diamine(tdmtn) was prepared by previously reported method [26]. Azo-coupled salicylaldehyde precursor was prepared according to the well-known literature procedure [27].

Scheme 1. Synthesis of sensor 1.

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Instrumentation The structure of all synthesized compounds was confirmed by H NMR spectra, recorded on a Bruker Avance III 400 MHz spectrometers. FT-IR spectra were recorded as pressed KBr disks, using Unicom Galaxy Series FT-IR 5000 spectrophotometer in the region of 400–4000 cm1. Melting points were determined on Electrothermal 9200 apparatus. C. H. N. analyses were performed on a Vario EL III elemental analyzer. Electronic spectral measurements were carried out using Optizen 3220 UV spectrophotometer in the range of 200–900 nm. 1

Fig. 1. Color changes of sensor 1 (1  105 mol L1 in DMSO) after addition of 10      equiv. of different anions. From left to right: none, H2PO 4 , NO2 , NO3 , HSO4 , F , Cl ,   + Br, I, ClO 4 , AcO and N3 (TBA salts).

Synthesis of sensor 1 A solution of N,N,N0 ,N0 -tetrakis(2-aminoethyl)2,2-dimethyl propane-1,3-diamine (0.19 g, 0.25 mmol) in absolute EtOH (10 mL) was added to a stirring solution of azo-coupled precursor (0.27 g, 1 mmol) in absolute EtOH (50 mL) during a period of 20 min at 60 °C. The solution was heated in water bath for 3 h at 80 °C with stirring. The mixture was filtered whil hot and the obtained precipitate was washed with hot ethanol (three times) and then with diethyl ether. The resulted product was dried in air. Yield: 96%, m. p. 232–234 °C. 1H NMR (DMSO-d6, 400 MHz, ppm): d 0.81 (s, 6H), 2.40 (s, 4H), 2.81 (bs, 8H), 3.71 (bs, 8H), 6.69 (d, 4H, J = 9.20 Hz), 7.76 (d, 8H, J = 8.80 Hz), 7.82 (dd, 4H, J = 9.20 Hz and 2.4 Hz), 7.85 (d, 4H, J = 2.4 Hz), 8.22 (d, 8H, J = 8.80 Hz), 8.56 (d, 4H, J = 7.60 Hz), 14.03 (br, 4H). IR (KBr, cm1); 1641 (C@N), 1587 (phenol ring), 1516 (NO2), 1421 (N@N), 1341 (NO2), 1290 (CAO), 1105, 854 and 754. Anal. Calcd. for C65H62N18O12: C, 60.65; N, 19.59; H, 4.85. Found: C, 59.91; N, 19.10; H, 4.96%. kmax (nm) (e (M1 cm1)): 268 (56,200), 430 (89,000) and 455 (89,300) in DMSO.

Fig. 2. UV–Vis absorption spectra of 1 (1  105 mol L1 in DMSO) in the presence of 10 equiv. of various anions.

N

N

N HC O

O N O

CH

N

H

H

N

N

N

O N O

A-

O

N

N

N

HC

CH O

N O

N

O H H

N N

O

O N

N AFig. 3. Charge-transfer transition occurring upon complexation between 1 and anions.

O

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Fig. 4. UV–Vis absorption spectra of sensor 1 (1  105 mol L1 in DMSO) upon the addition of TBAF (0–10 equiv.). Inset showing the binding isotherm at selected wavelengths in DMSO.

Fig. 5. Benesi–Hildebrand plot of 1 binding with F anion associated with absorbance change at 549 nm.

Result and discussion New four armed dendrimer based azo-azomethine receptor, 1, has been synthesized via condensation reaction of N,N,N0 ,N0 -tetrakis(2-aminoethyl)-2,2-dimethyl propane-1,3-diamine with 1-(3formyl-4-hydroxyphenylazo)-4-nitrobenzene in ethanol in good yield (Scheme 1). The resultant product was characterized using standard spectroscopic techniques (see Supplementary data). The total absence of t(C@O) absorption band of azo-coupled salicylaldehyde precursor in the IR spectra of azo-azomethine compound, 1, together with the appearance of a new absorption band at 1641 cm1 obviously indicate that Schiff-base, C@N, has formed. Also, the infrared spectrum of 1 exhibits strong bands at 1341 and 1516 cm1 assigned to the NO2 groups [28]. In the 1H NMR spectrum of 1, the CH@N protons exhibit a doublet resonance at 8.56 ppm which may be due to the formation of intra-molecular interaction between OH and hydrogen atom of HC@N groups [29]. Moreover, the slightly broad signal at 14.03 ppm, assigned to the OH protons, as was confirmed by deuterium exchange when D2O was added to DMSO-d6 solution. Furthermore, the C, H, N elemental analyses are also in accordance with the molecular formula. As an initial test, we examined anion-induced color changes of 1 in DMSO upon the addition of 10 equiv. of tetrabutylammonium

Fig. 6. Job’s plot for sensor 1 with tetrabutylammonium fluoride using UV–Vis.

    (TBA) salts of F, Cl, Br, AcO, I, H2PO 4 , ClO4 , N3 , NO2 , NO3 and HSO 4 . As shown in Fig. 1, dramatic color changes were observed from orange to dark blue, purple and light brown in the presence of fluoride, acetate and dihydrogenphosphate, respectively. In contrast, other anions failed to cause any conspicuous color changes. To further consideration of chromogenic behavior of 1, UV–Vis absorption experiments were carried out. The changes in UV–Vis absorption spectrum of receptor 1 (1  105 mol L1 in DMSO) upon the addition of 10 equiv. of tetrabutylammonium salts of anions are shown in Fig. 2. Free sensor indicated an intense absorption band (a twin band) centered at 443 nm attributed to the p ? p⁄ transition of the chromophores. Addition of the relatively basic anions such as F, AcO and H2PO 4 had a profound effect on the electronic spectrum of 1; the extent of changes for F was much more in compared with acetate and dihydrogenphosphate. As it is obvious, a large bathochromic shift was observed from 443 nm to ca. 550 nm for mentioned anions, though other anions apparently produced no spectral changes. Moreover, it is worth mentioning that sensor 1 is capable of allowing a rough discrimination of mentioned anions on the basis of multicolor response capability concomitant different electronic spectra in the presence of various anionic guests.

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Fig. 7. UV–Vis absorption spectra of sensor 1 (1  105 mol L1 in DMSO) upon the addition of TBAOH (0–10 equiv.).

The significant bathochromic shifts can be ascribed to the charge transfer interactions occurred between the electron-rich donor units (oxygen of azophenol) to the electron-deficient acceptor units (ANO2), Fig. 3. This suggests that after complexation of 1 with anions, the excited state would be more strongly stabilized, leading to a bathochromic shift in the absorption maxima as well

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as a noticeable color change [30]. It is noteworthy that progressive addition of protic solvents such as methanol or water triggered turning the solution color to original host color (orange). Correspondingly, the absorption band was recovered from ca. 550 nm to 443 nm (see Supplementary data). This phenomenon can be attributed to the fact that protic solvents would compete with anions for binding sites of the host and destroy the hydrogen bonding interactions between host and guest [31]. In order to assess the further details of binding characteristics of the prepared sensor, we carried out UV–Vis spectroscopic titrations upon the addition of standard solutions of F, AcO and + H2PO 4 as their n-Bu4N salts to dry DMSO solution of the receptor (1  105 mol L1). Fig. 4 depicts the UV–Vis titration of 1 with standard solution of TBAF. As shown in Fig. 4, on incremental increase of F, the absorption band at 430 nm slightly decreased and the band at 455 nm red shifted and coalesced into a new intensified tailing band appeared as charge transfer interaction at 549 nm, indicating the formation of hydrogen bonding complex between receptor and fluoride anion. Moreover, analogous investigations were carried out for addition of TBAOAc and TBAH2PO4 to the solution of 1 in dry DMSO (see Supplementary data). Furthermore, the stoichiometry between the sensor 1 with anions (F, AcO and H2PO 4 ) was determined from UV–Vis spectral changes with the help of Benesi–Hildebrand method [32]. As

Fig. 8. 1H NMR spectra of sensor 1 in DMSO-d6 (5  103 mol L1) in the absence and in the presence of different amounts of TBAF.

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shown in Fig. 5, the plot of 1/[A  A0] vs 1/[F]2 for the titration of 1 with TBAF provided a straight line, corroborating that F interacts with 1 in a 2:1 stoichiometry with binding constant of 1.83  109 M2. Similar trend of UV–Vis spectral changes have been observed in case of acetate and dihydrogenphosphate with binding constants of 1.56  109 and 8.59  108 M2, respectively (see Supplementary data). The stoichiometry of host–guest interaction was also confirmed by use of Continuous-variation plots (Job’s plots). The variation of measured absorbance at 549 nm reaches to a maximum value when the molar fraction of 1/[F] + 1 is 0.4, indicating 1:2 stoichiometric ratio of the sensor 1 to the fluoride anion, Fig. 6 [33]. Similar results were also obtained for AcO and H2PO 4 (see Supplementary data). Moreover, judging from titration findings, it has been established that the estimated detection limits of sensor 1 toward F, AcO and 6 H2PO , 6.63  107 and 2.46  106 mol L1, 4 are 1.27  10 respectively. It has been postulated that the selectivity and sensitivity of the sensor 1 toward the F, AcO and H2PO 4 are ascribed to the structural representations and basicity of the anions [34,35]. Among various anions tested, fluoride and acetate with higher basicity are anticipated to form strong complexes with sensor 1 and consequently exhibit remarkable color changes as well as UV–Vis spectral changes. In addition, the excellent affinity of 1 toward both fluoride and acetate can also be attributed to the possible double hydrogen bonding of the Y-shaped anion causing stronger binding. On the contrary, H2PO 4 with lower basicity and inability to engage the receptor in similar fashion regarding to its tetrahedral geometry constructs weaker complex and furthermore induce minor spectral changes [14,36]. In order to verify the deprotonation process during the anion complexation with sensor 1, the UV–Vis titration experiment of 1 with a standard solution of tetrabutylammonium hydroxide (TBAOH) was performed. The titration profiles afforded similar spectral pattern as observed for other basic anions, suggesting deprotonation of phenolic OH protons [17]. The OH induced spectral changes of 1 are shown in Fig. 7. To further look into the nature of interaction between receptor 1 and anions, 1H NMR titration experiments were performed. Fig. 8 indicates the 1H NMR spectra of 1 with different amounts of TBAF in DMSO-d6. As it is obvious, the OH protons of the azo phenol moieties at 14.03 ppm thoroughly disappeared when only 1 equiv. of F was added into DMSO solution of 1. This indicates the formation of strong hydrogen bonds between fluoride anion and active OH groups, as previously reported [17]. With increasing further F amount, the signals of Ha and Hb showed progressive upfield shifts, whereas the signal of Hc exhibited progressive reverse shift toward downfield. Simultaneously, a downfield shift of the imine proton (Hd) from 8.56 to 8.64 ppm was also observed. The evidence above can be attributed to the two possible effects which are responsible for observed chemical shifts upon OH-fluoride hydrogen bond complex formation: (1) through-bond effects, which increase the electron density on the phenyl ring and promote an upfield shift of the CAH protons and (2) through-space effects which polarize the CAH bonds proximal to the OH-fluoride moiety to induce downfield shift due to the partial positive charge [37]. However, deprotonation of the sensor can possibly occur. Interestingly, a new triplet at 16.1 ppm (1J HF  124 Hz) appeared which is ascribed to the FHF dimer [8]. The existence of FHF confirms the deprotonation of OH groups. Consequently, the 1H NMR titration experiments corroborated that F recognition occurs through initial hydrogen bond formation between anion and sensor, followed by deprotonation event. Moreover, it should be noted that 1H NMR titration of 1 in the presence of AcO and H2PO 4 as their TBA slats induced similar shift patterns (see Supplementary data).

Conclusion To sum up, we present the synthesis, characterization and anion binding behavior of a new four armed dendrimer based azo-azomethine receptor. The mentioned sensory system allows facile recognition of bioactive anions such as F, AcO and H2PO4 without any spectroscopic instrumentation. Interestingly, the introduction of four electron withdrawing ANO2 groups into the backbone of the sensor realized a significant enhancement in anion recognition ability without disturbing the capability of receptor to discriminate between anions. The selectivity trends in the binding affinities of sensor toward studied anions can be attributed to the guest basicity and structural geometry. The recognition details of anion sensing were also studied using 1H NMR technique. The 1H NMR experiments confirmed that the color response was considered to be the direct consequence of hydrogen bond formation between phenolic OH groups and anions, followed by deprotonation event. Acknowledgement We are grateful to the Arak University for financial support of this work. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.05.049. References [1] P.A. Gale, S.E. Garcia-Garrido, J. Garric, Chem. Soc. Rev. 37 (2008) 151–190. [2] Q.S. Lu, L. Dong, J. Zhang, J. Li, L. Jiang, Y. Huang, S. Qin, C.W. Hu, X.Q. Yu, Org. Lett. 11 (2009) 669–672. [3] H.J. Schneider, A. Yatsimirsky, Chem. Soc. Rev. 37 (2008) 263–277. [4] A. Basu, S.K. Dey, G. Das, RSC Adv. 3 (2013) 6596–6605. [5] S. Sharma, M.S. Hundal, G. Hundal, Tetrahedron Lett. 54 (2013) 2423–2427. [6] X. Yong, M. Su, W. Wan, W. You, X. Lu, J. Qu, R. Liu, New J. Chem. 37 (2013) 1591–1594. [7] S. Kumar, R. Saini, D. Kaur, Sens. Actuators, B 160 (2011) 705–712. [8] X. Zhuang, W. Liu, J. Wu, H. Zhang, P. Wang, Specterochim. Acta Part A 79 (2011) 1352–1355. [9] Q.Q. Wang, V.W. Day, K. Bowman-James, Chem. Sci. 2 (2011) 1735–1738. [10] R.J. Fitzmaurice, G.M. Kyne, D. Douheret, J.D. Kilburn, J. Chem. Soc., Perkin Trans. 1 (2002) 841–864. [11] S. Sen, M. Mukherjee, K. Chakrabarty, I. Hauli, S.K. Mukhopadhyay, P. Chattopadhyay, Org. Biomol. Chem. 11 (2013) 1537–1544. [12] W.N. Lipscomb, N. Strater, Chem. Rev. 96 (1996) 2375–2434. [13] R. Sakai, E.B. Barasa, N. Sakai, S. Sato, T. Satoh, T. Kakuchi, Macromolecules 45 (2012) 8221–8227. [14] A. Okudan, S. Erdemir, O. Kocyigit, J. Mol. Struct. 1048 (2013) 392–398. [15] R.M. Duke, E.B. Veale, F.M. Pfeffer, P.E. Kruger, T. Gunnlaugsson, Chem. Soc. Rev. 39 (2010) 3936–3953. [16] X. Yong, M. Su, W. Wang, Y. Yan, J. Qu, R. Liu, Org. Biomol. Chem. 11 (2013) 2254–2257. [17] C. Jin, M. Zhang, C. Deng, Y. Guan, J. Gong, D. Zhu, Y. Pan, J. Jiang, L. Wang, Tetrahedron Lett. 54 (2013) 796–801. [18] J.-Q. Li, T.-B. Wei, Q. Lin, P. Li, Y.-M. Zhang, Specterochim. Acta Part A 83 (2011) 187–193. [19] M. Khandelwal, I.C. Hwang, P.C.R. Nair, J. Fluorine Chem. 135 (2012) 339–343. [20] J. Ma, Z. Li, Y. Zong, Y. Men, G. Xing, Tetrahedron Lett. 54 (2013) 1348–1351. [21] Y. Zhao, Y. Li, Z. Qin, R. Jiang, H. Liu, Y. Li, Dalton Trans. 41 (2012) 13338–13342. [22] J. Isaad, A.E. Achari, Tetrahedron 67 (2011) 4939–4947. [23] S. Das, D. Saha, C. Bhaumik, S. Dutta, S. Baitalik, Dalton Trans. 39 (2010) 4162– 4169. [24] C. Yang, J. Xu, J. Ma, D. Zhu, Y. Zhang, L. Liang, M. Lu, Polym. Chem. 3 (2012) 2640–2648. [25] D.H. Lee, K.H. Lee, J.I. Hong, Org. Lett. 3 (2001) 5–8. [26] H. Khanmohammadi, S. Amani, H. Lang, T. Rüeffer, Inorg. Chim. Acta 360 (2007) 579–587. [27] H. Khanmohammadi, M. Darvishpour, Dyes Pigm. 81 (2009) 167–173. [28] H. Khanmohammadi, A. Abdollahi, Dyes Pigm. 94 (2012) 163–168. [29] P.V. Rao, C.P. Rao, E.K. Wegelius, K. Rissanen, J. Chem. Crystallogr. 33 (2003) 139–147. [30] P. Thiampanya, N. Muangsin, B. Pulpoka, Org. Lett. 14 (2012) 4050–4053. [31] L. Wang, X. He, Y. Guo, J. Xu, S. Shao, Org. Biomol. Chem. 9 (2011) 752–757.

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