Selective Detection of Fe3+, F-, and Cysteine by a Novel Triazole-Linked Decaamine Derivative of Pillar[5]arene and Its Metal Ion Complex in Water.

Appropriately functionalized pillar[n]arenes are elegant supramolecular hosts for ion and molecule sensing. A water-soluble decaamine derivative of pillar[5]arene (APA) bearing triazole and amide moieties is synthesized. The ion and molecular recognition properties of APA are studied by fluorescence, UV-visible, and 1H nuclear magnetic resonance (NMR) spectroscopy. The APA selectively detects Fe3+ among 11 studied ions, which are important in several biological processes. Moreover, the in situ prepared Fe3+ complex of APA (FeAPA) exhibits the highest responsiveness toward F- (∼12-fold) among 11 anions and cysteine (∼120-fold) among the 20 naturally occurring amino acids by a fluorescence turn-on mechanism.

Introduction

Detection of ions and molecules is paramount in biology because of their involvement in various physiological processes. Owing to the existence of more than one oxidation state, transition-metal ions, such as iron, the most important trace element in the human body, are involved in a number of electron transfer as well as oxidoreductase processes.[1,2] Both deficiency and excess accumulation of iron are harmful to life and lead to a number of diseases in humans.[3,4] Similarly, fluoride ions have several advantages, especially in preventing tooth decay as well as in the treatment of osteoporosis, while their excess intake may be detrimental to life.[5,6] Being the fundamental unit of proteins and an integral part of numerous biological processes, amino acids play vital roles in biology. Among the 20 naturally occurring amino acids, l-cysteine plays an important role in living systems, and its deficiency may cause several medical conditions, such as lethargy, liver damage, skin lesions, weakness, etc.[7−9] Hence, easy detection of biologically relevant ions and molecules have gained increasing attention among researchers.

Several macrocycles such as crown ethers, cyclodextrins, calixarenes, cucurbiturils, and pillararenes are known for selective sensing of ions and molecules in both organic and aqueous solvents.[10−16] Their unique shape, ease of synthesis, versatile functionalization, tunable solubility, and ability to form host–guest complexes account for their significance in supramolecular chemistry.[17−20] Pillararenes are relatively new additions into the family of macrocycles, and their unique pillarlike shape and the presence of two reactive phenolic groups at the para positions rendered them inevitable in molecular recognition and in the fabrication of supramolecular systems.[19,21] Recently, pillararene-based systems have found excellent applications in the areas of sensors, supramolecular polymers, molecular devices, drug delivery, etc.[21−29] Recent studies demonstrated the detection of Fe3+ by a 2-mercaptobenzothiazole derivative of pillar[5]arene, and in another case, the sensing was achieved by a supramolecular host–guest complex of a water-soluble pillar[5]arene conjugate and a perylenediimide derivative.[30,31] Huang and co-workers reported the selective sensing of Fe3+ with an anthracence-appended copillar[5]arene in dimethyl sulfoxide (DMSO) solution using different spectral techniques.[32] Wei and co-workers demonstrated dual sensing of Fe3+ and F– in H2O/DMSO (1:9) by a copillar[5]arene and its iron complex, respectively.[33] Similarly, Zhang and co-workers have shown the sequential fluorescence sensing of Fe3+ and F– in DMSO/H2O (8:2) using an imine derivative of pillar[5]arene.[34] Although there are several pillararene-based receptors to recognize Fe3+ and F–,[35−39] to the best of our knowledge, the receptors for amino acids are limited to methionine, tryptophan, arginine, and lysine.[40−45]

Sequential sensing of cations and anions by pillar[5]arene conjugates has been reported in the literature, while a single pillar[n]arene derivative capable of detecting multiple ions and amino acids is rather rare.[46] In addition to this, it is important to note that the majority of biological and environmental processes occur in an aqueous system. Hence, the host system used for the detection of ions and molecules should be water soluble. Other challenges remaining in developing a selective sensor molecule are the interference by other guest ions and the high hydration enthalpy of guest species in water, which, in turn, reduces the binding between the host and guest species. We have successfully overcome all of these challenges, and herein, we report the synthesis of a novel triazole-linked amino derivative of pillar[5]arene, APA, and its selective sensing of Fe3+ among 11 biologically relevant ions in aqueous solution. Further, we have used an in situ prepared Fe3+ complex of APA for the selective sensing of F– among 11 halides and cysteine among the 20 naturally occurring amino acids.

Results and Discussion

A pillar[5]arene bearing 10 primary amine groups through a triazole and amide linkage has been synthesized by a four-step reaction procedure (Scheme ). In the first step, hydroquinone, a commercially available starting material, was treated with propargyl bromide and potassium carbonate in acetone under refluxing conditions, leading to the formation of a disubstituted derivative of hydroquinone, 1a. The cyclized pillar[5]arene derivative, 1b, was obtained by reacting 1a with paraformaldehyde and BF3·OEt2 in dichloromethane. A 1,3-diploar cycloaddition reaction was carried out using 1b and ethyl azidoacetate in the presence of CuSO4·5H2O and ascorbic acid in dimethylformamide (DMF) to afford a decaester derivative of pillar[5]arene, 1c. Finally, the fluorescent probe, APA, was synthesized by the reaction of 1c with an excess amount of ethylene diamine at 80 °C. The structure of the decaamine derivative, APA, and its precursors was confirmed by 1H nuclear magnetic resonance (NMR), 13C NMR, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and elemental analysis (Figure S1).

Scheme 1

Synthesis of the Decaamine Derivative of Pillar[5]arene, APA

APA is decorated with three different functional groups, such as amide, triazole, and primary amine, on each of its arms. Hence, the decaamine-functionalized APA provides a flexible binding core during its interaction with various ions and molecules. The ion and amino acid detection of APA was carried out by fluorescence and UV–visible spectroscopy in water. We excited APA at 290 nm and studied its fluorescence emission from 300 to 420 nm. The sensor molecule, APA, exhibited fluorescence quenching upon interaction with Fe3+, while no change in the fluorescence intensity was observed upon titration with other ions, viz., Na+, K+, Ca2+, Mg2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ (Figure ). APA showed fluorescence quenching of ∼265-fold during the titration with Fe3+, indicating the selectivity of APA toward Fe3+ over other studied metal ions. The detection limit of the sensor, APA, for Fe3+ was 689 ppm.

Figure 1

(a) Fluorescence spectral traces obtained during the titration of APA with increasing concentrations of Fe3+, (b) plot of relative fluorescence intensity (I/I0) of APA versus mole ratios of Fe3+, and (c) histogram representing the fluorescence quenching (I0/I) obtained during the titration of APA with 11 metal ions.

UV–visible spectral titrations were also carried out to support the binding of Fe3+ with APA. During the titration, the concentration of APA was kept constant at 10 μM and the concentration of Fe3+ was increased gradually from 2 to 100 equiv. When APA was titrated against Fe3+, a marginal increase was observed in the absorbance band at 290 nm, indicating the complex formation of APA with Fe3+. Plots of absorbance versus wavelength and absorbance versus mole ratio, [Fe3+]/[APA], are given in Figure .

Figure 2

(a) Absorption spectra obtained for the titration of APA by Fe3+, and (b) plot of absorbance versus mole ratio, [Fe3+]/[APA].

(a) Absorption spectra obtan class="Chemical">ined for the titration of APA by Fe3+, and (b) plot of absorbance versus mole ratio, [Fe3+]/[APA].

The interaction between APA and Fe3+ was studied by 1H NMR titrations in D2O/CD3OD (9.2:0.8). During the titration, the concentration of APA was kept constant and the concentration of Fe3+ was varied to afford mole ratios, [Fe3+]/[APA], of 0.5, 1.5, and 3.0 (Figure ). The 1H NMR signals of aliphatic protons (protons 7 and 8) exhibited a marginal downfield shift during the titration with Fe3+, implying the interaction of Fe3+ with the terminal groups of APA. A minimal or no change in chemical shift was observed for the other protons of APA during the titration with Fe3+. A similar kind of chemical shift was reported in the literature for the interaction of pillar[n]arene conjugates with metal ions during complexation.[47] Hence, in addition to the fluorescence and UV–visible titrations, 1H NMR studies further supported the interaction of Fe3+ with APA.

Figure 3

1H NMR spectra obtained during the titration of APA (3.4 mM) with different mole ratios of Fe3+ in D2O/CD3OD (9.2:0.8): (a) 0, (b) 0.5, (c) 1.5, and (d) 3.0. The asterisk denotes the solvents.

1H NMR spectra obtan class="Chemical">ined during the titration of APA (3.4 mM) with different mole ratios of Fe3+ in D2O/CD3OD (9.2:0.8): (a) 0, (b) 0.5, (c) 1.5, and (d) 3.0. The asterisk denotes the solvents.

Further, we explored the secondary sensing properties of an in situ prepared Fe3+ complex of APA (FeAPA) with anions and amino acids. The initial fluorescence intensity of APA was quenched by the addition of 50 equiv of Fe3+. During the titration of FeAPA with anions, the fluorescence intensity of FeAPA was increased by a gradual addition of F–, and it showed a fluorescence enhancement of ∼12-fold (Figures and S2). None of the other anions, viz., Cl–, Br–, I–, CO32–, HCO3–, HSO4–, H2PO4–, OAc–, NO3–, and SO4–, produced any change in the fluorescence intensity of FeAPA even after the addition of 200 equiv of anions. The titration results clearly indicated the selectivity of FeAPA to F– over other studied ions. The fluorescence enhancement observed during the titration of FeAPA with F– was caused by the displacement of Fe3+ from the FeAPA complex, leaving free APA in the titration solution. Hence, upon complete removal of Fe3+ by F–, the initial fluorescence intensity of APA was restored, as indicated by the fluorescence enhancement (Figure ). The minimum concentration of F– that can be detected by FeAPA in water was found to be 434 ppm. The secondary sensing ability of FeAPA with F– was further confirmed by absorption spectral titrations. During the titration, the absorbance of the in situ prepared FeAPA was monitored by increasing the concentration of F–. It was found that the initial absorbance of the FeAPA complex was decreased by the addition of F–, and finally, the absorption spectrum showed characteristics similar to that of free APA (Figure S3). These results support the displacement of Fe3+ from the FeAPA complex and the formation of free APA with higher equivalents of F– in the solution.

Figure 4

(a) Plot of relative fluorescence intensity (I/I0) of FeAPA versus mole ratio ([F–]/[FeAPA]) of added F– in water, and (b) histogram representing the fluorescence response of FeAPA after the addition of 200 equiv of F–, Cl–, Br–, I–, CO32–, HCO3–, HSO4–, H2PO4–, OAc–, NO3–, and SO4– in water.

In addition to the selective sensing of FeAPA toward F–, we further explored the secondary sensing ability of FeAPA upon interaction with the 20 naturally occurring amino acids. The in situ prepared complex, FeAPA, was prepared by mixing APA and Fe3+ in a ratio of 1:100 in water. During the titration of FeAPA with amino acids, the fluorescence intensity of FeAPA increased exponentially with the addition of cysteine and the maximum intensity was obtained at 80 equiv of cysteine (Figures and S4) with a fluorescence enhancement of ∼112-fold. The regeneration of the fluorescence intensity of FeAPA during the titration with cysteine is attributed to the displacement of Fe3+ from the binding core of FeAPA and the formation of free APA. We also tested the interaction of all remaining 19 amino acids with FeAPA and found that none of the amino acids brought about any significant change in the fluorescence intensity of FeAPA. Hence, our result demonstrates the efficacy of FeAPA in detecting cysteine over other naturally occurring amino acids by fluorescence enhancement. The detection limit of Cys by FeAPA was estimated to be 1740 ppm. The displacement mechanism during the detection of Cys was further established by absorption spectral titration of FeAPA with Cys. The initial absorbance of the in situ prepared FeAPA complex was gradually decreased upon the addition of Cys. Finally, the absorbance of the complex was found to be almost the same as that of free APA. This result suggests the disruption of the FeAPA complex followed by the removal of Fe3+ by Cys, leaving free APA in the solution (Figure S5).

Figure 5

(a) Plot of relative fluorescence intensity (I/I0) of FeAPA versus mole ratio of ([Cys]/[FeAPA]) of added Cys in water, and (b) histogram representing the fluorescence of FeAPA after the addition of 200 equiv of the 20 naturally occurring amino acids.

In summary, a novel functionalized pillar[5]arene, APA, was synthesized and its ion and amino acid sensing capability was demonstrated using fluorescence, absorption, and 1H NMR spectroscopy. Water-soluble APA could act as a sensor for Fe3+ among 11 biologically relevant ions with a minimum detection limit of 689 ppm. The in situ prepared ensemble, FeAPA, could act as a turn-on sensor for fluoride among the 11 anions studied by fluorescence and absorption spectroscopy. The sensitive and selective sensing of cysteine among the 20 naturally occurring amino acids was achieved by FeAPA through fluorescence enhancement (∼120-fold) in water. The minimum detection limits of FeAPA for F– and Cys were 434 and 1740 ppm, respectively. Selective detection of F– and Cys was achieved by the displacement mechanism where Fe3+ was dechelated from the binding core of FeAPA, leaving APA alone.

Materials and Physical Methods

The perchlorate salts used for this study, viz., Mn(ClO4)·6H2O, Fe(ClO4)2·xH2O, Fe(ClO4)3·6H2O, Co(SO4)2·7H2O, Ni(ClO4)2·6H2O, Cu(ClO4)2·6H2O, Zn(ClO4)2·6H2O, NaClO4·H2O, KClO4, Ca(ClO4)2·4H2O, and Mg(ClO4)2·6H2O, were procured from Sigma-Aldrich Chemical Company. Among the salts of anions, Bu4NF and Me4NCl were procured from Otto Chemie Pvt. Ltd., and Bu4NBr, Bu4NI, Bu4NHSO4, Bu4NPO4, Me4NNO3, Bu4OAc, Bu4H2PO4, Na2CO3, NaHCO3, and Na2SO4 were procured from Spectrochem Pvt. Ltd., India. All of the 20 naturally occurring amino acids, except lysine (TCI Chemicals (India) Pvt. Ltd.) and histidine (Avra Synthesis Pvt. Ltd., India), were procured from Spectrochem Pvt. Ltd., India. All solution studies were carried out in high-performance liquid chromatography (HPLC) grade water. The solvents used for recording NMR spectra were procured from Sigma-Aldrich Chemical Company. 1H and 13C NMR spectra were measured on a Bruker Ascend 400 spectrometer working at 400 MHz. The mass spectra were recorded on a Bruker UltrafleXtreme MALDI-TOF mass spectrometer. The absorption and steady-state fluorescence spectra presented in this article were measured on a Varian Cary 100 Bio and a Horiba Scientific FluoroMaz-4, respectively. The elemental analysis was carried out using a PerkinElmer 2400 SeriesII CHNS.

General Procedure for Fluorescence Experiments

All fluorescence titrations were carried out in HPLC grade water. The bulk solution of APA (6 × 10–4 M) was prepared in water/DMSO, and the total concentration of DMSO present in the final solution used for titration studies was 0.6%. The salts of cations and anions, and the amino acids, were dissolved in water. During the fluorescence titration, the final concentration of APA was kept at 20 μM and the concentration of metal salts was increased gradually to get the required mole ratios of APA/M. The final concentration of APA was kept at 10 μM for UV–visible spectral titrations. The limit of detection (LOD) was calculated using the equation, LOD = 3σ/m.[46]

Experimental Section

Synthesis of 1c

A mixture of 1b(48) (1.0 g, 1.2 mmol), ethyl azidoacetate[49] (2.3 g, 17.8 mmol), CuSO4·5H2O (71 mg, 0.28 mmol), and ascorbic acid (0.37 g, 1.9 mmol) in DMF (40 mL) was kept at 90 °C for 24 h. The reaction mixture was cooled to 25 °C, and the solvents were evaporated. The solid was dissolved in dichloromethane (100 mL), washed with water (2 × 50 mL) and brine (2 × 50 mL), and dried with Na2SO4. The solvent was evaporated, and the product was purified by chromatography (silica gel; dichloromethane/methanol) to afford 1c as a white solid (1.6 g, 59%). 1H NMR (400 MHz, DMSO-d6): δ 8.33 (s, ArH, 10H), 6.95 (s, ArH, 10H), 5.36 (s, OCH2, 20H), 5.06 and 4.75 (br, NCH2, 20H), 4.15–4.04 (m, OCH2, 20H), 3.67 (s, ArCH2Ar, 10H), 1.17 (t, J = 6.90 Hz, CH3, 30H) ppm. 13C NMR (100 MHz): δ 167.1, 148.8, 143.4, 128.1, 125.5, 114.3, 61.47 (ArC × 2), 50.3, 28.7, 13.8 ppm. Anal. Calcd for C110H130N30O30·8.65H2O: C, 52.67; H, 5.92; N, 16.75, found C, 52.00; H, 5.26; N, 17.44.

Synthesis of APA

A mixture of 1c (1.0 g, 0.43 mmol) and ethylene diamine (15 mL, 225 mmol) was kept at 80 °C for 24 h. The reaction mixture was cooled to 25 °C, and the product was precipitated by the addition of diethylether (50 mL). The precipitate was filtered and purified from methanol/diethylether to afford APA (0.95 g, 92%) as a light brown solid. 1H NMR (400 MHz, DMSO-d6): δ 8.29 (br, NH, 10H), 8.24 (s, ArH, 10H), 6.95 (s, ArH, 10H), 5.07–4.98 (m, CH2, 30H), 4.70 (d, J = 10.7 Hz, CH2), 3.65 (s, ArCH2Ar, 10H), 3.07 (br, CH2, 20H), 2.58 (br, CH2, 20H) ppm. 13C NMR (100 MHz): δ 165.6, 148.8, 142.9, 128.0, 125.7, 114.2, 61.2, 51.5, 42.6, 41.0, 28.7 ppm. MALDI-TOF calcd for C105H140N50O20 ([M]+) 2422.55, found 2422.27.