Multiple Sensor Array of Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+

Sep 14, 2012 - detection limit experiments for the metal ions were carried out by taking the L to metal ion ratio of 1:1 and in case of amino acids, t...
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Multiple Sensor Array of Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ Complexes of a Triazole Linked Imino-Phenol Based Calix[4]arene Conjugate for the Selective Recognition of Asp, Glu, Cys, and His Rakesh K. Pathak, Jayaraman Dessingou, and Chebrolu P. Rao* Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India S Supporting Information *

ABSTRACT: The triazole linked o-imino phenol appended calix[4]arene conjugate (L) has been synthesized and characterized. The structure of L has been established based on single crystal XRD. The binding and recognition behavior of conjugate, L toward the transition metal ions, such as Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, has been demonstrated using fluorescence, absorption and ESI-MS techniques. The in situ prepared complexes of these metal ions, namely, [Mn2L], [Fe2L], [Co2L], [Ni2L], [Cu2L], and [Zn2L] have shown recognition toward Glu, Asp, His and Cys. Hence L provides a multiple sensing molecular tool where the response for the recognition of biologically active amino acids of metalloproteins is elicited by the presence of specific metal ion.

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ransition metal ions, such as Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, are integral parts of biology1 because of their role as cofactor in various metalloproteins and metalloenzymes.2 These ions bind to different amino acid residues resulting in specific coordination geometry at the active site of these proteins, according to their metal/amino acid preferences, to exhibit particular enzymatic/catalytic activity/function.3 The side chains of a few amino acids, such as His, Cys, Asp, and Glu, are most commonly involved in metal binding in the proteins.4 Among these, His and Cys have the maximum contribution to the primary coordination sphere of Ni2+, Cu2+, and Zn2+ in their proteins.5 Some of these metal ions are also present as free pool or bound with labile ligands of proteins in cells and body fluids and their relative concentrations are being controlled by peptides and proteins.6 Imbalance in the homeostasis of these metal ions and the corresponding amino acids can cause several diseases.7 Therefore, the recognition of these species is of prime importance in medical sciences to develop future diagnostic tools for the diseases.8 Recently, metal bound fluorescent organic molecular systems emerged as receptors to recognize particular amino acid because of their specific metal/amino acid binding characteristics such as that present in the natural systems.9 Among these, calix[4]arene based supramolecular systems possessing preorganized binding cores formed by two arms on the calix[4]arene platform were developed as suitable molecular scaffolds to bind to metal ions by exhibiting appropriate changes in their fluorescence signals upon recognition.10,11 The amino acid competes for the transition metal ion present in the complex of the conjugate during the recognition events and thereby triggers a change in the optical properties and hence can be used to recognize the amino acid selectively.12 Recently, our research group reported selective © 2012 American Chemical Society

detection of amino acids using calix[4]arene conjugates bearing transition-metal ions.13 By bringing marginal modifications in the binding motif topology, the calix[4]arene platform can provide selectivity toward a particular metal ion, and the corresponding complex in turn can act as a responsible ensemble for exhibiting secondary selectivity, namely, toward amino acids.13g−i Herein, we report a multisensor array to detect and quantify amino acids in a highly sensitive and selective manner by using the in situ prepared transition metal ion (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+) complexes of oimino phenol appended triazole linked calix[4]arene conjugate (L).

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EXPERIMENTAL SECTION General Information and Materials. Fluorescence emission spectra were measured on a Perkin-Elmer LS55 spectrofluorimeter by exciting the samples at 360 nm, and the emission spectra were recorded in 370−650 nm range. The bulk solutions of L, metal perchlorate salts, and amino acid were prepared in methanol. All the measurements were made in a 1 cm quartz cell, and the effective concentration of L was maintained at 10 μM. During the amino acid recognition experiments, the complexes of L were made by in situ adding a 1: 2 mixture of L and metal perchlorate. During the titration, the concentration of amino acid was varied accordingly to result in the requisite mole ratios of amino acid to [M2L, M = Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+] and the total volume of the Received: June 30, 2012 Accepted: September 4, 2012 Published: September 14, 2012 8294

dx.doi.org/10.1021/ac301821c | Anal. Chem. 2012, 84, 8294−8300

Analytical Chemistry

Article

Scheme 1. Synthesis of the Calix[4]arene Conjugate, La

Synthesis of L: (a) Propargyl bromide, K2CO3, acetone, reflux, 24 h; (b) 3-(azidomethyl)-5-tert-butyl-2-hydroxybenzaldehyde, CuSO4·5H2O, and sodium ascorbate in dichloromethane: water (1:1) rt, 12 h; (c) 2-amino phenol, methanol, rt, 4 h. R = tert-butyl.

a

Figure 1. (a) Stereo view of molecular structure of the conjugate, L as drawn at 50% probability ellipsoids. (b) A part of the lattice showing intermolecular O−H···N hydrogen bonds.

C, 73.71; H, 7.34; N, 7.77. Crystal parameters for L: empirical formula, C44.50H50N4O5; Mr = 720.89; temperature/K = 150(2); radiation, Mo Kα; wavelength/Å = 0.71073; crystal system = monoclinic, C2/c; unit cell dimensions a = 17.7170(8), b = 21.2858(10), c = 21.9638(9) (Å); α = 90, β = 103.048(2), γ = 90 (°); V = 8069.1(6) (Å3); Z = 8; Dcalcd= 1.187 (g/cm3); μ = 0.078 (1/mm); F = 3080 (000); θ = 3.3106, 32.8415 (min, max); Nref = 7088; Npar = 504; Robs = 0.0533, wR2obs = 0.1512; GOF = 0.967.

solution was maintained constant at 3 mL in each case by adding an appropriate volume of solvent. For the absorption titrations, solutions were prepared by following the similar procedure as given for fluorescence studies. Minimum detection limit experiments for the metal ions were carried out by taking the L to metal ion ratio of 1:1 and in case of amino acids, the [M2L] and [Amino acids, aa] mole ratio was 1:5. Color change experiments were carried out at 25 μM concentration of L in 1:2:10 (L/M2+/aa) mole ratio. Synthesis and Characterization of L. A mixture of 2 (1.0 g, 0.839 mmol) and 2-aminophenol (0.183 g, 1.679 mmol) in 10 mL methanol was stirred for 4 h. The resultant precipitate was filtered and washed with cold methanol to get pure reddish yellow solid product, L. 1H NMR (DMSO-d6, 400 MHz): 14.60 (s, 2H, Sal−OH), 9.87 (s, 2H, imino-phenol−OH), 9.03 (s, 2H, imine-H), 8.06 (s, 2H, Ar−OH), 7.95 (s, 2H, triazoleH), 7.59 (d, 2H, Sal-H, J = 2.25 Hz), 7.51 (d, 2H, Sal-H, J = 2.25 Hz), 7.36 (d, 2H, imino-phenol-H, J = 7.78 Hz), 7.12 (t, 2H, imino-phenol-H, J = 7.52 Hz), 6.98 (d, 2H, imino-phenolH, J = 7.78 Hz), 6.97 (s, 4H, Ar−H), 6.95 (s, 4H, Ar−H), 6.86 (t, 2H, imino-phenol-H, J = 7.52 Hz), 5.69 (s, 4H, Ar-OCH2), 5.12 (s, 4H, Sal-CH2), 3.93 (d, 4H, Ar−CH2−Ar, J = 12.54 Hz), 3.11 (d, 4H, Ar−CH2−Ar, J = 12.79 Hz), 1.23 (s, 18H, Ar−C(CH3)3), 1.13 (s, 18H, Sal-(CH3)3), 1.01 (s, 18H, Ar− C(CH3)3). 13C NMR (CDCl3, 100 MHz) δ (ppm): 163.12, 156.61, 150.00, 150.42, 149.80, 147.53, 144.73, 142.24, 141.8, 136.60, 132.69, 131.86, 129.93, 128.60, 127.60, 125.82, 125.14, 123.58, 121.52, 120.60, 119.30, 119.18, 117.60, 70.37, 47.80, 34.10, 33.90, 31.80, 31.32, 31.08, 27.05. HRMS m/z Calcd for C86H100N8O8: 1373.7742, Found 1373.7697. Elem anal. Calcd for C86H100N8O8.2CH3OH: C, 73.51; H, 7.57; N, 7.79. Found:

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RESULTS AND DISCUSSION The calix[4]arene conjugate, L has been synthesized as per that shown in Scheme 1 by the condensation of 2 with o-amino phenol. All the synthesized molecules at every stage in the Scheme 1 were characterized by analytical and spectral techniques, namely, 1 H and 13 C NMR and ESI MS (Experimental Section and Supporting Information, Figure S1−S2). NMR spectra supports the cone conformation for the calix[4]arene conjugate. This has been further confirmed from the single crystal XRD structure of L. Single Crystal XRD Structure of L. Single crystals of L suitable for X-ray diffraction were obtained by slow evaporation of methanolic solution of L. It crystallizes in monoclinic system with C2/c space group (Experimental Section). The calixarene unit adopts cone conformation by extending cyclic intramolecular O−H···O hydrogen bonds at the lower rim. The triazole linked o-imino phenol arms were bent toward the direction of the upper rim of calixarene (Figure 1a). In the lattice, the structure is stabilized by the intermolecular hydrogen bonds extended between the triazole nitrogens and 8295

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the o -imino-phenolic−OHs of neighbor molecules as can be seen from Figure 1b. Metal Ion Titrations. The o-imino phenol appended triazole linked bifunctionalized calix[4]arene conjugate, L containing imino-phenolic binding core has been studied toward different transition metal ions by fluorescence, absorption, and ESI MS titrations. Fluorescence and Absorption Titrations of L with Metal Ions. The conjugate, L shows weak emission at ∼535 nm when excited at 360 nm in methanol. Titration of L with Zn2+ shows fluorescence enhancement up to the addition of 5 equivalents, beyond which there is a minimal decrease in the fluorescence intensity. Similar titrations carried out with other metal ions, namely, Mn2+, Fe2+, Co2+, Ni2+, and Cu2+ exhibit quenching in the fluorescence of L (Figure 2b and Supporting

titrations indicating transition between the unbound and the complexed species (Figure 2 and Supporting Information, Figure S5). For example, the isosbestic points were observed at 478, 380, and 312 nm in case of Cu2+ titration (Figure 2c). Comparison of the absorbance of the ∼415 nm band (Figure. 2d) exhibits the classical Irving−William order (Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+). The stoichiometry of the complexes were found to be 1:2 between L and M2+ based on the Job’s plot (Supporting Information, Figure S6) using absorption data. ESI MS and 1H NMR Titrations. The complex formed by these metal ions with L has also been studied by ESI MS titrations. ESI MS spectrum obtained for the in situ prepared complexes of Mn2+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+ exhibits 1:2 complex with peaks at m/z 1480.6, 1482.8, 1488.8, 1488.8, 1497.8, and 1501.8, respectively (Supporting Information, Figure S7−S12). The isotopic peak pattern supports the presence of metal ions in these complexes. The binding is further supported by carrying out the 1H NMR titrations of L with Zn2+ owing to its nonparamagnetic nature (Supporting Information Figure S13). Upon the interaction of Zn2+ with L, the imine−H, salicyl−H and −OH, bridged- and arm −CH2, and triazole−H showed minimal to marginal changes in their δ values during the titration. The significant downfield shifts observed in the o-imino phenolic −H and −OH supports the binding of the imino-phenolic core. Hence, all these changes are suggestive of 1:2 complex formation of L with Zn2+ through utilizing both of its binding motifs (Supporting Information, Figure S13). Recognition of Amino Acids by the Metal Ion Complexes. To achieve multiple sensing for the recognition of amino acids, the in situ metal ion complexes of L, namely, M2L (M = Mn2+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) have been used as the “chemosensing ensemble” (CsE). The CsE has been prepared by mixing 1:2 mol ratio of the conjugate L and the corresponding metal ion in methanol and this has been used for all the recognition studies reported in this paper. Secondary Sensing Properties of [M2L; M = Mn2+, Fe2+, and Co2+]. The recognition behavior of [M2L] has been evaluated by carrying out the fluorescence and absorption titrations with all the twenty amino acids. Out of these in situ complexes, [Fe2L] does not show any fluorescence revival with any of the amino acid suggestive of the strong complexation nature of Fe2+ with L (Figure 3 and Supporting Information, Figure S14−S16). [Co2L] shows a marginal to significant fluorescence revival with three amino acids in the order, His > Asp > Cys, with a minimum detection concentration {in ppm (μM)} of, 0.53 (3.44), 0.34 (2.5) and 0.68 (5.65) respectively (Figure 3 and Supporting Information, Figure S17−S19). Whereas, the quenched fluorescence of [Mn2L] has been

Figure 2. (a) Fluorescence spectra obtained during the titration of L with Zn2+ in methanol, λex = 360 nm; inset shows the relative fluorescence intensity (I/I0) as a function of [Zn2+]/[L] mole ratio at 495 nm band. (b) Histogram showing the fluorescence response of L with M2+. [L] = 10 μM and [Mn+] = 20 μM. (c) Absorption spectra obtained during the titration L with Cu2+, inset shows the plot of absorbance vs. [Cu2+]/ [L] mole ratio for different bands. (d) Histogram shows the absorbance of L at 415 nm band when titrated with different M2+.

Information, Figure S3) and the Stern−Volmer quenching constants follow an order, Mn2+< Ni2+< Fe2+< Co2+≪ Cu2+. All the metal ions show saturation in fluorescence intensity ∼2 equivalents with a blue shift of 40 nm from 535 to 495 nm in support of the complex formation. The sensitivity of L for these metal ions has been further evaluated by measuring the lowest concentration that can be detected. The fluorescence titration carried out between L and [M2+] by maintaining a 1:1 ratio clearly demonstrates that L responds up to a minimum concentration {in ppb (μM)} 98 (1.8), 65 (1.02), 117 (1.99), 117 (1.99), 67 (1.05) and 50 (0.76) for Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, respectively (Supporting Information, Figure S4). To understand the binding of these metal ions with L, absorption titrations were carried out. The absorption spectra of L in methanol exhibited a band at ∼355 nm and a shoulder at 480 nm. Upon the titration of L with these metal ions, a new band appears at 415−426 nm depending upon the metal ion. The absorbance of two other bands, namely, ∼355 and ∼480 nm decreases successively. The isosbestic points were observed in the

Figure 3. Histogram showing the fluorescence response of [M2L; M = Mn2+, Fe2+ and Co2+] with amino acids. 8296

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resurged successively upon addition of Asp or Glu selectively and the increase in intensity saturates at ∼15−20 [amino acid]/ [Mn2L] mole ratio. All the other amino acids do not show such resurgence in fluorescence intensity. The detectable minimum concentration {in ppm (μM)} of Glu and Asp by [Mn2L] were, 2.8 (19.0) and 2.67 (20.2) respectively as derived from the fluorescence titrations (Figure 3 and Supporting Information, Figure S20−S22). To support the results obtained from the fluorescence studies, similar titrations were carried out even by absorption spectroscopy. Titration of [Mn2L] with Asp or Glu exhibits marginal increase in the absorbance of the band at ∼355 nm which corresponds to the free receptor. The absorbance of the free receptor band increases as a function of increase in the concentration of amino acid added. Other amino acids do not show such changes in the absorption of [Mn2L] suggesting the binding nature of Asp and Glu with Mn2+ by exhibiting selective sensing (Supporting Information, Figure S23−S24). Upon the titration of [Co2L] with all the amino acids show marginal absorption changes only in case of His, Asp and Cys and not with the others, and the changes follow similar trends as that observed in the fluorescence spectra (Supporting Information, Figure S25−S26). However, there is no change observed in the absorption spectra with the amino acids when the titration of [Fe2L] was performed with the amino acids (Supporting Information, Figure S27). Thus, [Fe2L] is found to be nonresponsive toward all the twenty amino acids, the [Co2L] shows marginal selectivity only for His, Cys, and Asp and that of the [Mn2L] shows selectivity only for Asp and Glu. Secondary Sensing Properties of [Ni2L]. The in situ prepared [Ni2L] complex exhibits fluorescence quenching owing to the presence of Ni2+ in the complex. The fluorescence emission of [Ni2L] increases in case of His followed by Asp, while all the other amino acids show no significant changes (Figure 4 and Supporting Information, Figure S28−S30). The fluorescence increase observed in case of His saturates at ∼20 equiv through exhibiting almost complete revival of the fluorescence intensity of conjugate, L and hence supports the dechelation of Ni2+ to form His complex of this ion. The absorption spectrum of [Ni2L] exhibits a broad band ∼430 nm (Supporting Information, Figure S5). Upon the titration of this complex with amino acids, His followed by Asp exhibits decrease in the absorbance of ∼430 nm band besides showing two new bands as a function of the added concentration (Figure 4c and Supporting Information, Figure S31). At saturation, the spectrum of {[Ni2L] + His} exhibits similar absorption characteristics as that observed for simple L. All this strongly support the dechelation of Ni2+ followed by the formation of its His complex and thus demonstrates the selective sensing of His. This conclusion has been further strengthened by observing the His bound nickel species in ESI MS (Supporting Information, Figure S32). A distinguishable color change has been observed only in case of His. The color of the {[Ni2L] + His} mixture was found similar to that observed for L, further supporting the dechelation (Supporting Information, Figure S32). The detectable minimum concentration {in ppm (μM)} of His and Asp were 0.35 (3.0) and 1.06 (7.9), respectively, as derived from the fluorescence titrations (Supporting Information, Figure S33). Therefore, the sensitivity and selectivity for His has been demonstrated based on the CsE of [Ni2L]. Secondary Sensing Properties of [Cu2L]. Fluorescence titrations were also carried out for the copper complex,

Figure 4. (a) Fluorescence spectra obtained during the titration of [Ni2L] with His in methanol, λex = 360 nm; inset shows the relative fluorescence intensity (I/I0) as a function of [His]/[Ni2L] mole ratio at 535 nm band. (b) Histogram showing the fluorescence response of [Ni2L] with amino acids. (c) Absorption spectra obtained during the titration [Ni2L] with His; inset shows the plot of absorbance vs. [His]/ [Ni2L] mole ratio for different absorption bands. (d) Histogram shows the absorbance of [Ni2L] at 425 nm band when titrated with different amino acids.

[Cu2L] with all the twenty amino acids where the initial fluorescence of L is completely quenched owing to the presence of Cu2+ (Supporting Information, Figure S3). The quenched fluorescence of [Cu2L] is regained only in case of Cys as a function of the increase in the concentration of Cys added (Figure 5). All the other amino acids showed no change

Figure 5. (a) Fluorescence spectra obtained during the titration of [Cu2L] with Cys in methanol, λex = 360 nm; inset shows the relative fluorescence intensity (I/I0) as a function of [Cys]/[Cu2L] mole ratio at 535 nm band. (b) Histogram showing the fluorescence response of [Cu2L] with amino acids. (c) Absorption spectra obtained during the titration [Cu2L] with Cys; inset shows the plot of absorbance versus [Cys]/[Cu2L] mole ratio for different absorption bands. (d) Histogram shows the absorbance of [Cu2L] at 425 nm band when titrated with different amino acids. 8297

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in the fluorescence intensity (Figure 5b and Supporting Information, Figure S34−S35). Increase or regain in fluorescence intensity in the presence of Cys can be ascribed mainly to the interaction of Cys with [Cu2L] followed by the dechelation of Cu2+ from the coordination sphere of this complex to form adduct with Cys, namely, {Cys·Cu}. The selectivity in the sensing can be reconciled from the fact that in most of the metalloproteins, Cu2+ is bound to the Cys residues. Therefore, the metal ion responsible for quenching has been taken away by the Cys releasing the free L, which shows its original fluorescence emission. The detectable minimum concentration of Cys is 0.42 ppm (3.45 μM) based on fluorescence titration (Supporting Information, Figure S36). The other amino acids which cannot pull Cu2+ from the coordination sphere, shows no change in the fluorescence intensity (Figure 5). To further support the fluorescence results, absorption titrations of [Cu2L] with all the amino acids were carried out. Upon the titration of [Cu2L] with Cys, the absorbance of the band at ∼426 nm decreases and the absorbance of the new band observed at ∼360 nm increases because this arises from free L (Figure 5c). However, other amino acids do not show any change in 425 nm band, as well do not show the two new bands corresponding to the free L (Figure 5d and Supporting Information, Figure S37). Therefore, the changes observed only in case of Cys is imputable to the displacement of Cu2+ from [Cu2L]. This has been further supported by observing a Cys complex of copper in ESI MS and also by observing color change only in case of Cys supporting the release of L (Supporting Information, Figure S38). Secondary Sensing Properties of [Zn2L]. The in situ prepared Zn2+ complex of L shows strong fluorescence emission at ∼495 nm upon excitation at 360 nm (Figure 2). The titrations were carried out with all the 20 amino acids. The fluorescence of [Zn2L] has been completely quenched in the presence of His and only to a marginal extent in presence of Cys and Asp (Figure 6). All the other amino acids do not show any significant fluorescence quenching (Figure 6 and Supporting Information, Figure S39−S41). Hence, the trend in fluorescence quenching of [Zn2L] follows an order, namely, His ≫ Asp ≫ Cys. This may be ascribed mainly to the dechelation of the Zn2+ from the coordination sphere of [Zn2L], followed by the formation of His complex using its side chain imidazole. The minimum detectable concentration of His is 0.33 ppm (2.15 μM) based on fluorescence titration (Supporting Information, Figure S41). Therefore, the CsE of [Zn2L] is useful as selective and sensitive receptor for His. The Zn2+ displacement from [Zn2L] by His has been further studied by carrying out absorption titrations. Upon the titration of [Zn2L] with amino acids only His exhibits the decreases in absorbance of the ∼415 nm band and that of the new bands, namely, ∼355 and ∼485 nm absorbance is increases. The new bands correspond to the free L (Figure 6). The isosbestic points observed at ∼380 and 460 nm indicates the transition between the zinc bound and unbound species. However, other amino acids do not show such absorbance changes (Supporting Information, Figure S42). This has been further supported by observing the zinc complex of His in ESI MS and also by observing the color change (Supporting Information, Figure S43 and S44). The final color of the {[Zn2L] + His} mixture is same as that of the L supporting the dechelation. Therefore, the significant changes observed only in case of His are ascribed to

Figure 6. (a) Fluorescence spectra obtained during the titration of [Zn2L] with His in methanol, λex = 360 nm; inset shows the relative fluorescence intensity (I/I0) as a function of [His]/[Zn2L] mole ratio at 495 nm band. (b) Histogram showing the fluorescence response of [Zn2L] with amino acids. (c) Absorption spectra obtained during the titration [Zn2L] with His; inset shows the plot of absorbance versus [His]/[Zn2L] mole ratio for different absorption bands. (d) Histogram shows the absorbance of [Zn2L] at 415 nm band when titrated with different amino acids.

the displacement of Zn2+ from the complex and form its His complex while providing selective sensing.

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CONCLUSIONS AND CORRELATIONS The calix[4]arene conjugate, L has been synthesized and characterized and the structure has been determined based on single crystal XRD. The binding characteristics of the conjugate, L has been studied toward the transition metal ions, such as, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ which are integral part of biology as cofactors in various metalloproteins and metalloenzymes. Among these metal ions, Zn2+ shows enhancement in the fluorescence intensity of L, whereas Mn2+, Fe2+, Co2+, Ni2+, and Cu2+ shows quenching. Hence, the conjugate, L exhibits distinguishable fluorescence and absorption changes with these metal ions through forming 1:2 “L to metal ion” ratios as observed from the ESI MS. In all these, the calix[4]arne platform is indispensable, since the studies carried out with noncalixarene analogues failed to show such metal ion selectivity.13 The in situ complexes of these metal ions, namely, [Mn2L], [Fe2L], [Co2L], [Ni2L], [Cu2L], and [Zn2L] have been used as chemosensing ensemble for the recognition of amino acids via complete demetalation. On the basis of the spectral data, it is reasonable to conclude that the oxidation state of the metal ion remains same during sensing. Based on these, the selective sensing behavior follows as [Mn2L] for Glu and Asp, [Fe2L] for none, [Co2L] for His, Cys, and Asp, [Ni2L] for His and Asp, [Cu2L] for Cys through “turn on” fluorescence, and [Zn 2 L] for His through “turn off” fluorescence (Scheme 2). The minimum detectable concentration of these amino acids has been given as table in Scheme 2a. The results obtained in the current findings exhibit significant similarities with the biological systems where the affinity of a particular amino acid toward a metal ion plays crucial role for its selectivity followed by activity of a metalloenzyme. As an example in the metalloprotiens, Mn2+ 8298

dx.doi.org/10.1021/ac301821c | Anal. Chem. 2012, 84, 8294−8300

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Scheme 2. (a) Minimum Detectable Concentrations for Amino Acids by the [M2L] Based CsE and (b) a Flow Chart Representation for the Metal Ion Preferences for the Given Amino Acids As Found in Current Findings where the Middle Portion Represents the Multiple Sensing Arrays of [M2L] for Selected Amino Acids

binds preferably with the Asp and Glu, Co2+ binds with the His, Cys, and Asp, Ni2+ binds with the His and Asp, and Cu2+ and Zn2+ binds with Cys and His. While these are involved mainly in the primary coordination, few other amino acids supports to complete the coordination sphere. Thus the proteins rich in His would have high preference toward Co2+, Ni2+, and Zn2+; those rich in Asp would prefer to have Mn2+, Co2+, and Ni2+ with a greater affinity of Glu toward Mn2+; and the proteins with rich binding sites of Cys prefers to have Cu2+, Zn2+, and Co2+ with a high preference toward Cu2+ followed by Zn2+ (Scheme 2b). In fact these findings resemble the results obtained from the studies carried out in the analysis of amino acids found in the primary coordination sphere of the corresponding metalloproteins. Hence L can multiply sense biologically active amino acids of metalloproteins, in the presence of different biologically essential transition metal ions, by eliciting appropriate fluorescence response to result in a multiarray molecular tool.

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*Phone: 91 22 2576 7162. Fax: 91 22 2572 3480. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.P.R. acknowledges the financial support from DST, CSIR, and DAE-BRNS. R.K.P. acknowledges C.S.I.R for his fellowship. J.D. acknowledges DRDL, Hyderabad for allowing him to pursue the Ph.D. program at IIT Bombay.

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REFERENCES

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