A fluorescent di-zinc(II) complex of bis-calix[4]arene conjugate as chemosensing-ensemble for the selective recognition of ATP

A triethylene glycol di-imine locked triazole linked bis-calix[4]arene conjugate L has been synthesised and characterised. Conjugate L exhibits high fluorescence enhancement towards Zn2+ among the 13 metal ions studied down to a lower detection limit of ∼12 ppb. The absorption and visual colour change experiments differentiated the Zn2+ from the other metal ions studied. The isolated zinc complex, [Zn2L] has been used as a chemo-sensing ensemble for the recognition of anions based on their binding affinities towards Zn2+. [Zn2L] was found to be sensitive and selective towards phosphate-bearing species and in particular to adenosine triphosphate (ATP2 − ) among the other 20 anions studied as observed based on the changes occurred in the fluorescence intensity. The selectivity of the ATP2 − has been shown on the basis of the changes observed in the emission and absorption spectral studies. The lowest detectable concentration for ATP2 − with the chemo-sensing ensemble [Zn2L] is 348 ppb in methanol. The fluorescence quenching by the phosphate-based anions has been modelled by molecular mechanics studies and found that the anions possessing two or more phosphate moieties can only bridge between the two zinc centres, and hence those possessing only one phosphate moiety (H2PO4− and AMP2 − ) are ineffective.


Introduction
Ion and molecular recognition in the complex chemical and/or simulated biological medium received momentum over the past couple of decades and this turns out as one of the important areas of suprmolecular chemistry (1). Among the available scaffolds, the calix[4]arene plays pivotal role as platform in the design and development of molecular sensors in order to understand the cascade of biological events (2). Transition metal ion, especially Zn 2þ , is one of the most plenteous ions present in the human tissue to deliver its service to maintain various biological pathways (3). Therefore, the imbalance in its concentration can be of positive indication of certain diseases. On the other hand, the biologically abundant anions, such as chloride, phosphate and carbonate contributes significantly to the maintaining of scrupulous amount of Zn 2þ required in the biological fluids. Pyrophosphate (PPi 42 ) and ATP 22 are the two strongly chelating anions among the phosphates and show potent binding affinity to Zn 2þ (4). Therefore, the recognition of Zn 2þ and phosphates using the same molecular system can be of great advantage to understand their homeostatic nature (5a, b) in several malignancies (5c). However, it is not an easy task to design a molecular system which can selectively produce signals for a particular cation or anion of interest (6). Hence, the design and topological features of the molecular ensemble play an important role to determine the selectivity for a particular analyte. Recently, our group and others have designed several metal ionbased fluorogenic molecular systems for the selective recognition of anions and amino acids (7). To our knowledge, no molecular sensor based on a bis-calix [4] arene has ever been reported for sensing ATP 22 . Thus, in this paper, we present the synthesis and characterisation of 1,3-triazole linked bis-calix[4]arene conjugate L and its Zn 2þ complex as a molecular ensemble for the recognition of ATP 22 .

Titrations of L with metal ions
The interaction of the biologically and environmentally important metal ions with L has been studied by fluorescence and absorption spectroscopy. The fluorescence studies have been carried out by exciting the solutions of L at 325 nm and measuring its emission spectra in the range 335 -600 nm, while the l max of the emission is centred , 450 nm. Among the 13 ions studied, viz. Na þ , K þ , Mg 2þ , Ca 2þ , Mn 2þ , Fe 2þ , Co 2þ , Ni 2þ , Cu 2þ , Zn 2þ , Cd 2þ , Hg 2þ and Pb 2þ , only Zn 2þ exhibits significant fluorescence enhancement with L ( Figure 1(a), (b)) (S11 Supplementary Information, available online). The fluorescence titration results were similar even when the excitation was centred at 370 nm, which is a charge transfer band that is developed upon Zn 2þ binding in its absorption spectrum. The fluorescence emission intensity increases as a function of the addition of Zn 2þ and saturates , 2 equivalents with an overall enhancement of , 38^3 fold when compared to the simple conjugate L. The quantum yields of L and its complex with Zn 2þ are 0.004 and 0.226, respectively, with reference to the quinine sulphate. The 1:2 stoichiometry of L to Zn 2þ in the complex formed during the titration was obtained from the Job's plot, and the species were confirmed by ESI-MS.
The binding of Zn 2þ to L with K a ¼ (5.3^0.6) £ 10 4 M 21 has been derived based on the Benesi -Hildebrand Equation (S13 Supplementary Information, available online). The minimum concentration of Zn 2þ that can be detected by L is 12^0.5 ppb (0.18 mM) (S15 Supplementary Information, available online) as determined by the fluorescence titration carried out at a mole ratio of L to Zn 2þ of 1:2, but by varying the concentration of the individual species. The Zn 2þ sensing of L has been further supported by observing the intense blue fluorescence colour only in case of Zn 2þ and not in the presence of any other M nþ under an incident light of 365 nm ( Figure 1(c)).
In order to confirm the binding mode of L with different metal ions, absorption spectral studies were carried out in methanol ( Figure 2). During the titration, the absorbance of the bands observed at 277 and 375 nm increases while those of 322 and 425 nm decreases. The changes observed in the absorbance of these bands cease upon addition of two equivalents of Zn 2þ , supporting the formation of 1:2 complex between L and Zn 2þ . Thus, the spectral changes and the isosbestic points observed at 286, 338, and 404 nm clearly suggests the complex formation between L with Zn 2þ . The strength of the binding of Zn 2þ to L having K a ¼ 5.6 £ 10 4 M 21 is in agreement with that observed in case of fluorescence titration (S17 Supplementary Information, available online).

Anion recognition studies of the Zn 21 complex [Zn 2 L]
As the [Zn 2 L] complex exhibits high fluorescence intensity, it would be of interest to study the utility of this complex in the recognition of anions including phosphates. Therefore, the isolated zinc complex has been used as a chemosensing ensemble for the recognition of anions in methanol.

Synthesis, isolation and characterisation of [Zn 2 L]
The Zn 2þ complex of L, i.e. [Zn 2 L] has been prepared by mixing L with zinc(II) acetate in methanol followed by refluxing the reaction mixture for 5 h (see 'Experimental details' section). The resultant complex has been isolated as solid product and was well characterised by 1 H and 13 C NMR and HRMS. 1

Fluorescence titrations
The utility of the [Zn 2 L] for the recognition of anions has been evaluated by carrying out the fluorescence and absorption titrations with different anions, viz. F 2 , Cl 2 , Br 2 , I 2 , N 3 2 , CO 3 (Figure 5(a)). Similarly, when [Zn 2 L] was titrated with other anions, no significant fluorescence quenching was observed with anions other than the phosphates (S19 Supplementary Information, available online).
All this clearly suggests that the [Zn 2 L] acts as a sensitive chemosensing ensemble for phosphate-based anions, in particular for ATP 22 , among the 20 anions studied ( Figure 5(b)). The minimum detection limit for the ATP 22 with [Zn 2 L] has found 348 ppb (S21 Supplementary Information, available online).

Absorption titrations
In order to verify the results obtained from the fluorescence, absorption titrations of [Zn 2 L] were carried out with different anions. Upon the titration of [Zn 2 L] with  In the minimised structures of these complexes, the X is settled through bridging the two Zn 2þ centres (Zn a and Zn b ) to result in distorted octahedral geometry with a binding core of N 2 O 4 where two of the oxygens are from the X. In bridging between the two Zn 2þ centres, the X brings changes in the structural features of the precursor, [Zn 2 L 0 ], to different extents depending upon the nature of the X, though the Zn a to Zn b distance changes from 7.6 Å to 6.1 -6.3 Å in case of the three complexes. All these changes can be clearly seen by comparing the stereo views of the corresponding complexes of [Zn 2 L 0 X] (Figure 7). Such complex formation by X through bridging, viz. [Zn 2 L 0 X], results in quenching the fluorescence intensity as observed experimentally with the precursor di-zinc complex. In the complexes of [Zn 2 L 0 X], the metric parameters about each Zn 2þ differ, thereby disrupting any symmetry that may have been present in the precursor (Figure 8). One of the Zn 2þ centres of the complexes, viz. Zn a , exhibits trans-angles in the range of 151 -1638 and the other angles in the range of 65 -1188 suggesting distorted zinc centre and this trend is almost same in all the three cases. However, in case of Zn b , the trans-angles are in the range of 137 -1568 and the other angles are in the range of 63 -1308. The trans-angle turns out to be more acute on going from PPi 42 to ADP 22 to ATP 22 owing to their respective increase in the bulkiness of these moieties (S27 Supplementary Information, available online).
However, the control species, viz. HPO 4 22 and AMP 22 , possessing single phosphate moiety cannot span across the two zinc centres to bridge and hence no complex was observed in the MM computational results in these cases.
Thus, this result obtained from the computation is in agreement with the fluorescence data, wherein the fluorescence intensity is not affected by the titration of HPO 4 12 and AMP 22 , unlike the other three, viz. PPi 42 , ADP 22 and ATP 22 where in the fluorescence intensity is quenched due to the capability of these ions to bridge the two zinc centres.

Conclusions and correlations
A triethylene glycol di-imine-locked triazole linked biscalix[4]arene conjugates L and its di-Zn 2þ complex [Zn 2 L] have been synthesised and well characterised by various spectral techniques, viz. 1 H, 13 C NMR and HRMS. In the precursor, L as well as its di-Zn 2þ complex exhibit cone confirmation for the calixarene as evident from the corresponding 1 H NMR spectra. L showed high fluorescence enhancement towards Zn 2þ among the 13 metal ions, viz. Na þ , K þ , Mg 2þ , Ca 2þ , Mn 2þ , Fe 2þ , Co 2þ , Ni 2þ , Cu 2þ , Zn 2þ , Cd 2þ , Ag þ and Hg 2þ studied by eliciting an emission signal at , 450 nm. The absorption spectra further confirmed the binding of Zn 2þ to L. Both the fluorescence enhancement and the absorption spectral changes revealed an association strength of K a ¼ (5.3^0.6) £ 10 4 M 21 for the binding of Zn 2þ to L. The fluorescence studies also yielded a minimum detection of 12 ppb (0.18 mM) for Zn 2þ by L. The ESI-MS confirmed the formation of 1:2 complex for L to Zn 2þ based on the corresponding m/z peak and the isotopic peak pattern. 1 H NMR spectrum of L and the isolated [Zn 2 L] in CDCl 3 were undertaken to further determine the complexation of L with Zn 2þ and the corresponding spectral differences can be seen in Figure 3.The absorption spectral features of L are similar in both their solid and solution states. The same is true even for the [Zn 2 L]. In case of fluorescence study, the peak position for [Zn 2 L] shifts from 450 to 467 nm on going from its solution to the solid state, thus exhibiting a red shift of , 17 nm in the solid state. The isolated [Zn 2 L] complex showed selectivity towards phosphate-bearing anions among all the anions, viz. F 2 , Cl 2 , Br 2 , I 2 , N 3 2 , CO 3 22 , NO 2 2 , NO 3 2 , SCN 2 , SO 4 22 , ClO 4 2 , HSO 4 2 , HCO 3 2 , CH 3 COO 2 , HPO 4 22 , H 2 PO 4 2 (Pi) and P 2 O 7 42 (PPi), AMP 22 , ADP 22 and ATP 22 studied by fluorescence and absorption spectroscopy. While the fluorescence showed quenching of the emission intensity in case of anions having di-phosphate moiety, the absorption spectra showed appropriate changes due to the interaction. The displacement of Zn 2þ from the [Zn 2 L] has been further supported by observing peaks at m/z ¼ 575, 1309 which corresponds to the formation of adduct of {Zn.ATP} and the free L, respectively, in the ESI-MS spectra. The minimum detection limit for the ATP 22 with [Zn 2 L] is 348 ppb. The binding of the anions containing diphosphate moieties, viz. PPi 42 , ADP 22 and ATP 22 through bridging the two Zn 2þ centres present in the complex has been demonstrated by MM computations. The H 2 PO 4 2 and AMP 22 possessing single phosphate moiety does not bind to the complex as demonstrated by the computational studies, which is in agreement with the fact that these ions are unable to quench the fluorescence intensity of the di-Zn 2þ complex.

General information
Bulk solutions of L and the metal salts were prepared in MeOH by initially dissolving L in 50 mL of CHCl 3 .The concentration of the bulk solution of L has been maintained as 10 24 M throughout the preparations for all the titrations. All the fluorescence titrations were carried out by exciting L at 325 nm of the solutions taken in 1 cm quartz cell by maintaining a final [L] at 1 mM in a total volume of 3 mL achieved by diluting with MeOH. All the metal ions used for the fluorescence and absorption titrations were used as their perchlorate salts. L] and the anions were maintained as 6 £ 10 24 M. Absorption studies were carried out in a similar manner as in fluorescence studies.

Synthesis and characterisation of L
A mixture of P 2 (1.00 g, 0.839 mmol) and 2,2 0 -(ethylenedioxy)bis(ethylamine) (0.125 g, 0.839 mmol) in methanol was stirred for 4 h. The solvent was removed under vacuum to get yellow solid which was further recrystalised using chloroform and diethyl ether to get pure solid product. 1

Computational studies
In order to understand the interactions present between [Zn 2 L] and the phosphate-based species, MM computations using MM þ force field in Hyperchem (8). Prior to assuming the initial guess model for computational calculations, L was independently optimised by using semi-empirical method taking the coordinates from the crystal structure (3b) reported by us by bringing the following modifications: (i) removal of n-butyl and Zn 2þ ; (ii) protonation of salicyl O 2 and (iii) construction of biscalixarene by adding the 2,2 0 -(ethane-1,2-diylbis(oxy)) diethanamine moiety to the ortho position of salicyl OH. Before optimisation, the L 0 was built simply by replacing each of the tert-butyl group of L by a hydrogen. The L 0 thus obtained was optimised by using PM3. The deprotonated form of the optimised L 0 was used for the complexation with two equivalents of Zn 2þ ions. The optimisation of the di-zinc complex formation, viz.
[Zn 2 L 0 ], has been carried under PM3 semi-empirical method. In order to make the complexes of [Zn 2 L 0 ] with phosphate possessing species (X ¼ PPi 42 , ADP 22 and ATP 22 ), further computational studies were carried out by MM using MM þ force field.
Supporting information 1 H and 13 C NMR, and mass spectral data for L and [Zn 2 L]; fluorescence and absorption spectra of all the metal ion titrations with L and all the anion titrations with [Zn 2 L] and the computational data have been given in the supporting information. This material is available free of charge via the Internet.