Combined photophysical, NMR and theoretical (DFT) study on the interaction of a multi component system in the absence and presence of different biologically and environmentally important ions

Abstract

A simple multi-component system, comprising fluorophore (dansylamide), spacer (propyl) and receptor (dimethylamino) units, has been synthesized in order to investigate the receptor–analyte binding interactions in the presence of both cations and anions within a single molecular system. While the acidic N–H proton of the dansylamide group is expected to interact with a basic anion, the dimethylamino group is expected to interact with metal ions. The photophysical behaviour of this system in the absence and presence of various metal ions and anions is investigated in acetonitrile (ACN) medium. The absorption and fluorescence spectra of the system in ACN consist of broad bands, typical of the intramolecular charge transfer (ICT) nature of the system. A significant reduction in the fluorescence intensity of the system in the presence of fluoride has been observed. No significant changes in the optical properties of the system have been noticed in the presence of other commonly encountered anions. The interaction of fluoride with the present system has also been monitored through ¹H NMR experiments. The formation of the HF₂⁻ species due to the abstraction of the acidic proton by F⁻ is believed to be responsible for the fluoride ion selective signalling behaviour. In the case of cations, a bathochromic shift in fluorescence, as well as a concomitant decrease in fluorescence intensity, of the present system has been observed in the presence of transition metal ions. Interestingly, no bathochromic shifts in absorbance or fluorescence, were observed when a similar experiment was carried out employing a structurally similar compound which is devoid of the dimethylamino group. The cation signalling event has also been monitored through FTIR and ¹H NMR experiments. Spectroscopic results indicate a ground state 1 : 2 complex formation between the present system and the transition metal ions. Theoretical calculations have also been carried out in the presence of various analytes to throw more light on the binding interactions. Additionally, through fluorescence microscopy studies, it has been observed that the present system is non-toxic in a cellular environment, and has the potential to be used in live-cell imaging studies.

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1. Introduction

The increasing amount of pollutants in air, water and soil is a major concern facing the human race today. In an atmosphere with an increasing number of unwanted chemical entities, the development of optical sensors or chemosensors for different analytes in environmental analysis and medical diagnosis applications is of great importance.^1–22 Even though different metal and non-metal ions are important in biology,⁴ they pose a serious environmental concern when present in uncontrolled amounts.^23–25

The rapid growth of supramolecular chemistry and cutting-edge technologies has enabled researchers to develop optical sensing devices, such as sensors, molecular logic gates, etc.^1–14 Signaling via optical means and fluorescence, in particular, have attracted more attention compared to classical techniques due to their ability to monitor in situ analyte concentrations in real time and in real space.²⁶ Single molecule level detection is now also possible.²⁷

Signalling systems have been designed by employing organic chromophores/luminophores, inorganic–metal frameworks, coordination frameworks or by a suitable combination of any of these. This allows one to obtain various structural motifs where the signaling by optical means can be achieved in the presence of various chemical inputs such as ions, neutral molecules, etc.^1–6 Furthermore, the design of a system is such that the binding of anions and cations to their corresponding receptors will be capable of altering the electron transfer or charge transfer between the donor and the acceptor. These binding events are monitored through changes in the absorbance and fluorescence response upon the binding of anions and cations to the respective receptors. Another important aspect of sensor design includes the signalling of the binding events of metal ions through ratiometric changes in the photophysical behaviour of the sensor system.^15,16 In the case of metal ions, electron rich ligand centres have been used such that an effective metal–ligand (receptor) coordinative interaction is responsible for the binding of cations.^5–15 While for targeting anions, molecular systems having polarized N–H fragments that are capable of forming H-bonds with anions have generally been used as receptors for recognition and sensing in aprotic solvents.^17,22

Even though a large number of reports on selective chemosensors are present in the literature, studies that focus on the mechanistic details of fluorophore–analyte interactions are limited. Investigation of the details of the sensing mechanism is fundamental to the design and development of new and improved sensors with higher sensitivity and selectivity. Also, theoretical calculations are required to corroborate the experimental results in order to gain deeper insights at the molecular level.^17,28–32 Though several sensor systems have already been developed for various analytes, still there is a huge demand for sensors which are selective for an analyte of interest and which can also be exploited for the simultaneous detection of cations and anions.

Developing a simple molecular system which can detect both cations and anions is challenging and reports on the photophysical study of such molecules are limited. In the present work, we report two dansyl based fluorophores, 3,3-dimethylaminopropyl dansylamide (DANSn2) as a fluoroionophore and pentyl dansylamide (DANSp) as a structurally similar reference compound (Scheme 1), where the detection of analytes (cation and anions) is possible through receptor–analyte interactions. In the present system, while the anion binding event is expected to be mediated through hydrogen bonding interactions, cation signalling occurs through coordination of the N-atom lone pair of the dimethylamino group to the metal ions. DANSp was purposefully chosen to monitor the effect of the terminal dimethylamino group (attached to the propyl spacer) on the binding event.

Scheme 1 Molecular structures of the target molecule DANSn2 and parent molecule DANSp.

Quantum mechanical calculations based on density functional studies have been carried out to understand the fluorophore–analyte interactions responsible for the selectivity and specificity of the system. Further confirmation of the mechanistic details is obtained from NMR studies of the system in the presence of varying concentrations of analyte. The outcome of the present combined experimental and theoretical study of receptor–analyte interactions is expected to be useful in interpreting the subtleties of the signalling mechanism and consequently designing more efficient chemosensors for a wide range of analytes.

2. Experimental section

2.1. Materials

Dansyl chloride, 1,3-diaminopropane, 3-(dimethylamino)-1-propylamine, 4-chloro-7-nitrobenzoxadiazole and the solvents used were purchased from Sigma-Aldrich. In the titration experiments all anions, in the form of tetrabutyl ammonium (TBA) salts, and all cations, in the form of perchlorate salts, were purchased from Sigma-Aldrich. For titration purposes, the solvents were dried and distilled using published procedures.³³ The deuterated solutions of chloroform (CDCl₃) and dimethylsulfoxide [(CD₃)₂SO] used for NMR titrations, were also purchased from Sigma-Aldrich.

2.2. Synthesis of the molecular systems

2.2.1. Synthesis and characterization of 3-(dimethylamino)-1-propyldansylamide (DANSn2). The compound 3-(dimethylamino)-1-propyldansylamide was earlier synthesized by Ceroni et al.³⁴ Similar methodologies have been employed to synthesize the present compound. To a solution of 3-(dimethylamino)-1-propylamine (1.7 g, 16.68 mmol) in DCM (25 mL), dansyl chloride (100 mg, 0.38 mmol) in dichloromethane (DCM) (25 mL) was added dropwise at 0 °C and the reaction was allowed to stir for 1 hour, while allowing the solution to gradually attain room temperature. The reaction mixture was then extracted with a 1 M aqueous solution of HCl (3 × 30 mL). The aqueous phase was basified with 5 M NaOH solution. Again the aqueous phase was extracted with DCM. The collected organic phase was dried over anhydrous Na₂SO₄ and the organic phase was then evaporated under reduced pressure to obtain a yellowish-white product (Scheme 2).³⁴ The final product, DANSn2 was characterized by ¹H NMR spectroscopy and mass spectrometry.

Scheme 2 Synthetic route for DANSn2.

¹H NMR; (CDCl₃): δ = 8.44 (d, 1H), 8.27 (d, 1H), 8.08 (d, 1H), 7.60 (d, 1H), 7.58 (d, 1H), 7.24 (d, 1H), 2.81 (s, 6H), 2.78 (t, 2H), 1.96 (t, 2H), 1.87 (s, 6H) and 1.35 ppm (q, 2H). ESI-MS: 336.46 (M + H⁺). Yield = 97%.

2.2.2. Synthesis and characterization of pentyldansylamide (DANSp). To a solution of pentylamine (1.5 g, 16.78 mmol) in DCM (15 mL), dansyl chloride (50 mg, 0.19 mmol) in DCM (15 mL) was added dropwise at 0 °C and the reaction was allowed to stir for 1 hour, while allowing the solution to gradually attain room temperature. The reaction mixture was then extracted with a 1 M aqueous solution of HCl (3 × 30 mL). The aqueous phase was basified with 5 M NaOH solution. Again the aqueous phase was extracted with DCM. The collected organic phase was dried over anhydrous Na₂SO₄ and the organic phase was then evaporated under reduced pressure to obtain a yellowish-white product (Scheme 3). The final product, DANSp, was characterized by ¹H NMR spectroscopy and mass spectrometry.

Scheme 3 Synthetic route for DANSp.

¹H NMR; (CDCl₃): δ = 8.29 (d, 1H), 8.26 (d, 1H), 8.24 (d, 1H), 7.59 (d, 1H), 7.53 (d, 1H), 7.20 (d, 1H), 2.91 (s, 6H), 2.88 (t, 2H), 1.36 (t, 2H), 1.13 (d, 2H), 1.11 (d, 2H) and 0.74 ppm (t, 3H). ESI-MS: 321.45 (M + H⁺). Yield = 94%.

2.3. Fluorescence quantum yield measurements

For the quantum yield measurements, optically matched solutions (at the excitation wavelength) were used. The quantum yield was obtained by measuring the integrated area under the emission curves and using eqn (1),³⁵ where, ϕ is the quantum yield, I is the integrated emission intensity, OD is the optical density at the excitation wavelength, and η is the refractive index. The fluorescence quantum yields of DANSn2 were measured using anthracene (in cyclohexane, ϕ = 0.36)³⁶ as a standard.

2.4. Binding constant evaluation from fluorescence titration

The binding constants for the analytes were also determined from the fluorescence data using a Benesi–Hildebrand plot³⁷ derived from eqn (2), where F₀ is the fluorescence intensity in the absence of analytes, F is the fluorescence intensity in the presence of analytes, F_f is the fluorescence intensity following the final addition of analyte, K is the binding constant and [M] is the analyte concentration.

2.5. Computational methods

The ground-state structures of DANSn2 were optimized using the hybrid density functional M06-2X^38,39 and 6-31+G(d)⁴⁰ and LANL2DZ (metal) basis sets.⁴¹ This level of theory is known to be successful in predicting the structure and molecular properties in different chemical reactions.^42,43 All positive vibrational frequencies obtained at the same level of theory ensure that the located geometries represent true minima. The interaction energies of the DANSn2–metal ion complexes (DANSn2⋯Mⁿ⁺) were obtained by calculating, ΔE_int = E_{(DANSn2⋯Mⁿ⁺)} − E_(DANSn2) − E_Mⁿ⁺, where E_{(DANSn2⋯Mⁿ⁺)}, E_(DANSn2), and E_Mⁿ⁺ represent the energies of the metal complex, DANSn2, and metal ion, respectively. All the calculations were carried out using the Gaussian 09 suite of programs.⁴⁴

3. Instrumentation

For ¹H NMR spectra, a Bruker Avance 400 NMR spectrometer was used with tetramethylsilane (TMS) as an internal standard at ambient temperature. ESI-MS spectra were obtained with a Bruker micrOTOF-QII spectrometer. All the steady-state absorption spectra and fluorescence spectra were collected using a Cary 100 Bio spectrophotometer and a Perkin-Elmer, LS 55 spectrofluorimeter respectively. A time-correlated single-photon counting (TCSPC) spectrometer (Edinburgh, OB920) with a light emitting diode (LED) source (λ_exc. = 330 nm, FWHM = 700 ps) was used for all time-resolved measurements. A micro channel plate (MCP) photomultiplier (Hamamatsu R3809U-50) was used as the detector (response time 40 ps) for all time resolved measurements. A dilute ludox colloidal dispersion in water was employed as the scatterer to measure the source profile. Decay curves were analyzed using the nonlinear least-squares iteration process using F900 decay analysis software. The quality of the fit was judged using the chi square (χ²) values, and the weighted deviation was obtained by fitting.

4. Results and discussion

4.1. Molecular design of DANSn2

The design of the target molecule (DANSn2) was based on a [receptor (cation)–fluorophore–receptor (anion)–spacer–receptor (cation)] architecture. The dansylamide moiety was consciously chosen mainly because of its high fluorescence quantum yield.¹⁵ The two terminals of the propyl spacer were expected to be responsible for the binding interactions, with one end having a dimethylamino group for cation binding and the other end having a sulfonamide moiety for anion binding. The polarisable sulfonamide N–H proton can participate in hydrogen bonding interactions with anions.⁴⁵ Additionally, the dimethylamino group present in the dansylamide moiety also has the ability to bind cations through coordination of the N-atom lone pair. The fluorescence excitation spectrum of the probe at λ_emi = 538 nm, is reported in the ESI, Fig. S1,† in order to confirm the purity of the molecule. The excitation spectrum and absorption spectrum of the molecule are observed to be very similar to each other.

4.2. Photophysical properties DANSn2

4.2.1. Steady-state UV-visible absorption spectra. The representative absorption and emission spectra of DANSn2 in acetonitrile (ACN) are shown in Fig. 1. The spectral data corresponding to the absorption and emission maxima of DANSn2 in several solvents of varying polarity are provided in Table 1. Free DANSn2 shows a broad absorption band with a peak maximum at 338 nm and a broad fluorescence band with a maximum at 530 nm in ACN (Fig. 1). Upon increasing the polarity of the medium from cyclohexane to ACN, bathochromic shifts of both the absorption (∼9 nm) and emission (∼69 nm) maxima are observed (Table 1). From this solvatochromic response of the DANSn2 molecule, it can be inferred that the broad absorption and emission bands of the present system are due to the intramolecular charge-transfer (ICT) transition from the dimethylamino nitrogen (present in the dansylamide moiety) to the sulfonamide moiety of DANSn2, which is consistent with other dansylamide based systems.^15–20

Fig. 1 Absorption and emission spectra (λ_exc. = 338 nm) of DANSn2 in ACN. All spectra are normalized at their corresponding peak maximum.

Table 1 Absorption and fluorescence spectral data of DANSn2 in cyclohexane, tetrahydrofuran (THF), and ACN

	Cyclohexane (32.4)^a	THF (37.4)^a	ACN (45.6)^a
a Quantities in the parentheses indicate the micro-polarity parameter [ET (30)] values of the solvents.
λ abs(max)	329 nm	334 nm	338 nm
λ flu(max)	446 nm	482 nm	515 nm

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Fig. 2 Fluorescence decay curve of DANSn2 in ACN at 298 K at λ_exc. = 330 nm. The black line denotes the line fitted to the decay curve.

The fluorescence quantum yield of DANSn2 in ACN medium measured from the steady state emission spectra is found to be 0.52 (using anthracene as the standard, ϕ_std = 0.36 in cyclohexane).³⁶ This value is quite comparable with the fluorescence quantum yield of its structurally similar analogue, DANSp [ϕ = 0.51 (using anthracene as the standard, ϕ_std = 0.36 in cyclohexane)³⁶]. Thus in the present system the possibility of through space photo-induced electron transfer from the dimethylamino group to the dansylamide moiety can be excluded. Perhaps the orientation of the dialkyl amino group (attached to propyl spacer) with respect to the dansylamide moiety in the present system is not favourable for electron transfer communication. The fact that the relative orientation of the donor and acceptor moieties plays an important role in the electron transfer process is well documented in the literature.^46,47

The time-resolved fluorescence decay behaviour of DANSn2 has also been investigated in ACN medium. For this purpose, DANSn2 was excited at 330 nm and its emission was monitored at 525 nm. The time-resolved emission decay profile of DANSn2 is shown in Fig. 2. The decay profile is fitted to a single exponential function and the lifetime is estimated to be 12.04 ns which is also comparable to the lifetime value (12 ns) of DANSp. This observation also demonstrates that in the present system, excited state phenomenon such as PET is absent.

Fig. 3 Absorption (upper panel) and emission (lower panel) spectra of DANSn2 (∼1.4 × 10⁻⁵ M) in ACN upon the progressive addition of TBAF. The concentrations of TBAF in the various solutions range from 0 M to 5.58 × 10⁻³ M.

4.2.2. Anion interaction with DANSn2. The fluorophore–analyte interaction with various biologically important anions has been studied using spectrophotometric as well as fluorometric titration experiments in ACN medium. All titrations were carried out by adding freshly prepared tetrabutylammonium salts of the corresponding anion (∼10⁻⁴ M) to a dilute solution of DANSn2 (∼10⁻⁵ M) in a stepwise manner. Notably, upon progressive addition of fluoride salt, the intensity of the band at ∼338 nm, corresponding to charge transfer transition, decreases gradually and a new band at 323 nm starts to develop (Fig. 3). The band at ∼323 nm, in principle can arise due to the formation of a receptor–F⁻ complex through hydrogen-bonding interactions between the NH proton of the sulfonamide moiety of DANSn2 and F⁻. If the polarisable fragment is sufficiently acidic it may also be possible for fluoride to deprotonate the acidic proton (Scheme 4).⁴² In that scenario, investigations based on NMR and theoretical studies would be required to establish the binding events (vide infra).

Scheme 4 Receptor–fluoride complex formation through hydrogen-bonding interaction.

In the fluorescence response, with the progressive addition of F⁻ to the solution of DANSn2, a significant decrease in the intensity of the 525 nm fluorescence band takes place (Fig. 3). A small blue shift in the fluorescence band (Fig. S2, ESI†) upon the progressive addition of TBAF is also observed. An increase in electron density around the sulphonamide moiety, due to the deprotonation of the NH proton of sulfonamide by F⁻, perhaps inhibits charge transfer from the dialkyl amino moiety to the sulphonamide group which causes the blue shift. In this context, the variation in the quantum yield of DANSn2 with the addition of F⁻ has been provided in Fig. S3 (ESI†). The variation of the relative fluorescence intensity of DANSn2, with and without the addition of fluoride is also shown in Fig. S4 (ESI†). The monotonous decrease in the emission intensity of DANSn2 upon addition of F⁻ without any appreciable shift in the steady-state absorption spectrum indicates that the final species that is formed due to the binding of F⁻ to DANSn2 is very weakly fluorescent in nature.

To test the selective behaviour of the present system, we have carried out similar experiments with other commonly encountered anions using UV/Vis absorption and fluorescence titration measurements. The system did not show any appreciable changes in the fluorescence intensity with other commonly encountered anions, except for a small change that was observed for the acetate ion. The selective behaviour of the system towards fluoride is clearly manifested when we plot the fluorescence response of the system towards the anions in a bar diagram (Fig. 4).

Fig. 4 The bar diagram showing the fluorescence response of DANSn2 towards selected anions in ACN. F₀ stands for the fluorescence intensity of the free molecules (at the emission maximum 525 nm), and F stands for the fluorescence intensity in the presence of the anions (at the same wavelength). The concentration of all the anions is 5.5 × 10⁻³ M.

The binding constant of DANSn2 with F⁻ is calculated from the fluorescence spectra using the Benesi–Hildebrand equation.³⁷ The binding constant is estimated to be 1.53 × 10⁴ M⁻¹ (Fig. S5†).

To throw more light on the binding interactions of DANSn2 with F⁻, we have carried out FTIR and NMR studies. Fig. 5 represents the FTIR spectra of DANSn2 in the absence and presence of F⁻. It can be seen that the characteristic stretching frequency for the N–H bond of the sulfonamide group, that appears at 3420 cm⁻¹, becomes broadened and appears at a lower energy (3380 cm⁻¹) in the presence of F⁻. This broadening and decrease in the vibrational frequency also support the weakening of the N–H bond due to the hydrogen bonding interaction between the fluoride ion and N–H proton.

Fig. 5 FTIR spectra of DANSn2 in the absence and presence of TBAF.

To confirm further whether the DANSn2–F⁻ interaction is taking place via a hydrogen bonding interaction or by a complete proton abstraction mechanism, we have carried out NMR titration experiments with varying concentrations of F⁻ (Fig. 6). In the NMR spectra, the NH proton of the dansylamide group appears at 7.81 ppm. Upon addition of F⁻, the intensity of the NH proton at 7.81 ppm gradually decreases and at relatively high concentrations of F⁻ the signal at 7.81 ppm is found to be extremely broadened and merges into the base line (Fig. 6). Interestingly, Fig. 6 also demonstrates that a new broad NMR signal at 16 ppm appears at higher concentrations of F⁻. The band at 16 ppm bears the characteristic signature of the HF₂⁻ species.⁴³ Thus the present observation clearly indicates the formation of the HF₂⁻ species, which must be due to the abstraction of the acidic NH proton from the present system by F⁻.

Fig. 6 ¹H NMR spectra of DANSn2 in DMSO-d₆ in the presence of (a) 0 eq., (b) 0.2 eq., (c) 0.5 eq., (d) 1.0 eq., (e) 3 eq. and (f) 4 eq. of TBAF. The inset (zoomed in spectra) shows the gradual decrease in the NMR signal at 7.81 ppm with the appearance of a signal at 16 ppm.

4.2.3. Interaction of cations with DANSn2. The interaction between DANSn2 and metal ions in ACN has been investigated using UV/Vis as well as fluorescence titration experiments. Spectrophotometric titrations have been carried out on a dilute solution of DANSn2 by adding freshly prepared metal perchlorate salt solutions in a stepwise manner. Representative absorption and emission spectra of DANSn2 in the absence and presence of Zn²⁺ are shown in Fig. 7.

Fig. 7 Absorption (upper panel) and emission (lower panel) spectra of DANSn2 (∼1.4 × 10⁻⁵ M) in ACN upon the progressive addition of Zn(ClO₄)₂. The concentration of Zn(ClO₄)₂ in the various solutions ranges from 0 M to 1.8 × 10⁻³ M.

As can be seen from Fig. 7, upon progressive addition of the metal salt, the absorption band at 338 nm that corresponds to the dansylamide moiety is significantly affected and the intensity of the band is found to decrease with the simultaneous formation of a new band at 286 nm. The observed spectroscopic result is a clear indication of the binding of metal ions to the N,N-dimethylamino nitrogen atoms of the dansylamide group. The new band in the blue region (higher energy) resembles the absorption band of an unsubstituted naphthalene moiety (devoid of a dimethylamino group) due to the inhibition of charge transfer from the N,N-dimethylamino nitrogen atoms of the moiety to the dansylamide group upon metal ion binding.

Interestingly, when DANSn2 is excited at 338 nm (corresponding to the charge transfer absorption band), a considerable (∼10–14 fold) decrease in the fluorescence quenching is observed upon the gradual addition of the metal ions (Fig. 7). This quenching of fluorescence can be attributed to the formation of a DANSn2⋯Mⁿ⁺ complex, where the lone electron pair on the nitrogen atom of the dimethylamine group is utilized for metal ion binding, which in turn inhibits electron transfer from dimethylamine to the fluorophore, and as a result, the intensity of the broad intramolecular charge transfer (ICT) emission band at ∼530 nm decreases. It may also be noted here that there is the possibility of formation of a stable six membered chelate ring upon metal binding, where the sulfonamide nitrogen and the dimethylamino nitrogen of the propyl spacer participate in complex formation with the metal ion (Scheme 5). Such interaction, which is thermodynamically feasible, would enhance the charge transfer from the dimethylamino group to the sulphonamide group. Therefore, this chelate complex formation may explain the observation of a red shift in both the absorption as well as the emission band as shown in Fig. 7.

Scheme 5 Most probable binding interaction mode of DANSn2 with a metal ion (Mⁿ⁺).

To confirm further the fact that an interaction between a metal ion and the dimethylamino nitrogen group that is attached to propyl moiety is indeed taking place, we have carried out similar photophysical studies with a reference compound DANSp, which does not contain a dimethylamino group in the spacer unit. We have observed that the photophysical properties of DANSp are similar to that of DANSn2 in the absence of any analyte, and in the presence of transition metal ions, DANSp demonstrates similar changes in its absorption as well as emission spectra to that demonstrated by DANSn2 without experiencing any shift (Fig. 8). This kind of behaviour is expected for DANSp as it does not bear any receptor moiety on the alkyl chain and hence no complexation or chelate formation through the involvement of the dimethylamino group on the alkyl chain is taking place.

Fig. 8 Absorption spectra (upper panel) and emission spectra (lower panel) of DANSp (∼1.2 × 10⁻⁵ M) in ACN upon the progressive addition of Zn(ClO₄)₂. The concentration of Zn(ClO₄)₂ in the various solutions ranges from 0 M to 2.1 × 10⁻³ M.

Similarly, the interactions of DANSn2 in ACN with other cations, for example, other transition metal ions, alkali metals and alkaline earth metals, have also been explored using UV/Vis absorption and fluorescence titration measurements. Among these cations, transition metal ions show significant (∼13 times) fluorescence quenching. However, alkali and alkaline earth metal ions fail to cause any noticeable changes in the emission of DANSn2. The fluorescence quenching responses of different cations in ACN are provided in Fig. S6† and are also compared in Fig. 9.

Fig. 9 Ratiometric plots showing the cation binding ability of DANSn2.

The binding constants for DANSn2 with alkali metals, alkaline earth metals and transition-metal ions were calculated from the fluorescence emission spectra using the Benesi–Hildebrand equation.³⁷ The binding constants for the transition-metal ions are presented in Table 2. As can be seen from Table 2, copper(II) binds to DANSn2 with the strongest affinity followed by Fe²⁺, Zn²⁺, Mn²⁺, and Cd²⁺.

Table 2 Binding constants for transition metal ions with DANSn2

Transition metal ions (M^2+)	Binding constant (1 : 2) (×10^4 M^−2)
Cu	1.45
Zn	0.65
Fe	0.67
Mn	0.32
Cd	0.57

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The relative intensity plots (Fig. 9) also support the observed binding of the transition-metal ions.

In the present case, the stoichiometry (1 : 2) of the complex (DANSn2/Cu²⁺) is confirmed via a Jobs plot (Fig. 10). The Jobs plot also reveals a 1 : 2 stoichiometry for the complex formed between DANSn2 and the Zn²⁺ ion (Fig. S7, ESI†). The mode of binding between the ligand (DANSn2) and the metals ions (Zn²⁺ and Cu²⁺) is again confirmed through FTIR and NMR experiments (vide infra). The binding constant value calculated from steady-state emission measurements for Cu²⁺ is 1.45 × 10⁴ M⁻¹ (Fig. S8†). This value is found to be very similar to the binding constant value (1.32 × 10⁴ M⁻¹) estimated from the steady-state absorption measurements (Fig. S9†). This observation is interesting in the sense that it also provides evidence in favour of the fact that quenching of the fluorescence is related to the formation of a ground state complex upon metal ion binding.

Fig. 10 Jobs plot for the complexation of DANSn2 with Cu²⁺ ions in ACN (λ_exc. = 338 nm).

To observe the suitability of the present system for practical applications in aqueous medium, the interaction of the present system with Cu²⁺ metal ions has also been investigated (Fig. 11) in a mixture of ACN/H₂O (5 : 95, v/v). As seen in Fig. 11, DANSn2 behaves as an excellent sensor, even in aqueous medium, and thus is useful for practical applications.

Fig. 11 Emission spectra of DANSn2 (1.0 × 10⁻⁵ M) in ACN/H₂O (5 : 95, v/v) upon the progressive addition of Cu²⁺. The conc. of Cu²⁺ varies from 1.5 × 10⁻⁴ M to 2.0 × 10⁻³ M (λ_exc. = 338 nm).

To investigate the cation binding ability of DANSn2 further, time resolved fluorescence decay measurements of the present system in the absence and presence of transition metal ions (Cu²⁺ and Zn²⁺) have been performed in ACN medium. The average lifetime of DANSn2 is observed to remain unaffected with the gradual addition of metal ions. It can be concluded that the metal–fluorophore interaction (complexation) is in fact a ground state phenomena. We have earlier shown that in the present molecule any excited state phenomenon such as PET is absent.

To further illustrate the binding interactions of DANSn2 with Cu²⁺, we have carried out FTIR and NMR studies. Fig. 12 presents the IR spectra of DANSn2 in the absence and presence of Cu²⁺. It can be seen that the characteristic stretching of the N–H bond of the sulfonamide group present in the dansyl moiety, which appears at 3420 cm⁻¹, becomes broadened and appears at a lower energy in the presence of Cu²⁺. This broadening and the decrease in vibrational frequency also indicate the weakening of the N–H bond, which supports the fact that an interaction between the metal ion and the dimethylamino nitrogen of the propyl spacer is indeed taking place.

Fig. 12 FTIR spectra of DANSn2 in the absence and presence of Cu(II) perchlorate.

In order to confirm the binding site of the metal ions, a ¹H NMR study of DANSn2 in the absence and presence of metal ions has been carried out. It has been observed that the NMR peak corresponding to the six dimethyl protons of the dimethylamino group of the propyl spacer and of the dansylamide moiety undergo a downfield shift from 1.87 to 2.01 ppm and 2.81 to 2.84 ppm respectively, upon the addition of Cu²⁺ ions to the DANSn2 solution (Fig. 13). This shift of the dimethyl protons is expected as the binding of metal ions to the dimethylamine moiety causes a deshielding of the dimethyl protons, and thus a downfield shift in the proton NMR signal. The downfield shift is greater in the case of the aliphatic dimethylamino group (present in the propyl spacer) due to stronger binding interaction with the metal ions. The peaks corresponding to the CH₂ protons adjacent to the dimethylamino group present in the spacer and the N–H proton of the sulfonamide group of DANSn2, show downfield shifts of 0.13 and 0.02 ppm respectively. This observation clearly indicates that both of the N,N-dimethyl groups (one attached to the arene ring, the other attached to the propyl spacer) of DANSn2 are involved in the binding interaction with Cu²⁺ in a 1 : 2 (DANSn2/Cu²⁺) fashion as shown in Scheme 5. Significant broadening of the aromatic protons is observed with the further addition of Cu²⁺ ions to the solution due to the paramagnetic nature of the copper ion. Similarly, with the addition of Zn²⁺ ions to the solution of DANSn2, a downfield shift (Δδ = 0.011) for the N–H proton of the sulfonamide group is observed, whereas the peaks corresponding to the N–CH₃ protons showed a shift of Δδ = 0.027 (for those present on the dansylamide moiety) and Δδ = 0.17 (for those present on the propyl spacer) (Fig. S10, ESI†).

Fig. 13 Zoomed in ¹H NMR spectra of DANSn2 in DMSO-d₆ in the presence of (a) 0 eq., (b) 0.2 eq., (c) 0.5 eq., and (d) 1.12 eq. of copper(II) perchlorate.

4.2.4. Cooperative effect. Finally, in order to investigate whether the binding of one ion affects the binding ability of another ion (i.e. a cooperative effect), we have performed photo-physical studies of DANSn2 when fluoride is added in the presence of zinc (Fig. 14), and when zinc is added in the presence of fluoride (Fig. S11†). The binding constants of the anions in the absence and presence of cations (Table 3), and the binding constants of the cations in the presence and absence of anions (Table S1†) are determined. It has been observed that the binding constants of ions obtained in the presence of other ions are lower compared to those for the ion-free DANSn2. This decrease in the binding ability of an analyte (in the presence of another analyte) is also known as a negative cooperative effect and indicates the possibility of ion pair formation during the titration process. So, even in the presence of zinc cations, fluoride is able to bind to the fluorophore and cause quenching of the fluorescence, leading to fluoride detection.

Fig. 14 Change in the emission profile of DANSn2 in acetonitrile, upon the addition of fluoride.

Table 3 Binding constants for DANSn2 with F⁻ in the presence of Zn²⁺

Complexes	Binding constant (10^−4)
DANSn2 + F^−	1.528 M^−1
DANSn2 + F^− (in the presence of Zn^2+)	0.256 M^−1

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4.3. Theoretical calculations

Theoretical (DFT) calculations have also been carried out to obtain a molecular level understanding of the binding interactions between the analytes and DANSn2. We have modelled and investigated the interactions of Cu²⁺, Zn²⁺, Fe²⁺, Cd²⁺ and F⁻ with DANSn2 to understand the nature of the binding of transition-metal cations and fluoride ions with the respective receptors.

The geometries of DANSn2 and a DANSn2⋯Mⁿ⁺ complex are shown in Fig. 15, and their structural parameters and interaction energies are given in Table 4. The Cartesian coordinates of the optimized geometries of DANSn2 and its complex with Cu²⁺ and F⁻ are provided in the ESI (Table S2–S4†). It has been found that one of the metal ions binds to the receptor through both the nitrogen atom of the dimethylamine group and an sp² carbon atom in the arene ring, while the second metal ion is involved in the formation of a chelate structure, as shown in Fig. 15 (lower panel).

Fig. 15 Optimized geometry of DANSn2 (upper panel) and the mode of interaction of Mⁿ⁺ ions with DANSn2 in a DANSn2⋯Mⁿ⁺ complex (lower panel).

Table 4 Structural parameters and cation-binding energies of the DANSn2⋯Mⁿ⁺ complexes

DANSn2⋯M^n+ complex	Bond distance (in Å)					Bond angles (in degrees)		ΔEint (in kcal mol^−1)
	r 1	r 2	r 3	r 4	r 5	θ 1	θ 2
DANSn2⋯Cu^2+	1.96	2.01	2.11	3.14	1.99	101.94	48.31	−523.3
DANSn2⋯Zn^2+	2.01	2.35	2.07	2.28	2.06	104.99	83.53	−446.2
DANSn2⋯Fe^2+	1.92	2.04	1.91	2.25	2.05	108.95	65.43	−510.4
DANSn2⋯Cd^2+	2.21	2.67	2.29	2.99	2.22	95.21	73.96	−368.6

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It can be observed from Table 4 that Cu²⁺ binds DANSn2 with the highest affinity (ΔE_int = −523.3 kcal mol⁻¹), followed by Fe²⁺, Zn²⁺ and Cd²⁺.

As shown in Scheme 4, DANSn2 can bind to a F⁻ ion via its sulfonamide NH proton. From theoretical calculations, the short HF bond distance is found to be 1.04 Å in the DANSn2⋯F⁻ complex (Fig. 16). Calculations also show that the fluoride ions bind very strongly to the NH protons, leading to a proton abstraction mechanism. Due to this, the NH bonds elongate considerably from 1.02 Å in DANSn2 to 1.41 Å in the DANSn2⋯F⁻ complex. A large interaction energy of −65.09 kcal mol⁻¹ has been observed for the binding of F⁻ in the DANSn2⋯F⁻ complex.

Fig. 16 Optimized geometry of the DANSn2⋯F⁻ complex. Selected bond distances (in Å) and angles (in degrees) are shown.

Finally, we have investigated the utility of DANSn2 for live-cell imaging through fluorescence microscopy studies at different probe concentrations (Fig. S12†). We have also performed toxicity studies (Fig. S13†) in a tetrahymena cell culture. It has been observed that the present system is non-toxic in a cellular environment and has the potential to be used in live-cell imaging studies.

5. Conclusion

In the present study, we have demonstrated the photophysical and NMR studies on the interactions of a simple multi-component system in the absence and presence of different cations and anions. The absorption and fluorescence spectra of the system in different solvents of varying polarity demonstrate the intramolecular charge transfer (ICT) nature of the transition. While a significant reduction in the fluorescence intensity of the system in the presence of fluoride has been observed, no significant changes in the optical properties of the system have been observed in the presence of other commonly encountered anions. The binding interaction of fluoride with the present system has also been monitored through FTIR and ¹H NMR experiments. The formation of the HF₂⁻ species due to the abstraction of the acidic proton by F⁻ is primarily responsible for the fluoride ion selective signalling behaviour. In the case of cations, a bathochromic shift of fluorescence as well as a concomitant decrease in the fluorescence intensity of the present system has been observed. Interestingly, no bathochromic shift in the absorbance or fluorescence has been observed when a similar experiment was carried out employing a structurally similar compound devoid of the dimethylamino group attached to the propyl moiety. The cation signalling event was also monitored through FTIR and ¹H NMR experiments. The dimethylamino moieties attached to both the naphthalene ring and propyl spacer are observed to interact with the metal ions. These interactions are primarily responsible for the observed change in the photophysical behaviour of the present system in the presence of metal ions. Theoretical calculations also support the experimental observations. Fluorescence microscopy studies have demonstrated that the present system is cell-permeable, non-toxic, and can be used in live-cell imaging studies.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

M. S. thanks the National Institute of Science Education and Research (NISER), Bhubaneswar for funding. G. J. is thankful to the Department of Science and Technology (DST), New Delhi for providing the Kishore Vaigyanik Protsahan Yojana (KVPY) fellowship. P. K. S is thankful to NISER, Bhubaneswar for the fellowship awarded to him. Authors thank Dr Himanshu Biswal, Assistant Professor, School of Chemical Sciences, NISER, for his help in carrying out DFT calculations.

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Footnote

† Electronic supplementary information (ESI) available: Figures for the fluorescence response of DANSn2 upon the progressive addition of fluoride and copper ions, Benesi–Hildebrand plots for determination of the binding constants of DANSn2 with fluoride and copper ions (using both absorption and emission), fluorescence response of DANSn2 in the absence and presence of different metals, Jobs plot for the complexation of DANSn2 with Zn²⁺ ions in ACN, and the zoomed in ¹H NMR spectra of DANSn2 in DMSO-d₆ in the presence of different concentrations of zinc(II) perchlorate. The Cartesian coordinates of the optimized geometries of the DANSn2 and its complex with Cu²⁺ and F⁻. Fluorescence excitation spectra of DANSn2 and fluorescence microscopy studies of DANSn2 in tetrahymena cells. See DOI: 10.1039/c5ra10414a

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