Abhas Kumar
Bhoi
,
Prabhat Kumar
Sahu
,
Gaurav
Jha
and
Moloy
Sarkar
*
School of Chemical Sciences, National Institute of Science Education and Research, Bhubaneswar 751005, India. E-mail: moloysarkar@gmail.com; Fax: +91-674-2302436; Tel: +91-674-2304037
First published on 29th June 2015
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.
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.
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.
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%.
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%.
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Fig. 1 Absorption and emission spectra (λexc. = 338 nm) of DANSn2 in ACN. All spectra are normalized at their corresponding peak maximum. |
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Fig. 2 Fluorescence decay curve of DANSn2 in ACN at 298 K at λexc. = 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, ϕstd = 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, ϕstd = 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.
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).
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.
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−.
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.
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.
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.
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+.
Transition metal ions (M2+) | Binding constant (1![]() ![]() |
---|---|
Cu | 1.45 |
Zn | 0.65 |
Fe | 0.67 |
Mn | 0.32 |
Cd | 0.57 |
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.
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.
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Fig. 11 Emission spectra of DANSn2 (1.0 × 10−5 M) in ACN/H2O (5![]() ![]() |
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.
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†).
Complexes | Binding constant (10−4) |
---|---|
DANSn2 + F− | 1.528 M−1 |
DANSn2 + F− (in the presence of Zn2+) | 0.256 M−1 |
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).
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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). |
DANSn2⋯Mn+ 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⋯Cu2+ | 1.96 | 2.01 | 2.11 | 3.14 | 1.99 | 101.94 | 48.31 | −523.3 |
DANSn2⋯Zn2+ | 2.01 | 2.35 | 2.07 | 2.28 | 2.06 | 104.99 | 83.53 | −446.2 |
DANSn2⋯Fe2+ | 1.92 | 2.04 | 1.91 | 2.25 | 2.05 | 108.95 | 65.43 | −510.4 |
DANSn2⋯Cd2+ | 2.21 | 2.67 | 2.29 | 2.99 | 2.22 | 95.21 | 73.96 | −368.6 |
It can be observed from Table 4 that Cu2+ binds DANSn2 with the highest affinity (ΔEint = −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.
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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.
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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