Dipicolylamine coupled rhodamine dyes: new clefts for highly selective naked eye sensing of Cu²⁺ and CN⁻ ions

Abstract

The dipicolylamine (DPA) motif which is known as a binder of Zn(II) ions, has been utilized in devising rhodamine labelled compounds 1 and 2. Compound 1 acts as a FRET sensor and shows excellent selectivity for Cu(II) ions over a series of other cations in CH₃CN/H₂O by exhibiting a colour change (colourless to pink) of the solution. The spectral and colour changes are recovered in the presence of CN⁻ ions and thus, the ensemble 1·Cu²⁺ in CH₃CN/H₂O is established as the medium for selective detection of CN⁻ ions. In contrast, the modified compound 2 with the dipicolylamine motif as the principal binding site has been established as the colorimetric sensor of Cu(II) ions and the fluorometric sensor of Hg(II), Zn(II) and Cd(II) ions. Both the compounds 1 and 2 are cell permeable and are successfully employed for the detection of intercellular metal ions through bright field and fluorescence imaging.

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Introduction

Copper is an essential transition metal ion that plays an important role in environmental, biological and chemical systems.¹ It is toxic to biological systems when the level of Cu²⁺ ions exceeds the cellular needs. Overloading of this ion can cause oxidative stress and neurological disorders including Alzheimer's, Parkinson's and Wilson's diseases.² Thus rapid sensing of this ion by simple systems is desirable. In this regard, numbers of fluorescent as well as colorimetric sensors for copper ion have been reported in the past few years.^3,4 Of the different chemosensors, rhodamine-labelled sensors draw attention because of visualization of sensing through both color and fluorescence changes.

Rhodamine B and its derivatives exhibit excellent photo-physical properties such as high fluorescence quantum yield, large molar extinction coefficient and visible wavelength excitation. It is further mentionable that the rhodamine B and its derivatives in the spirolactam form are non fluorescent (colourless). Upon complexation of metal ions/proton the spirolactam ring is opened and shows strong emission as well as color. Considering this chemistry of rhodamine B, researchers have used this platform in conjunction with different binding sites to construct different metal ion sensors.⁵

Careful scrutiny of the literature reveals that use of dipicolyl amine (DPA) motif as binding site onto the rhodamine unit is less explored in metal ion sensing.⁶ Usually this motif binds Zn²⁺ ions. The wide spread use of DPA in devising receptors of different architectures for Zn²⁺ ion is worth mentioning. Lippard et al., have reported dipicolylamine coupled some rhodamine derivatives for sensing of Zn²⁺ ions.^6a,b Not only Zn²⁺ ion but also the complexation of Al³⁺ and Pb²⁺ ions using dipicolyl motif in rhodamine platform is known in few cases.⁷ Inspection of such different reports indicates that the disposition of dipicolylamine motif around the rhodamine part is crucial for tuning the metal ion selectivity. In relation to this, design and synthesis of new dipicolylamine coupled rhodamine derivatives thus draws attention.

In this manuscript, we report a new rhodamine-based structure 1 (Fig. 1) that senses Cu²⁺ ion through color change involving dipicolyl amine as the binding site. In addition, the ensemble of 1 with Cu²⁺ ion is observed to detect CN⁻ ion with significant sensitivity. In an effort to understand the role of anthracene in 1 other than FRET, compound 2 was designed and synthesized (Fig. 1). Under identical conditions, compound 2 while selectively senses Cu²⁺ ions through color change, it fluorometrically detects some multiple ions such as Hg²⁺, Zn²⁺ and Cu²⁺ ions without showing any color change. To explain the role of DPA unit in 1 or 2, the model compound 3 (Fig. 1) was synthesized. Compound 3, under identical conditions, did not show any selectivity in the recognition process.

Fig. 1 Chemdraw structures (left) of 1–3 and XP diagram (30% ellipsoids, right) of 1. Hydrogen atoms were omitted for clarity.

Results and discussion

The synthesis of compound 1 was achieved according to the Scheme 1. Initially, 9-anthraldehyde was transformed into the Schiff base 3 on reaction with 2-picolylamine. Reduction of the Schiff base 3 using NaBH₄ afforded the amine 4. The amine was reacted with 2-pivaloylamide-6-bromomethyl pyridine which was obtained from 2-amino-6-methylpyridine by reported procedure⁸ to obtain the compound 5. Hydrolysis of the amide bond in 5 using KOH in EtOH resulted in amine 6 (Scheme 1a). For compound 2, Scheme 1b was pursued to obtain the precursor amine 10 like amine 6. In this case, reaction of pyridine-2-aldehyde with n-butylamine in dry MeOH gave Schiff base 7 which on in situ reduction with NaBH₄ yielded amine 8. The amine was reacted with 2-pivaloylamide-6-bromomethyl pyridine to yield the compound 9. Hydrolysis of the amide bond in 9 using KOH in EtOH resulted in amine 10 (Scheme 1b). For model compound 3, m-nitrobenzaldehyde was converted to the Schiff base 11 which on reduction yielded the amine 12. Benzylation of the amine 12 using benzyl bromide followed by reduction of nitro group furnished the amine 14 in appreciable yield (Scheme 1c). Finally, rhodamine B was converted into its acid chloride 15 with POCl₃ in 1,2-dichloroethane and was coupled with the amines 6, 10 and 14 to give the desired compounds 1, 2 and 3, respectively in appreciable yields (Scheme 1d). The structures of 1, 2 and 3 were unambiguously characterized by ¹H, ¹³C NMR and HRMS. For 1, single crystal X-ray analysis further confirmed the structure.

Scheme 1 (a). (i) 2-Picolylamine, dry MeOH, reflux 6 h; (ii) NaBH₄, dry MeOH, reflux 6 h; (iii) 2-pivaloylamide-6-bromomethyl pyridine, dry K₂CO₃, CH₃CN, reflux 8 h; (iv) KOH, EtOH, reflux 6 h; (b). (v) n-Butyl amine, dry MeOH, reflux, 6 h; (vi) NaBH₄, dry MeOH, reflux, 6 h; (vii) 2-pivaloylamide-6-bromomethyl pyridine, dry K₂CO₃, CH₃CN, reflux, 8 h; (viii) KOH, EtOH, reflux, 6 h; (c). (ix) n-Butylamine, dry C₆H₆, reflux, 6 h, (x) NaBH₄, dry MeOH, reflux, 6 h; (xi) benzyl bromide, dry K₂CO₃, CH₃CN, reflux 6 h; (xii) SnCl₂, EtOH, reflux 3 h; (d). (xiii) POCl₃, 1,2-dichloroethane, 8 h; (xiv) 6, Et₃N, dry CH₂Cl₂, 6 h; (xv) 10, Et₃N, dry CH₂Cl₂, 6 h; (xvi) 14, Et₃N, dry CH₂Cl₂, 6 h.

Single crystal of 1 was grown from slow evaporation of acetonitrile solution and was characterized using X-ray crystallography (Fig. 1).⁹ In the crystal, compound 1 crystallized in the space group P1̄ and shows the expected orthogonal arrangement of the xanthene unit towards the lactam ring. The angle between the two planes was found to be 89.8°. The nitrogen atom N5 shows sum of the respective C–N–C angles of ΣN1^CCC = 332.6°. In the packing diagram (see ESI, Fig. 1S†) hydrogen bonding interactions involving the carbonyl group C=O and the C–H units of the diethylamino moieties were found (C–H⋯O 2.517 Å and 2.761 Å, respectively). Moreover weak π–π-interactions (∼3.48 Å) between two adjacent pyridine groups were detected. No relevant π–π-interactions involving the anthracene moieties were observed.

The optical responses of compound 1 to various metal cations viz. Hg²⁺, Cu²⁺, Cd²⁺, Fe²⁺, Mg²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ag⁺, Fe³⁺, Mn²⁺, Na⁺, Pd²⁺, Al³⁺ and Pb²⁺ (taken as their perchlorate salts) were investigated in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) through UV-vis and fluorescence spectroscopic methods. In this context, a high content of water in aqueous measuring solution was desirable, but such an approach was constrained due to the limited solubility of receptor in water. As a reasonable negotiation, CH₃CN : H₂O (4 : 1, v/v) was used in the study. It is to note that compound 1 was also found to be soluble in EtOH–H₂O (7 : 3, v/v). However, the UV spectrum of 1 in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) exhibited an intensive band centered at 367 nm which was attributed to the characteristics of anthracene unit. However, addition of Cu²⁺ ions produced a new band centered at 552 nm which underwent significant change on progression of titration (Fig. 2). On addition of Cu²⁺ ions, the receptor solution became pink in color.

Fig. 2 (a) Change in absorbance of 1 (c = 2.5 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) upon gradual addition of 2 equiv. amounts of Cu²⁺ (c = 1 × 10⁻³ M), (b) change in absorption intensity with the addition Cu²⁺ ions.

As reason, the appearance of new peak at 552 nm is ascribed to the binding-induced spirolactam ring opening of rhodamine unit in 1. UV-vis titrations of 1 under similar conditions with other metal ions exhibited negligible perturbation in the spectra showing no peak at 552 nm as well as no color change of the solution (ESI, Fig. 2S†). Thus receptor 1 provides a naked eye detection of Cu²⁺ ions among the tested metal ions. Fig. 3 highlights the change in absorbance ratio of 1 and Fig. 4 displays the change in color of the receptor solution in presence of the metal ions examined.

Fig. 3 Change in absorption ratio (A − A₀/A₀) of 1 (c = 2.5 × 10⁻⁵ M) at 552 nm upon addition of 2 equiv. amounts of various metal ions in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer).

Fig. 4 Photographs showing the color change of the solution of 1 (c = 2.5 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) in presence of 2 equiv. amounts of metal ions studied.

To understand the interference of other metal ions in the sensing of Cu²⁺ ion, competition experiment was also carried out by adding Cu²⁺ ions to the solutions of 1 in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) in presence of other metal ions (Fig. 5). Results in Fig. 5 indicate that the sensing of Cu²⁺ ion by 1 is hardly affected by the ions considered in the study. The sensor 1 shows 1 : 1 stoichiometric interaction (Fig. 6a)¹⁰ with Cu²⁺ ion with an association constant¹¹ value of (2.01 ± 0.28) × 10⁴ M⁻¹ [K_d = (4.97 ± 3.57) × 10⁻⁵ M] (Fig. 6b). In the sensing of Cu²⁺ ions, the detection limit¹² is determined to be 1.97 × 10⁻⁶ M (ESI, Fig. 3S†).

Fig. 5 Competitive selectivity of 1 (c = 2.5 × 10⁻⁵ M) towards Cu²⁺ (c = 1 × 10⁻³ M) in presence of 2 equiv. of other metal ions in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer).

Fig. 6 (a) UV-vis Job plot of receptor 1 with Cu²⁺ at 552 nm in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) where [H] = [G] = 2.5 × 10⁻⁵ M; (b) binding constant curve from non-linear fitting of UV-vis titration data.

Compound 1 in EtOH–H₂O (7 : 3, v/v, pH = 7.2, 10 mM Tris–HCl buffer) also exhibited similar results as observed in aq. CH₃CN (ESI, Fig. 4S and 5S†).

The sensor 1 is a FRET system due to presence of the anthracene at the dipicolyl amine motif. The emission of the intermediate 5 (donor) overlaps with the absorbance of the rhodamine B (acceptor) (Fig. 7a) and thus demonstrates 1 as a FRET system. This FRET-based compound 1 on excitation at the absorbance wavelength 370 nm of anthracene in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) gave structured emission at 416 nm. This emission was due to the anthracene. A broad emission at ∼550 nm in addition to the monomer emission centered at 416 nm is presumed to be either due to intermolecular excimer between the pyridine rings or due to intermolecular exciplex between anthracene and pyridine. However, upon titration with the metal ions except Cu²⁺ the intensity of broad emission at ∼550 nm was reduced showing no splitting in the region 575 nm to 590 nm due to lactam ring opening of rhodamine part (ESI, Fig. 6S†). Under this condition, the FRET process remained off. In contrast, in presence of Cu²⁺ ions, the broad peak at 550 nm was split into two peaks of which the peak at ∼580 nm was due to lactam ring opening that activated the FRET process and gave sharp color change (Fig. 7b). However, the intensity of this peak was gradually reduced along with the emission of anthracene at 416 nm on progression of titration and no ratiometric behavior in the emission spectra was observed. This is believed to be due to paramagnetic effect of Cu²⁺ ion.¹³ Quenching of fluorescence occurs due to the excitation energy transfer from the ligand to the metal d-orbital and/or ligand to metal charge transfer (LMCT) in aqueous CH₃CN.¹⁴ This is in accordance with the observations of other researchers.¹⁴ The peak at 530 nm arising from splitting was finally abolished on progression of titration. Importantly, the compound 1 is a system where the sensing mechanism is an integration of PET (photoinduced electron transfer) and FRET processes (Scheme 2).¹⁵ At pH 7.2, the nitrogen atom of aromatic imino in rhodamine part as well as nitrogen atom of the tripodal centre quench the emission of anthracene to a certain extent. Upon complexation of Cu²⁺ ion at the DPA moiety involving the lactam part of rhodamine, the FRET process from anthracene to rhodamine moiety occurs. At the same time, the PET process occurring in between the copper complexed site and the excited state of anthracene remains activated that led to a non ratiometric change in the emission spectrum. This was realized from the recording of the emission spectra of 1 at different pHs.

Fig. 7 (a) FRET plot showing the overlapping of emission of donor 5 (λ_exc = 370 nm) and absorbance of acceptor rhodamine B (c = 2.5 × 10⁻⁵ M); (b) change in emission of 1 (c = 2.5 × 10⁻⁵ M) upon gradual addition of 10 equiv. amounts of Cu²⁺ (c = 1 × 10⁻³ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) (λ_exc = 370 nm).

Scheme 2 Proposed PET and FRET processes in 1.

In Fig. 7b, the appearance of the peak at 580 nm under the broad emission at ∼550 nm due to Cu²⁺-induced lactam ring opening was confirmed by recording the fluorescence spectra of 1 in presence of acid where H⁺-induced lactam ring opening in rhodamine system is usual. In this regard, the fluorescence spectra of 1 were recorded at different pHs. As can be seen from Fig. 8a, the sharp peak at 583 nm in fluorescence with considerable intensity was observed from pH 3 to pH 2. Fig. 8b demonstrates that ratio of intensities at 583 nm and 416 nm (I₅₈₃/I₄₁₆) increases with decrease in pH from 4.00 to 2.00. This observation clearly indicates that anthracene acts as FRET donor in receptor 1 and the receptor 1 behaves as an ideal ratiometric sensing platform at strong acidic condition. It is to note that in pH range 4–12, there was no peak at 583 nm for ring opening (ESI, Fig. 7S†). The UV-vis titration spectra with change in pH can be found in the ESI† where the absorbance of 1 at 551 nm at pHs 2 and 3 is in conformity with the opening of lactam ring that resulted in pink color (ESI, Fig. 7S†). Interestingly, a similar pH-response of 1 was observed in EtOH–H₂O (7 : 3, v/v, 10 mM Tris–HCl buffer) (ESI, Fig. 8S†).

Fig. 8 (a) Fluorescence spectra of 1 (c = 2.5 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, 10 mM Tris–HCl buffer) at different pH values (λ_exc = 370 nm); (b) change of fluorescence emission ratios (I₅₈₃/I₄₁₆) by pH values.

Thus the selective response of 1 toward Cu²⁺ ion in both UV and fluorescence at pH 7.2 is attributed to the coordination of Cu²⁺ ion at the dipicolyl moiety involving the lactam part. FTIR spectral analysis of 1 itself and with Cu(ClO₄)₂ reveals a shifting of carbonyl stretching frequency from 1700 cm⁻¹ to 1650 cm⁻¹ (ESI, Fig. 9S†). This indicates the participation of the amide carbonyl in complexation. In order to realize the participation of the pyridine rings in complexation, we tried to record the ¹H NMR of 1 itself and in presence of 1 equiv. amount of Cu(ClO₄)₂ in CD₃CN/D₂O (4/1, v/v). Unfortunately, in presence of Cu²⁺ the sharp signals for different protons of 1 became too broad to interpret. Then we performed the ¹H NMR titration of 1 with Cu²⁺ ion in pure CD₃CN solvent. In this solvent system, on progression of titration, signals for the three methylenes (types ‘a’, ‘b’ and ‘c’) around the picolyl motif exhibited downfield shifts of 0.07 ppm upon complexation of equivalent amount of Cu²⁺ ion. The signals for pyridyl ring protons became broad and underwent small downfield chemical shifts (ESI, Fig. 10S†). Addition of more than 2 equiv. amounts of Cu²⁺ ions caused broadening of the signals and limits the titration to continue.

This corroborated a weak ground state interaction of 1 with Cu²⁺ ion at the DPA moiety in the suggested mode shown in Fig. 9a. To be confirmed with the binding role of pyridyl ring nitrogens in 1, compound 3 which lacks of pyridine rings was further explored in binding study. In fluorescence and UV-vis titrations, compound 3 exhibited color change in presence of multiple metal ions (e.g., Cu²⁺, Hg²⁺, Fe³⁺ and Al³⁺) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) giving no selectivity in the sensing process (ESI, Fig. 11S and 12S†).

Fig. 9 (a) Suggested binding mode of 1 with Cu²⁺ ion; (b) DFT optimized geometry of the complex 1·Cu²⁺ in gas phase.

DFT optimization of the copper complex in gas phase (Fig. 9b) reveals that the pyridyl ring nitrogens along with the lactam amide oxygen are intimately involved in coordination of Cu²⁺ ion. It is important to be pointed out that the DPA unit which is usually the binder of Zn²⁺ ion, prefers the binding of Cu²⁺ ion in 1. We believe that this occurs due to the change in metal coordination environment around the dipicolyl motif. To realize this, we considered the intermediate compound 5 which in fluorescence clearly showed the preference for Zn²⁺ and Cd²⁺ ions (Fig. 10) like other systems reported in the literature.¹⁶

Fig. 10 (a) Change in emission ratio (I − I₀/I₀) of 5 (c = 2.5 × 10⁻⁵ M) at 424 nm upon addition of 2 equiv. amounts of various metal ions in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer), (b) change in emission of 5 (c = 2.5 × 10⁻⁵ M) upon gradual addition of 2 equiv. amounts of Zn²⁺ (c = 1 × 10⁻³ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) (λ_exc = 370 nm).

Careful scrutiny of the literature reveals that rhodamine coupled with 2-aminopyridine^17a shows responses towards Fe³⁺, Hg²⁺ and Pb²⁺ ions instead of Cu²⁺. On the other hand, the dipicolylamine motif in 1,8-naphthalimide system as reported by Yoon et al., is observed to bind Cu²⁺ ion instead of Zn²⁺ ion.^17b In addition, rhodamine-coupled 1,2,3-triazole functionalized 2-amino pyridine exhibits selective response to Zn²⁺ ions although there was no dipicolyl amine motif in the structure.^17c These observations thus validate the fact that the selection of a particular metal ion in a binding site is dependent on the coordination environments that regulate the binding of Cu²⁺ ions in present example.

In the selective binding of Cu²⁺ ion, the effect of the counter anions of different copper salts was observed to be negligible. This can be realized from Fig. 13Sa and b†. Furthermore, the reversibility of the binding process between 1 and Cu²⁺ was established by adding aqueous solution of Na₂EDTA to the solution of 1·Cu²⁺ in both CH₃CN/aqueous Tris–HCl buffer (4 : 1, v/v, pH = 7.2) and EtOH/aqueous Tris–HCl buffer (7 : 3, v/v, pH = 7.2) solvent systems. In the event, EDTA²⁻ caused demetalation of 1 and regeneration of the spirolactam ring with bleaching of absorption band at ∼552 nm (ESI, Fig. 13Sc and d†).

As practical application, the copper ion ensemble of 1 was explored in studying the interaction of different anions viz. F⁻, Cl⁻, Br⁻, I⁻, NO₃⁻, CN⁻, AcO⁻, ClO₄⁻, HSO₄⁻, HP₂O₇³⁻, H₂PO₄⁻ (counter catións: Bu₄N⁺), ATP, ADP, AMP, HPO₄²⁻, PO₄³⁻ and P₂O₇⁴⁻ (counter catións: Na⁺). The addition of different anions to the ensemble perturbed the emission to the different extents (Fig. 11a). Among the anions studied, only CN⁻ ion interacted strongly showing a sharp color change (pink to colorless) and recovery of the original absorbance characteristics of 1. This also indicated the reversibility in the binding of Cu²⁺ ion. In the event, pyrophosphate (P₂O₇⁴⁻) responded moderately. The CN⁻- induced color change from pink to colorless solution associated with complete decrease in absorbance at 552 nm of 1·Cu²⁺ complex with 1 : 1 stoichiometric composition suggested that Cu²⁺ ion was removed from the binding centre through its complexation with CN⁻ ion (Fig. 12a). However, the other tested anions were silent in the process (ESI, Fig. 14S†). Thus, 1·Cu²⁺ complex can be used as a potential ensemble for selective colorimetric naked eye sensing of CN⁻ over the other tested anions (Fig. 11b).

Fig. 11 (a) Change in absorbance ratio at 552 nm for ‘1·Cu²⁺’ ensemble upon addition of 12 equiv. of various anions (c = 1 × 10⁻³ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer); (b) photographs showing the color change of the solution of 1 (c = 2.5 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) in presence of 12 equiv. of (1) F⁻, (2) Cl⁻, (3) Br⁻, (4) I⁻, (5) NO₃⁻, (6) AcO⁻, (7) ClO₄⁻, (8) P₂O₇⁴⁻, (9) HP₂O₇³⁻, (10) CN⁻, (11) ATP, (12) ADP, (13) AMP, (14) H₂PO₄⁻, (15) HPO₄²⁻, (16) PO₄³⁻, (17) HSO₄⁻.

Fig. 12 (a) Emission spectra for 1·Cu²⁺ (1 : Cu²⁺ = 1 : 1 equiv.) ensemble upon gradual addition of 10 equiv. amounts of CN⁻ in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer); (b) change in detection limit of CN⁻ ion for different stoichiometric ensembles of 1·Cu²⁺.

It is important to be mentioned that the ensembles prepared from the mixing of 1 with more than 1 equiv. amount of Cu²⁺ responded to CN⁻ ion in a similar way to that of the ensemble 1·Cu²⁺ having 1 : 1 equiv. combination. Only there was a slight variation in detection limit of CN⁻ ion and this is due to minor interference of the free Cu²⁺ ion in the ensemble (Fig. 12b).

Considering the interaction of CN⁻ ion with the copper ensemble of 1, a logic operation was performed. Herein, the sequence dependant “off–on–off” switching of absorption was used to construct a INHIBIT logic gate using Cu²⁺ and CN⁻ as chemical inputs. In considering the INHIBIT logic gate properties of 1, Cu²⁺ and CN⁻ ions act as inputs while the absorbance at 552 nm (A₅₅₂) functions as output (Fig. 13a and b). The output is zero when (i) both the Cu²⁺ and CN⁻ are absent; (ii) CN⁻ alone is present, or (iii) both Cu²⁺ and CN⁻ are present, and the gate is OFF. The output is one only when Cu²⁺ alone is present and the gate is ON. Thus 1 can be used to construct a logic circuit mimicking an INHIBIT gate.

Fig. 13 (a) Logic circuit of receptor 1 with Cu²⁺ and CN⁻ as chemical inputs and A₅₅₂ as output of the INHIBIT gate, (b) truth table for the INHIBIT gate of 1 with Cu²⁺ and CN⁻ as chemical inputs and A₅₅₂ as output.

In order to understand the role of anthracene (other than FRET process) in the binding event, compound 2 was next explored in the study. The free receptor 2 (c = 3.72 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) exhibited almost negligible absorption peak at 554 nm. However, gradual addition of Cu²⁺ ion resulted in a significant enhancement of absorption centered at 554 nm and exhibited a sharp color change from colorless to pink (ESI, Fig. 15S†). Such increase in absorbance at 554 nm along with the color change is due to Cu²⁺-induced spirolactam ring of rhodamine B like the case of 1. The other tested metal ions did not perturb the absorption spectra of 2 and no color change was noticed. The bar plot in Fig. 15S† demonstrates the change in absorption ratio of 2 in presence of 2 equiv. amounts of different metal ions. Thus the binding study clearly reveals that the dipicolylamine motif either in 1 or 2 is sensible to Cu²⁺ ion.

Like 1, the competitive experiment as shown in Fig. 16Sa,† suggests that 2 is also able to selectively recognize Cu²⁺ in presence of other metal ions. The 1 : 1 stoichiometry of the complex was evaluated from the Job plot¹⁰ using absorbance data (ESI, Fig. 16Sb†). The association constant¹¹ of 2·Cu²⁺, derived from nonlinear curve fitting, was calculated to be (4.44 ± 0.14) × 10⁴ M⁻¹ [K_d = (2.23 ± 7.14) × 10⁻⁵ M] (ESI, Fig. 17S†). The detection limit¹² for 2 towards Cu²⁺ ion was found to be 8.45 × 10⁻⁷ M which is fairly good than the receptor 1 (ESI, Fig. 18S†).

Fluorescence titration experiments were performed to gain an insight into the binding interaction of 2 in the excited state. Sensor 2 (c = 3.72 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer), on excitation at 500 nm, displayed a weak emission at 577 nm. On gradual addition of Cu²⁺, Hg²⁺, Zn²⁺ and Cd²⁺, significant enhancements in emission of 2 were observed (Fig. 14). Interestingly, in case of addition of Cu²⁺, an increase in emission centered at 577 nm with concomitant color change of the solution from colorless to pink was observed. This suggested the chelation of Cu²⁺ ion at the DPA motif involving the amide ion of the spirolactam ring like the mode shown in Fig. 9a. Interestingly, in presence of Hg²⁺, Zn²⁺ and Cd²⁺ ions no color change of the receptor solution was observed. This indicated the intactness of the spirolactam ring during complexation and the increase in emission in such cases is attributed to the complexation of metal ions into the dipicolyl core leading to inhibition of PET process occurring in between the binding site and the excited state of rhodamine. The increment in emission intensity of 2 was significantly higher with the addition of Hg²⁺ ion (ESI, Fig. 19S†).

Fig. 14 Change in emission of 2 (c = 2.5 × 10⁻⁵ M) in CH₃CN–H₂O (4 : 1, v/v, pH = 7.2, 10 mM Tris–HCl buffer) upon addition of 10 equiv. of (a) Cu(ClO₄)₂, (b) Hg(ClO₄)₂, (c) Cd(ClO₄)₂, (d) Zn(ClO₄)₂, [concentration of metal salts were 1 × 10⁻³ M] (λ_exc = 500 nm).

However, excitation of 2 in fluorescence at 370 nm instead of 500 nm gave emission at ∼450 nm (presumably for the DPA motif) which underwent small increase in presence of Hg²⁺, Zn²⁺ and Cd²⁺ ions showing no color change (lactam ring intact) in the solution. Under the similar conditions, mere change in emission of 2 at 450 nm with a color change (opening of lactam ring) upon gradual addition of Cu²⁺ ions was due to paramagnetic effect of Cu²⁺ ion¹³ that quenched the emission (ESI, Fig. 20S†). This control experiment highlighted the key role of anthracene in 1 as FRET donor in the selective sensing of Cu²⁺ ions.

Like 1, receptor 2 was pH sensitive; the color change from colorless to pink was only observed at pHs 2 and 3 (ESI, Fig. 21S†). Receptor 2 was further explored in anion sensing through the use of its copper ensemble like 1. Among the various anions as considered in case of 1, only CN⁻ affected the spectral pattern of the ensemble 2·Cu²⁺, exhibiting a significant decrease in absorbance (ESI, Fig. 22Sa†). Cyanide ion-induced decrease in absorption of the ensemble 2·Cu²⁺ resulted in a sharp color change from pink to colorless. Other tested anions perturbed the absorption of the ensemble to the negligible extents (ESI, Fig. 22Sb†). Like 1, the ensembles prepared from mixing of 2 with 1 or more than 1 equiv. amounts of Cu²⁺ were sensitive to CN⁻ ion and gave a slight variation in detection limit of CN⁻ ion for minor interference of the free Cu²⁺ ion in the ensembles (ESI, Fig. 23S†).

Thus the experimental findings on structure 2 demonstrate that the replacement of anthracene part in 1 has a marked effect in sensing process. The anthracene part in 1 exerts a steric environment for which the dipicolylamine motif only allows the Cu²⁺ ion in the interaction process. In contrast, the butyl chain in 2 being less steric allows not only Cu²⁺ ion but also other ions such as Zn²⁺, Cd²⁺ and Hg²⁺ ions although the binding mechanism is different.

The potentiality of 1 and 2 in biological systems was evaluated for in vitro detection of Cu²⁺ ions in HepG2 cells. Images of bright field and fluorescence microscopy revealed that 1 and 2 treated cells did not show significantly detectable changes when compared to normal untreated control (Fig. 15a and 16a). But in cell sets of 1 and 2 treated with Cu²⁺, the cells were found to be red in coloration when observed under bright field (Fig. 15c and 16c). The images also clearly suggests that the receptors are not only permeable through cell membrane (cytoplasm was red in colour) but also receptor effectively cross the nuclear membrane (nucleus were also red in colour). The effect was greater for 2 + Cu²⁺ than that of 1 + Cu²⁺. However, no such changes were observed in fluorescent filter (Fig. 15d). On the other hand, cell sets incubated with either 2 + Cd²⁺, 2 + Zn²⁺ and 2 + Hg²⁺ although showed no significant changes in bright field imaging (Fig. 16e, g and i) but a significant increase in fluorescent intensity in red filter was observed in the following order: 2 + Cd²⁺ < 2 + Zn²⁺ < 2 + Hg²⁺ when compared to untreated control cell set (Fig. 16f, h and j).

Fig. 15 Fluorescence and bright field images of HepG2 cells: (a) bright field image of cells treated with receptor 1 (10 μM) for 25 min at 25 °C, (b) fluorescence image of cells treated with 1 (10 μM) for 25 min at 37 °C, (c) bright field image of cells upon treatment with receptor 1 (10 μM) followed by Cu(ClO₄)₂ (15 μM) for 1 h at 25 °C, (d) fluorescence image of cells upon treatment with 1 (10 μM) followed by Cu(ClO₄)₂ (15 μM) for 1 h at 37 °C [red filter is used, λ_ex = 480–580 nm].

Fig. 16 Fluorescence and bright field images of HepG2 cells: (a) bright field image of cells treated with 2 (10 μM) for 25 min at 25 °C, (b) fluorescence image of cells treated with 2 (10 μM) for 25 min at 25 °C, (c) bright field image of cells upon treatment with 2 (10 μM) followed by Cu(ClO₄)₂ (15 μM) for 1 h at 25 °C, (d) fluorescence image of cells upon treatment with 2 (10 μM) followed by Cu (ClO₄)₂ (15 μM) for 1 h at 25 °C, (e) bright field image of cells upon treatment with 2 (10 μM) followed by Cd(ClO₄)₂ (15 μM) for 1 h at 25 °C, (f) fluorescence image of cells upon treatment with 2 (10 μM) followed by Cd(ClO₄)₂ (15 μM) for 1 h at 25 °C, (g) bright field image of cells upon treatment with receptor 2 (10 μM) followed by Zn(ClO₄)₂ (15 μM) for 1 h at 25 °C, (h) fluorescence image of cells upon treatment with 2 (10 μM) followed by Zn(ClO₄)₂ (15 μM) for 1 h at 25 °C, (i) bright field image of cells upon treatment with 2 (10 μM) followed by Hg(ClO₄)₂ (15 μM) for 1 h at 25 °C, (j) fluorescence image of cells upon treatment with 2 (10 μM) followed by Hg (ClO₄)₂ (15 μM) for 1 h at 25 °C [red filter is used in fluorescence imaging, λ_ex = 480–580 nm].

This suggests that both the receptors 1 and 2 are permeable through cellular membrane and can assist in sensing the presence of Cu²⁺, even in a small concentration, in living tissues without imparting any significant cellular cytotoxicity. Even the receptor 2 in addition, is also able to detect the intercellular metal ions such as Cd²⁺, Zn²⁺ and Hg²⁺ ions effectively by exhibiting marked change in fluorescence.

Results of % cell viability assay suggest that the receptors 1 or 2 have no significant cellular cytotoxicity in HepG2 cells when compared to solvent treated cells (ESI, Fig. 24S†).

Conclusions

In conclusion, a new dipicolylamine coupled rhodamine B molecular sensor 1 has been designed and synthesized. The new sensor 1 displayed strong selectivity for Cu²⁺ ion over a series of other metal ions examined in CH₃CN/aqueous Tris–HCl buffer (4/1, v/v) at pH 7.2 by exhibiting a colour change (from colourless to pink). The sensor 1 has been detected to be insensitive to proton-induced-spirolactam ring opening in the pH range 4 to 12 and thus offers a large pH window for detection of metal ions. The ensemble of 1·Cu²⁺ has further been successfully used for naked eye detection of CN⁻ ion over a series of other anions examined in the study. Thus the present finding offers a new example of dipicolyl amine (DPA)-based rhodamine molecule for Cu²⁺ ion in addition to the existing few examples for other metal ions such as Zn²⁺, Al³⁺ and Pb²⁺ in the literature.^6,7 The role of DPA motif in 1 for selective binding of Cu²⁺ ion has been established by considering the model compound 3.

On the other hand, replacement of anthracene by butyl chain in receptor 2 shows that the receptor is able to detect Cu²⁺ colorimetrically and Cd²⁺, Zn²⁺ and Hg²⁺ ions fluorometrically. Like the case of 1, the ensemble 2·Cu²⁺ is also sensible to CN⁻ ion. Furthermore, the compounds 1 and 2 are cell permeable and can detect the intercellular mentioned metal ions through imaging process.

Experimental

Syntheses

1-(Anthracen-9-yl)-N-(pyridin-2-ylmethyl)methanamine (4). To a solution of 9-anthranaldehyde (1 g, 4.85 mmol) in dry methanol, 2-picolylamine (0.519 g, 4.85 mmol) was added and the reaction mixture was refluxed for 6 h. The Schiff base 3, formed in situ, was reduced by refluxing the mixture with NaBH₄ (0.600 g, 15.86 mmol) for 6 h. The progress of the reduction was monitored by thin layer chromatography (TLC) (petroleum ether–EtOAc, 1 : 1, v/v). After completion of the reaction, the solvent was evaporated under reduced pressure. Then, CHCl₃–water (2 : 1, v/v) was added to the residue and the compound was extracted with CHCl₃. The organic phase was dried over anhydrous Na₂SO₄ and evaporated in vacuo. The crude product was purified by column-chromatography using petroleum ether–EtOAc (1 : 1; v/v) as eluent to afford the amine 4 as a yellow gum (1.1 g, yield: 75.8%); ¹H NMR (400 MHz, CDCl₃): δ 8.59 (d, 1H, J = 8 Hz), 8.39 (s, 1H), 8.31 (d, 2H, J = 8 Hz), 7.99 (d, 2H, J = 8 Hz), 7.68–7.64 (td, 1H, J₁ = 8 Hz, J₂ = 4 Hz), 7.53–7.43 (m, 4H), 7.38 (d, 1H, J = 8 Hz), 7.20–7.17 (m, 1H), 4.76 (s, 2H), 4.16 (s, 2H), 2.16 (br s, 1H, NH); FTIR (KBr, cm⁻¹): 3411, 3050, 2856, 1589, 1432.

N-(6-(((Anthracen-9-ylmethyl) (pyridin-2-ylmethyl)amino)methyl)pyridin-2-yl)pivalamide (5). To a stirred solution of amine 4 (0.400 g, 1.34 mmol) in 20 mL dry CH₃CN, anhydrous K₂CO₃ (0.300 g, 2.17 mmol) was added and the mixture was stirred for 30 min. Then 2-pivaloylamide-6-bromomethyl pyridine (0.363 g, 1.34 mmol) was added and the reaction mixture was refluxed for 6 h and the progress of the reaction was monitored by TLC. After completion of the reaction CH₃CN was evaporated in a vacuum. CHCl₃–water (3 : 1, v/v) was added to the residue and the compound was extracted with CHCl₃. The combined organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated in vacuo. The crude product was purified through column chromatography using 35% EtOAc in petroleum ether as eluent to give 5 as a yellow solid (0.475 g, yield: 72.5%), mp 160 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.49 (d, 1H, J = 4 Hz), 8.39 (d, 2H, J = 8 Hz), 8.35 (s, 1H), 8.03 (d, 1H, J = 8 Hz), 7.95 (d, 2H, J = 8 Hz), 7.58–7.55 (m, 2H), 7.48–7.43 (m, 5H), 7.32 (d, 1H, J = 8 Hz), 7.12 (m, 1H), 7.01 (d, 1H, J = 8 Hz), 4.66 (s, 2H), 3.89 (s, 2H), 3.75 (s, 2H), 1.66 (s, 9H); FTIR (KBr, cm⁻¹): 3417, 2959, 2832, 1689, 1449.

6-((Anthracen-9-ylmethyl)(benzyl)amino)methyl)pyridin-2-amine (6). Compound 5 (0.400 mg, 0.81 mmol), taken in a 50 mL round-bottomed flask, was mixed with 4(N) KOH solution (4 mL) and ethanol (4 mL). The reaction mixture was then refluxed for 12 h. The completion of the reaction was monitored by TLC. Ethanol was distilled out and the product was extracted with ethyl acetate and dried over anhydrous sodium sulfate. Organic layer was evaporated to afford the compound 6 as yellow solid (0.250 g, yield: 75.7%), mp 186 °C. This was pure enough for the next step and for spectral analysis. ¹H NMR (400 MHz, CDCl₃): δ 8.46–8.40 (m, 3H), 8.35 (s, 1H), 7.95 (d, 2H, J = 8 Hz), 7.58 (t, 1H, J = 8 Hz), 7.47–7.41 (m, 4H), 7.39–7.34 (m, 2H), 7.10–7.07 (m, 1H), 6.75 (d, 1H, J = 8 Hz), 6.34 (d, 1H, J = 8 Hz), 4.64 (s, 2H), 4.39 (s, 2H), 3.86 (s, 2H), 3.71 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 158.0, 157.7, 148.5, 138.0, 136.1, 131.5, 131.3, 130.0, 128.8, 127.5, 125.4, 125.2, 125.0, 124.7, 123.5, 121.8, 113.6, 106.6, 60.6, 60.5, 50.8; FTIR (KBr, cm⁻¹): 3471, 3310, 2922, 1617, 1458.

N-(Pyridin-2-ylmethyl)butan-1-amine (8). To a solution of pyridine-2-aldehyde (0.500 g, 4.67 mmol) in dry MeOH, n-butyl amine (0.512 mL, 7.00 mmol) was added and the reaction mixture was allowed to reflux for 6 h. The Schiff base 7 formed in situ was reduced by refluxing the mixture with NaBH₄ (0.317 g, 8.40 mmol) for 4 h. The reaction was quenched by evaporation of the solvent under reduced pressure. CHCl₃–water (2 : 1) was added to the residue and extracted with CHCl₃. The organic phase was dried over anhydrous Na₂SO₄ and evaporated in vacuo. The crude product was purified by column-chromatography using petroleum ether/EtOAc, 10 : 1 (v/v) as eluent to afford the amine 8 as yellow liquid (0.650 g, yield: 84.8%), ¹H NMR (400 MHz, CDCl₃): δ 8.36 (d, 1H, J = 4.4 Hz), 7.47–7.42 (m, 1H), 7.11 (d, 1H, J = 8 Hz), 6.97 (t, 1H, J = 6.2 Hz), 3.70 (s, 2H), 2.46 (t, 2H, J = 7.2 Hz), 1.92 (brs, 1H), 1.36–1.29 (m, 2H), 1.21–1.12 (m, 2H), 0.71 (t, 3H, J = 7.4 Hz). FTIR (KBr, cm⁻¹): 3389, 2928, 1591, 1570.

N-(6-((Butyl(pyridin-2-ylmethyl)amino)methyl)pyridin-2-yl)pivalamide (9). To a stirred solution of amine 8 (0.600 g, 3.64 mmol) in 25 mL dry CH₃CN, anhydrous K₂CO₃ (0.757 g, 5.48 mmol) was added and the mixture was stirred for 30 min. Then 2-pivaloylamide-6-bromomethyl pyridine (0.990 g, 3.65 mmol) was added and the reaction mixture was refluxed for 6 h and the progress of the reaction was monitored by TLC. After completion of the reaction, CH₃CN was evaporated in vacuum. CHCl₃–water (3 : 1, v/v) was added to the residue and the compound was extracted with CHCl₃. The combined organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated in vacuo. The crude product was purified through column chromatography using 25% EtOAc in petroleum ether as eluent to give 9 as yellow gum (0.900 g, yield: 69.5%), ¹H NMR (400 MHz, CDCl₃): δ 8.44 (d, 1H, J = 4.4 Hz), 8.01 (d, 1H, J = 8 Hz), 7.95 (s, 1H), 7.60–7.55 (m, 2H), 7.45 (d, 1H, J = 8 Hz), 7.19 (d, 1H, J = 8 Hz), 7.06 (t, 1H, J = 8 Hz), 3.72 (s, 2H), 3.61 (s, 2H), 2.44 (t, 2H, J = 8 Hz), 1.46–1.43 (m, 2H), 1.25 (s, 9H), 1.23–1.17 (m, 2H), 0.78 (t, 3H, J = 8 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 177.0, 160.0, 158.4, 150.8, 148.8, 138.6, 136.3, 122.7, 121.8, 118.5, 111.8, 60.4, 60.0, 54.2, 39.7, 29.2, 27.4, 20.4, 13.9; FTIR (KBr, cm⁻¹): 3437, 2958, 1690, 1596.

6-((Butyl(pyridin-2-ylmethyl)amino)methyl)pyridin-2-amine (10). Compound 9 (0.650 mg, 1.83 mmol), taken in a 50 mL round-bottomed flask, was mixed with 4(N) KOH solution (8 mL) and ethanol (8 mL). The reaction mixture was then refluxed for 4 h. The completion of the reaction was monitored by TLC. Ethanol was distilled out and the product was extracted with ethyl acetate and dried over anhydrous Na₂SO₄. Organic layer was evaporated to afford the compound 10 as yellow liquid (0.330 g, yield: 66.56%). This was used directly in the next step without characterization.

N-(3-Nitrobenzyl)butan-1-amine (12). Compound 12 was obtained as yellow liquid (1.6 g, yield: 77.4%) from 3-nitrobenzaldehyde (1.5 g, 9.93 mmol) and n-butylamine (0.680 mL, 9.93 mmol) according to the procedure followed for the synthesis of 4. However, in this case, the intermediate Schiff base 11 was prepared in dry benzene. ¹H NMR (400 MHz, CDCl₃): δ 8.13 (s, 1H), 8.02 (d, 1H, J = 8 Hz), 7.61 (d, 1H, J = 8 Hz), 7.41 (t, 1H, J = 8 Hz), 3.82 (s, 2H), 2.55 (t, 2H, J = 8 Hz), 2.09 (s, 1H), 1.46–1.39 (m, 2H), 1.33–1.26 (m, 2H), 0.84 (t, 3H, J = 8 Hz); FTIR (KBr, cm⁻¹): 3411, 3050, 2856, 1589, 1432.

N-Benzyl-N-(3-nitrobenzyl)butan-1-amine (13). Compound 13 was obtained as yellow liquid (1.0 g, yield: 69.8%) from amine 12 (1.0 g, 4.80 mmol) and benzyl bromide (0.830 mL, 4.80 mmol) according to the procedure followed for the synthesis of 5. ¹H NMR (400 MHz, CDCl₃): δ 8.14 (s, 1H), 7.98 (d, 1H, J = 8 Hz), 7.61 (d, 1H, J = 8 Hz), 7.37 (t, 1H, J = 8 Hz), 7.28–7.21 (m, 4H), 7.16–7.13 (m, 1H), 3.53 (s, 2H), 3.49 (s, 2H), 2.35 (t, 2H, J = 8 Hz), 1.46–1.38 (m, 2H), 1.24–1.17 (m, 2H), 0.73 (t, 3H, J = 8 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 148.3, 142.6, 139.4, 134.7, 129.0, 128.7, 128.3, 127.0, 123.4, 121.9, 58.4, 57.5, 53.4, 29.2, 20.4, 14.0; FTIR (KBr, cm⁻¹): 2956, 2930, 1528, 1506, 1348.

3-((Benzyl(butyl)amino)methyl)aniline (14). To a stirred solution of 13 (0.700 g, 2.35 mmol) in 15 mL EtOH, SnCl₂ (1.78 g, 9.38 mmol) was added and the reaction mixture was refluxed for 3 h and the progress of the reaction was monitored by TLC. After completion of the reaction, EtOH was evaporated maximally in vacuum. Then the reaction mixture was basified with aq. NaHCO₃ solution and the organic compound was extracted with ethyl acetate (20 mL × 3). The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated in vacuo. The crude product was purified through column chromatography using 10% EtOAc in petroleum ether as eluent to give amine 14 as gummy product (0.430 g, yield: 68.2%). ¹H NMR (400 MHz, CDCl₃): δ 7.28 (d, 2H, J = 8 Hz), 7.23 (t, 2H, J = 8 Hz), 7.15–7.11 (m, 1H), 7.01–6.97 (m, 1H), 6.69–6.65 (m, 2H), 6.46 (dd, 1H, J₁ = 8 Hz, J₂ = 2.4 Hz), 3.46 (s, 2H), 3.38 (s, 2H), 2.32 (t, 2H, J = 4 Hz), 1.44–1.37 (m, 2H), 1.25–1.16 (m, 2H), 0.75 (t, 3H, J = 8 Hz) (signal for –NH₂ is not found due to broadening); ¹³C NMR (100 MHz, CDCl₃): δ 146.3, 141.3, 140.0, 129.0, 128.8, 128.1, 126.6, 119.2, 115.4, 113.6, 58.2, 53.1, 29.2, 20.5, 14.1 (one carbon in the aliphatic region is unresolved); FTIR (KBr, cm⁻¹): 3747, 2954, 2929, 1619, 1492.

Receptor 1. A solution of rhodamine B base (0.300 g, 0.62 mmol) in 1,2-dichloroethane (12 mL) was stirred, and phosphorus oxychloride (0.145 mL, 1.57 mmol) was added dropwise over 2 min. The solution was refluxed for 3 h. The reaction mixture was cooled and evaporated in vacuo to give rhodamine B acid chloride 15, which was not purified and used in the next step directly. The crude acid chloride 15 was dissolved in dry CH₂Cl₂ (25 mL) and was added to a solution of 6 (0.240 g, 0.59 mmol) in CH₂Cl₂ (15 mL) containing Et₃N (0.263 mL). The reaction mixture was stirred at room temperature for 3 h. After completion of reaction, solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using 35% EtOAc in petroleum ether as eluent to give receptor 1 as a yellow solid (0.290 g, yield: 55.9%), mp 110 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.42 (d, 1H, J = 8 Hz), 8.36 (d, 1H, J = 8 Hz), 8.31 (s, 1H), 8.09 (d, 2H, J = 8 Hz), 7.98 (d, 1H, J = 8 Hz), 7.92 (d, 2H, J = 8 Hz), 7.56 (t, 1H, J = 8 Hz), 7.46–7.36 (m, 7H), 7.10 (d, 1H, J = 8 Hz), 6.99–6.95 (m, 2H), 6.79 (d, 1H, J = 8 Hz), 6.43 (d, 2H, J = 8 Hz), 6.32 (d, 2H, J = 4 Hz), 5.94 (d, 1H, J = 4 Hz), 5.92 (d, 1H, J = 4 Hz), 4.18 (s, 2H), 3.57 (s, 2H), 3.50 (s, 2H), 2.95–2.82 (m, 8H), 0.85 (t, 12H, J = 4 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 168.6, 160.5, 156.7, 154.1, 153.1, 149.5, 148.2, 148.0, 137.0, 136.0, 133.5, 131.4, 131.3, 130.6, 129.9, 128.7, 127.9, 127.4, 127.1, 125.3, 125.2, 124.6, 124.3, 123.4, 123.1, 121.6, 119.5, 113.9, 108.6, 107.0, 97.7, 66.2, 59.9, 59.8, 50.1, 43.8, 12.4; FTIR (KBr, cm⁻¹): 3385, 3049, 2965, 1700, 1614, 1514; HRMS (TOF MS ES⁺): calcd for (M + H)⁺: 829.4230, found: (M + H)⁺: 829.4265.

Receptor 2. The rhodamine acid chloride 15 was dissolved in dry CH₂Cl₂ (35 mL) and was added to a solution of 10 (0.292 g, 1.08 mmol) in CH₂Cl₂ (20 mL) containing Et₃N (0.376 mL, 2.71 mmol). The reaction mixture was stirred at room temperature for 6 h. After completion of reaction, solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using 25% EtOAc in petroleum ether as eluent to give receptor 2 as pale pink solid (0.520 g, yield: 69.1%), mp 155 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.48 (d, 1H, J = 4 Hz), 8.31 (d, 1H, J = 8 Hz), 8.00 (d, 1H, J = 8 Hz), 7.57–7.47 (m, 4H), 7.33 (d, 1H, J = 8 Hz), 7.16 (d, 1H, J = 8 Hz), 7.10–7.04 (m, 2H), 6.43–6.38 (m, 4H, one doublet and one singlet are overlapped), 6.11 (dd, 2H, J₁ = 8 Hz, J₂ = 2.4 Hz), 3.61 (s, 2H), 3.47 (s, 2H), 3.32–3.22 (m, 8H), 2.30 (t, 2H, J = 8 Hz), 1.34–1.28 (m, 2H), 1.18–1.10 (m, 14H), 0.73 (t, 3H, J = 8 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 153.7, 153.6, 149.4, 148.6, 148.2, 137.0, 136.2, 133.4, 130.6, 128.0, 127.7, 124.4, 123.0, 122.8, 121.6, 117.6, 113.1, 108.8, 106.9, 97.6, 66.2, 61.0, 59.9, 54.4, 44.2, 29.4, 20.3, 13.9, 12.6 (two carbons in the aromatic regions are unresolved); FTIR (KBr, cm⁻¹): 3393, 3077, 2934, 1704, 1634, 1615; HRMS (TOF MS ES⁺): calcd for (M + H)⁺: 695.4073, found: (M + H)⁺: 695.4073.

Receptor 3. The rhodamine acid chloride 15 was dissolved in dry CH₂Cl₂ (35 mL) and was added to a solution of 14 (0.300 g, 1.08 mmol) in CH₂Cl₂ (20 mL) containing Et₃N (0.376 mL, 2.71 mmol). The reaction mixture was stirred at room temperature for 6 h. After completion of reaction, solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using 10% EtOAc in petroleum ether as eluent to give the receptor 3 as colourless gum (0.340 g, yield: 72.5%); ¹H NMR (400 MHz, CDCl₃): δ 7.95–7.92 (m, 1H), 7.42 (t, 2H, J = 8 Hz), 7.17–7.15 (m, 5H), 7.13–7.07 (m, 2H), 7.01 (t, 2H, J = 8 Hz), 6.68 (s, 1H), 6.59 (d, 1H, J = 8 Hz), 6.56 (d, 1H, J = 8 Hz), 6.20 (dd, 2H, J₁ = 8 Hz, J₂ = 2.4 Hz), 6.13 (d, 2H, J = 2.4 Hz), 3.26 (s, 2H), 3.24 (s, 2H), 3.21–3.14 (m, 8H), 2.13 (t, 2H, J = 8 Hz), 1.30–1.26 (m, 2H), 1.18–1.09 (m, 2H), 1.03 (t, 12H, J = 8 Hz), 0.71 (t, 3H, J = 4 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 167.6, 153.2, 153.1, 148.6, 139.9, 136.3, 132.7, 131.1, 128.89, 128.86, 128.2, 128.0, 127.9, 127.2, 126.5, 125.5, 124.0, 123.3, 108.0, 106.4, 97.7, 66.3, 57.9, 57.7, 52.6, 44.2, 29.7, 20.3, 14.0, 12.5 (two carbons in the aromatic regions are unresolved); FTIR (KBr, cm⁻¹): 3367, 2965, 2925, 1700, 1615; HRMS (TOF MS ES⁺): calcd for (M + H)⁺: 693.4169, found: (M + H)⁺: 693.4130.

X-ray diffraction

Data sets were collected with a Nonius KappaCCD diffractometer. Programs used: data collection, COLLECT (R. W. W. Hooft, Bruker AXS, 2008, Delft, The Netherlands); data reduction Denzo-SMN;¹⁸ absorption correction, Denzo;¹⁹ structure solution SHELXS-97;²⁰ structure refinement SHELXL-97 (ref. 21) and graphics, XP (BrukerAXS, 2000). Thermals ellipsoids are shown with 30% probability, R-values are given for observed reflections, and wR² values are given for all reflections.

Exceptions and special features. A badly disordered half benzene molecule was found in the asymmetrical unit and could not be satisfactorily refined. The program SQUEEZE²² was therefore used to remove mathematically the effect of the solvent. The quoted formula and derived parameters are not included the squeezed solvent molecule.

Computational details

Full geometrical optimizations of all the structures were carried out in the gas phase employing the Becke three-parameter hybrid density functional combined with the Lee–Yang–Parr correlation functional (B3LYP).^23–26 The DFT (B3LYP) calculations was performed with the 6-31G(d) basis set²⁷ for carbon, hydrogen, nitrogen and oxygen atoms and for the copper atom SDD basis set²⁸ was used. Frequency calculations were performed at the same level of theory to confirm that each stationary point was a local minimum (with zero imaginary frequency). All DFT calculations were performed with the Gaussian 09 suite of programs.²⁹

Material and methods for cell culture

Reagents. The reagents used in our study were of analytical grade and were procured from Sigma Aldrich, USA.

Cell culture. Human liver cancer cells (HepG2) were procured from National Centre for Cell Science, Pune, India. 5 × 10⁵ cells per mL were cultured in DMEM which was supplemented with 10% fetal bovine serum and 1% PSN antibiotic at 37 °C and 5% CO₂ supply.

% cellular cytotoxicity. Cellular cytotoxicity of 1 and 2 were assessed by following the standard protocol of Mossam,³⁰ where cells were plated in 96-well microplates for 24 hours along with either of 1 and 2 at different concentrations. At the end of incubation period MTT was added in each well and incubated for 4 h and then DMSO was further added for dissolving the formazan crystal. The absorbance was then measured at 595 nm in a microplate reader (Thermo scientific, Multiskan ELISA, USA).

Bright field and fluorescence microscopy. The cells were seeded and harvested at 10⁷ cells per plate and grown to 70% confluency for microscopic imaging. Untreated control set of HepG2 cells served as the standard to detect any changes brought about by addition of 1 and 2.³¹ The other set of cells include the following: (i) 1, (ii) 1 + Cu²⁺, (iii) 2, (iv) 2 + Cu²⁺, (v) 2 + Cd²⁺, (vi) 2 + Zn²⁺, (vii) 2 + Hg²⁺. In each case 40 μl of either of 1 and 2 were added and incubated for 25 min and then they were further incubated with either of 40 μl of Cu²⁺/Cd²⁺/Zn²⁺/Hg²⁺ and kept for 30 min. Images were then captured in bright field and fluorescence filter fitted microscope (Leica, Germany).

Acknowledgements

We thank DST and UGC, New Delhi, Govt. of India for providing facilities in the university under SAP program. DT thanks CSIR, New Delhi, India for a fellowship.

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Footnote

† Electronic supplementary information (ESI) available: Figures showing the change in fluorescence and UV-vis titrations of 1 and 2 with various cations, packing diagram, Job plots, binding constant and detection limit plots, MTT assay, spectral data. CCDC 1408733. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra05036k

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