Evaluation of electron or charge transfer processes between chromenylium-based fluorophores and protonated–deprotonated aniline

Abstract

Three functional dyes based on a chromenylium skeleton were prepared. The chromenylium-based fluorophores were regarded as acceptor parts and the aniline served as donor parts; their pH-dependent fluorescent responses were used to evaluate the photoinduced electron transfer (PET) or intramolecular charge transfer (ICT) processes between the chromenylium-based fluorophores and aniline. Two chromenylium–indole hybrid functional dyes (1a–b) respectively showed a fluorescence enhancement and decrease with increasing acidity, while chromenylium–coumarin hybrid dye (1c) gave an OFF–ON response towards a gradually decreasing pH. The three dyes gave emissions at 675–850 nm when they were excited at 650 nm, and the calculated pK_a values of 1a–c are 4.13, 3.15 and 2.48, respectively. The optical responses were also illustrated by (TD)DFT calculation; the OFF–ON emission of dye 1c was mainly controlled by the PET process, and both PET and ICT processes were found between the aniline parts and the chromenylium–indole parts in dyes 1a–b.

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Introduction

Near-infrared (NIR) fluorescent dyes emit in the 650–900 nm region, and they have been extensively studied in recent years.^1–5 Near-infrared light can penetrate deeply into tissue, reduce the damage to a biological sample, decrease the influence of background fluorescence in the test, and so on.^6–9 The emission of most NIR dyes is located at the near-infrared region, while a few of them have both NIR excitation and emission.^10,11 The fluorescent dyes with long excitation and emission wavelengths are more preferable for applications, and they have been used in the detection of biological species in in vivo assay.^9,12–14

Chromenylium is the moiety of pyronin with bicyclic pyrylium. Some fluorophores based on chromenylium have been developed in recent years; they retain the rhodamine-like fluorescence OFF–ON switching mechanism with a high pK_a, and their excitation and emission are both in the NIR region (Fig. 1, NIR 1–3).^15–17 Using a dye as a platform (NIR 1), one NIR fluorescent turn-on sensor, which was based on the spirocyclization-induced fluorescence switching mechanism, was capable of application in imaging for endogenously produced hypochlorous acid.¹⁵ A chromenylium-based ratiometric fluorescent probe (NIR 3) has been developed by the similar fluorescence switching ring-opening and ring-closing mechanism, and it provided a ratiometric fluorescent platform for NIR probes. Another ratiometric NIR fluorescent probe has been designed for sensing mercury(II) ions by this platform.¹⁷ The chromenylium derivatives keep some advantages of the original rhodamines, such as a high fluorescence quantum yield and light stability,^18–22 therefore, more chromenylium hybrid dyes have been researched by the spirocyclization-induced fluorescence switching mechanism.^12,23,24

Fig. 1 Reported chromenylium-based functional dyes: NIR 1, NIR 2, and NIR 3.

Photoinduced electron transfer (PET) and intramolecular charge transfer (ICT) are the basic design principles of fluorescent probes. There are unique advantages of the dyes based on the PET and ICT mechanisms, such as a satisfactory fluorescence response and clear mechanism explanation by frontier orbital theory.^25–30 In this paper, three typical chromenylium skeletons were selected as fluorophores, with the aniline group as a donor, which is usually used as the electron donor in PET and ICT-based fluorescent dyes.^31,32 Different from spirocyclization-based fluorescent dyes, such types of fluorescent dyes based on electron or charge transfer mechanisms usually have lower pK_a values with a protonated nitrogen atom.

Results and discussion

Design and preparation of the functional dyes

Compared with the reported design by carboxyl group participation in the reaction, the PET or ICT-based response needs two essential parts: an acceptor and a donor. The chromenylium-based fluorophores with a positive charge were designed as acceptor parts. Dyes 1a and 1b are chromenylium–indole hybrid fluorophores; more specifically, dye 1b is constructed with chromenylium and indole parts with conjugated double bonds, while dye 1a contains a non-conjugated cyclic structure. So the different stretching directions of the indole structures in 1a–b would have a different effect towards the optical response. A chromenylium–coumarin hybrid fluorophore was also selected in this design (1c); it is directly constructed with chromenylium and coumarin parts without any conjugated linkers. The aniline part served as a donor, since its donating ability could be varied during the proton-promoted protonation–deprotonation process. The PET process between the chromenylium-based fluorophores and aniline parts would be evaluated in different pH conditions.

The preparation of dyes 1a–c is shown in Scheme 1. Compound 4 was synthesized by a three-step procedure from 4-(diethylamino)benzoic acid, and it was respectively reacted with cyclohexanone and acetone to produce compounds 5 and 6. The required dyes 1a–b were further prepared with a Fischer aldehyde (7) and compounds 5–6, independently. Finally, dye 1c was synthesized from compound 4 and 3-acetyl-7-diethylaminocoumarin (8). The structures of all compounds were confirmed by ¹H NMR, ¹³C NMR, and high resolution mass spectroscopy (HRMS-ESI⁺).

Scheme 1 Preparation of dyes 1a–c.

Optical properties of the functional dyes with pH

Chromenylium–indole hybrid fluorophores (1a–b). Absorption and fluorescence pH titration experiments of dyes 1a and 1b (10 μM) were performed in Britton–Robinson (B–R) buffer (40 mM) containing 30% dimethyl sulfoxide (DMSO). The absorbance of dye 1a was slightly decreased at 719–723 nm from pH = 8.0 to pH = 2.8, and the color of the solution was accordingly changed from violet-blue to light sky blue under visible light (Fig. 2(a) and Table 1). The excitation wavelength was selected as 650 nm (Fig. 2(e)), and the pH-dependant fluorescence intensities showed a 6.2-fold enhancement immediately in acidic conditions, and the emission maxima slightly red-shifted from 749 nm to 758 nm (Fig. 2(b)). Meanwhile, the fluorescence quantum yield was increased from 2.0% in neutral conditions to 9.1% in acidic conditions. The absorption spectra of dye 1b are similar to those of dye 1a (Fig. 2(c)); the absorbance was reduced at 702–711 nm from pH = 8.0 to pH = 2.0. Compared with 1a, the pH-dependant emission spectra gave a reverse response; the fluorescent intensities were immediately reduced to 30% from 736 nm in neutral conditions to 742 nm in acidic conditions (Fig. 2(d)). The fluorescence quantum yield simultaneously dropped from 5.2% to 3.6%. The calculated pK_a values of dyes 1a–b with pH are 4.13 and 3.15 respectively (Fig. 2(f)). The basic photochemical data of 1a–b with different pH are listed in Table 1. Although dyes 1a–b gave optical changes towards pH variation, the increasing or decreasing ratios are too low for real applications, and other related fluorophores will be selected for the next stage to improve the PET process.

Fig. 2 Optical responses of dyes 1a–b (10 μM) with various pH values with Britton–Robinson buffer containing 30% DMSO: (a and b) dye 1a; (c and d) dye 1b; (a and c) absorption spectra (pH = 2.8–8.0 for 1a and pH = 2.0–8.0 for 1b), and the inset is a photograph of the pH-dependent samples; (b and d) emission spectra (λ_ex = 650 nm, slit width = 3 nm/3 nm, and pH = 2.8–8.0 for 1a, slit width = 3 nm/5 nm and pH = 2.0–8.0 for 1b); (e) excitation spectra of dyes 1a–b; (f) fluorescence intensity changes of dyes 1a–b with pH at maximum emission peaks.

Table 1 Photochemical properties of dyes 1a–c with pH

Dyes	pH^a	λabs^b	λem^b	ε^c	Stokes shift^b	Φ^d	pKa
a The photochemical properties measured in B–R buffer containing 30% DMSO.b Reported in nm.c Reported in M^−1 cm^−1.d Cyanine dye HPDITCP (Φ = 0.16 in ethanol, ref. 34).
1a	7.0	719	749	6.17 × 10^4	30	0.020	4.13
1a+H^+	3.0	723	758	4.60 × 10^4	35	0.091
1b	7.0	702	736	5.86 × 10^4	34	0.052	3.15
1b+H^+	2.0	711	742	2.83 × 10^4	31	0.036
1c	7.0	583	691	3.43 × 10^4	108	0.003	2.48
1c+H^+	1.6	668	719	3.50 × 10^4	51	0.023

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Chromenylium–coumarin hybrid fluorophore (1c). To improve the optical properties of the PET process based on the chromenylium skeleton, the chromenylium–coumarin hybrid structure was further selected. The direct conjugation of the two parts would make it a promising candidate for PET transformation, and the OFF–ON-type optical response would be shown. The absorption maxima of 1c at 583 nm were gradually red-sifted to 668 nm when the pH decreased from 7.0 to 1.6, and the solutions of 1c gave colorimetric changes from navy blue in neutral conditions to light blue under acidic conditions (Fig. 3(a)). The NIR emission peak of 1c at 719 nm was immediately enhanced with increasing acidity (Fig. 3(b)) when the excitation wavelength was selected as 650 nm (Fig. 3(c)). The emission spectra of dye 1c with pH showed a typical NIR OFF–ON fluorescence response with a 79-fold enhancement in the emission intensity; the fluorescence quantum yield was increased from 0.3% to 2.3% (Table 1). Based on the ratios of absorbance at 583 nm and 668 nm, the calculated absorbance-related pK^Abs_a is 2.71, and the calculated pK^FI_a value based on the emission property is 2.48 (Fig. 3(d)). Above all, dyes 1a–c keep some advantages like other chromenylium-based dyes, such as NIR excitation and emission and satisfactory fluorescence quantum yield, but the water solubility of them needs to be improved compared with reported pH probes.^18,33

Fig. 3 Optical responses of dye 1c (10 μM) with various pH with Britton–Robinson buffer containing 30% DMSO: (a) absorption spectra (pH = 1.6–7.0), and the inset is a photograph of the pH-dependent samples; (b) emission spectra (λ_ex = 650 nm, slit width = 3 nm/5 nm, pH = 1.6–7.0); (c) excitation spectra of dye 1c; (d) absorption ratios A_583nm/A_668nm of dye 1c at pH 1.6–7.0 and fluorescence intensity changes of dye 1c with pH at the maximum emission peak.

To verify the protonated structures of dyes 1a–c, the ¹H NMR spectra of dyes 1a–c under neutral and acidic conditions were obtained in DMSO-d₆ (dyes 1a–b in Fig. S1† and dye 1c in Fig. 4), which reveal that the chemical shifts of the protons on the N,N-diethylaniline ring were shifted to lower fields following the protonation of the amine.

Fig. 4 ¹H NMR of dye 1c in DMSO-d₆ (top) and acidic conditions (bottom).

Mechanism exploration

As discussed in the previous section, dyes 1a and 1b bearing chromenylium–indole hybrid fluorophores show enhanced (1a) and reduced (1b) fluorescence response with the increasing solution acidity respectively (Fig. 2). However, the chromenylium–coumarin hybrid fluorophore-based dye 1c, without any conjugated linkers, exhibits an OFF–ON fluorescent response (Fig. 3). In order to explore the mechanism, the geometric configurations of the ground and the lowest excited states of dyes 1a⁺–c⁺ and their protonated forms were fully optimized. The charge can only be defined on the whole molecule, not a particular atom in Gaussian, so the small differences between the resonance structures would only be given through bond lengths, bond angles, and other parameters, such as molecular energy etc. Take 1c for example: dyes 1c⁺-i and 1c⁺-ii possess exactly the same molecular energy (Table S1†) and the other bond parameters attributed to the fully conjugated molecular skeleton, and their protonated forms also express the same results. As shown in Fig. 5, dye 1c⁺ has two absorption peaks from S₀ to S₁ and from S₀ to S₂, however, when the electron in the excited state transitions back to the ground state, the HOMO energy of the electron donor (amine) was higher than that of the electron acceptor (fluorophore), so the PET process occurred (Fig. 5(b)), which led to the fluorescence quenching of dye 1c⁺. In acidic solution, the PET process was inhibited, so the fluorescence recovered (Fig. 5(c)). Because the conjugate degrees of 1a–b were less than that of dye 1c, the calculation results showed that the conjugated structures had very small energy differences (Table S1†), but they exhibited different charge transfer processes, such as 1a⁺-i and 1b⁺-ii showing the PET process (Fig. S2b and S3c†), while only part of the ICT process was found in 1a⁺-ii and 1b⁺-i (Fig. S2c and S3b†), which led to 1a–b and 1c showing a different fluorescence response. The detailed vertical excitation and emission-related parameters are listed in Tables S2 and S3 (ESI†). Calculation results are basically consistent with experiments.

Fig. 5 The resonance equilibrium of 1c⁺ and its protonated form (a), and frontier molecular orbitals (FMOs) involved in the vertical excitation and emission of dye 1c⁺ (b) and its protonated forms (c). CT stands for conformation transformation. Excitation and radiative processes are represented by solid lines and the non-radiative processes by dotted lines. For details please refer to Tables S2 and S3 (ESI†).

Selectivity and reversibility

The selectivity of dyes 1a–c for preferential binding to protons over other potential interferents under biological conditions was investigated in acidic and neutral conditions. For dyes 1a or 1b (10 μM), there was no significant change in the fluorescence intensity in the presence of common cations such as K⁺, Na⁺, Ca²⁺ and Mg²⁺, as well as most transition metal ions, such as Cd²⁺, Co²⁺, Mn²⁺, and Ni²⁺. Moreover, bioactive amino acids, such as Lys, Phe, Gly, Glu, Arg, Cys, Pro, Try, and His, were also investigated, and no obvious changes were observed. But when heavy metal ions such as Hg²⁺ were present under neutral conditions, the fluorescence intensity of dyes 1a–b slightly decreased. Moreover dye 1b also responded to Cu²⁺ in acidic solutions (Fig. S4 and S5†). This phenomenon may be caused by complexation of Hg²⁺, and Cu²⁺. As shown in Fig. 6, a significant change in the fluorescence intensity of dye 1c (10 μM) is also not observed in the presence of common cations, metal ions and amino acids. The results illustrate that dye 1c has a better selectivity response to pH than dyes 1a–b in the presence of potential interferents.

Fig. 6 Selectivity and reversibility of dye 1c (10 μM). (a and b) Fluorescence responses of dye 1c (10 μM) to different analytes, such as K⁺ (100 mM), Na⁺ (100 mM), Ca²⁺ (0.5 mM), Mg²⁺ (0.5 mM), Cd²⁺ (0.3 mM), Cu²⁺ (0.3 mM), Co²⁺ (0.3 mM), Hg²⁺ (0.3 mM), Mn²⁺ (0.3 mM), Ni²⁺ (0.3 mM), Cys (0.1 mM), Phe (0.1 mM), Gly (0.1 mM), Glu (0.1 mM), Arg (0.1 mM), Lys (0.1 mM), Pro (0.1 mM), Try (0.1 mM) and His (0.1 mM). (a) Tested in Britton–Robinson buffer (pH = 7.0); (b) tested in Britton–Robinson buffer (pH = 1.8). (c) The reversible optical response of dye 1c at different pH conditions.

The reversibility of dyes 1a–c was also evaluated. When a solution of dyes 1a–c was changed to acidic or neutral for five cycles, the emission spectra of them showed no significant changes (dyes 1a–b in Fig. S6†, and dye 1c in Fig. 6(c)). It can be deduced that dyes 1a–c have good reversibility.

Conclusions

In summary, three chromenylium-based NIR dyes 1a–c have been designed and synthesized. These dyes are based on the PET or ICT mechanism instead of the spirocyclization-induced fluorescence switching mechanism. Dyes 1a and 1b are chromenylium–indole hybrid fluorophores, but they have different effects for their optical response because of the different stretching directions of the indole structures. This phenomenon can be attributed to the fact that both PET and ICT processes exist in the aniline parts and the chromenylium–indole parts. Dye 1c is chromenylium–coumarin hybrid fluorophore, and it is a typical OFF–ON fluorescent dye mainly controlled by the PET process. All of these optical mechanisms are successfully verified by (TD)DFT calculations. Moreover, all these dyes have good reversibility, and dye 1c also has a better selectivity response to pH than dyes 1a–b. Therefore, dye 1c is expected to become an excellent near-infrared pH probe for the determination of the acidity of cancer cells.

Experimental section

Materials and apparatus

All reagents and solvents (analytical grade) were purchased from TCI Development Co., Ltd (Tokyo, Japan), Energy Chemical Co., Ltd (Shanghai, China) and Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and used directly. Flash chromatography was performed with silica gel (200–300 mesh). Britton–Robinson (B–R) buffer solution (40 mM, pH = 1.6–8.0) was used. The pH values were finally tested by a pH meter.

¹H NMR and ¹³C NMR spectra were recorded on a Varian 400 MHz spectrometer, and tetramethylsilane (TMS) or solvent peaks were used as an internal standard. High resolution mass spectra were recorded on a micrOTOF-Q III mass spectrometer in ESI⁺ mode. UV-vis spectra were obtained with a Shimadzu UV-1800 spectrophotometer, and fluorescence emission spectra were performed with a fused quartz cuvette (10 mm × 10 mm) on a Shimadzu RF-5301 PC spectroscope with an R-928 photomultiplier at room temperature. The pH values were measured with a Lei-Ci (pH-3C) digital pH meter (Shanghai, China) using a combined glass calomel electrode.

Synthesis and characterization

4-(Diethylamino)benzoyl chloride (2). To a solution of 4-N,N-diethylaminobenzoic acid (2.00 g, 10.3 mmol) in 20.0 mL dichloromethane, thionyl chloride (3.57 g, 30.0 mmol) and N,N-dimethylformamide (DMF, 1 drop) were added at room temperature. The resulting suspension was stirred for 20 h at 20 °C. The solvent was removed by evaporation to afford the acyl chloride product as a green solid (2.05 g, 97.0%). The product was used for the next step without further purification.

3-(Diethylamino)phenyl-4-(diethylamino)benzoate (3). To a stirred solution of 3-(diethylamino)phenol (3.30 g, 20.0 mmol), triethylamine (2.6 mL, 20.0 mmol), and dichloromethane (50.0 mL), a solution of 4-(diethylamino)benzoyl chloride (20.0 mmol) in dichloromethane (20.0 mL) was added dropwise at room temperature. The resulting solution was stirred for a further 5 hours at room temperature. The solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel eluting with hexane and ethyl acetate (8 : 1, v/v) to afford the product as a white solid (1.30 g, 19.0%). Mp: 54.0–55.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 8.5 Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 6.65 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 8.3 Hz, 1H), 6.46 (br, 2H), 3.41 (q, J = 6.9 Hz, 4H), 3.33 (q, J = 6.9 Hz, 4H), 1.19 (t, J = 7.2 Hz, 6H), 1.15 (t, J = 7.2 Hz, 6H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 165.6, 152.7, 151.3, 149.0, 132.2, 129.7, 115.5, 110.2, 108.8, 108.6, 105.2, 44.5, 44.4, 12.6, 12.5 ppm. HRMS (ESI⁺): m/z = 341.2221 (calcd for [M + H⁺]⁺, 341.2229).

(4-(Diethylamino)-2-hydroxyphenyl)(4-(diethylamino)phenyl)methanone (4). A mixture of anhydrous aluminum chloride (640.0 mg, 4.80 mmol) and 3 (412.0 mg, 1.20 mmol) was heated for 2 h at 175 °C. The reaction mixture was cooled to room temperature, and slowly hydrolysed with iced water and 1.0 mL concentrated hydrochloric acid. The mixture was stirred for 2 hours at room temperature, and then it was extracted with dichloromethane (20.0 mL × 3). The organic phase was removed by evaporation, and the residue was purified by column chromatography on silica gel eluting with dichloromethane to afford the product as a yellow oil (118.0 mg, 28.0%). ¹H NMR (400 MHz, CDCl₃) δ 13.19 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 8.5 Hz, 2H), 6.16 (br, 2H), 3.45–3.37 (m, 8H), 1.21 (t, J = 6.9 Hz, 12H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ 195.9, 165.3, 152.6, 149.5, 134.6, 131.3, 124.9, 109.7, 108.8, 102.4, 97.0, 44.10, 44.05, 12.21, 12.06 ppm. HRMS (ESI⁺): m/z = 341.2259 (calcd for [M + H⁺]⁺, 341.2229).

6-(Diethylamino)-9-(4-(diethylamino)phenyl)-1,2,3,4-tetrahydroxanthylium perchlorate (5). A solution of 4 (72.0 mg, 0.21 mmol) and cyclohexanone (22 μL, 0.21 mmol) in concentrated H₂SO₄ (2.0 mL) was stirred at 90 °C for 6 h. After cooling to room temperature, the reaction mixture was poured into cold water (10.0 mL), and then 70% perchloric acid (200 μL) was added. The solution was extracted with dichloromethane (30.0 mL × 3), and the organic layer was evaporated in a vacuum. The residue was purified by column chromatography on silica gel eluting with dichloromethane and methanol (25 : 1, v/v) to afford the product as a dark brown solid (70.0 mg, 52.0%). Mp: 211.0–212.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d, J = 9.6 Hz, 1H), 7.26 (d, J = 9.4 Hz, 2H), 7.13 (d, J = 9.5 Hz, 1H), 6.84 (br, 3H), 3.64 (q, J = 6.9 Hz, 4H), 3.49 (q, J = 6.8 Hz, 4H), 3.09 (t, J = 6.3 Hz, 2H), 2.68 (t, J = 5.3 Hz, 2H), 2.00–2.05 (m, 2H), 1.82–1.77 (m, 2H), 1.33 (t, J = 7.0 Hz, 6H), 1.26 (t, J = 7.0 Hz, 6H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 169.2, 163.0, 159.3, 155.0, 132.21, 132.16, 121.1, 119.65, 117.1, 116.9, 111.5, 110.9, 95.5, 46.0, 44.7, 29.7, 26.5, 22.2, 21.1, 12.6, 12.5 ppm. HRMS (ESI⁺): m/z = 403.2733 (calcd for [M − ClO₄⁻]⁺, 403.2749).

7-(Diethylamino)-4-(4-(diethylamino)phenyl)-2-methylchromenylium perchlorate (6). A solution of 4 (131.0 mg, 0.38 mmol) and acetone (200 μL, 2.70 mmol) in concentrated H₂SO₄ (3.0 mL) was stirred at 90 °C for 6 h. After cooling to room temperature, the reaction mixture was poured into cold water (20.0 mL), and then 70% perchloric acid (300 μL) was added. The solution was extracted with dichloromethane (50.0 mL × 3), and the organic layer was evaporated by evaporation. The crude product was purified by column chromatography on silica gel eluting with dichloromethane and methanol (25 : 1, v/v) to afford the product as a dark brown solid (35.0 mg, 21.0%). Mp: > 250.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 9.6 Hz, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 9.4 Hz, 1H), 7.15 (s, 1H), 6.85 (d, J = 8.8 Hz, 2H), 6.78 (s, 1H), 3.63 (q, J = 6.8 Hz, 4H), 3.51 (q, J = 6.8 Hz, 4H), 2.72 (s, 3H), 1.33 (t, J = 7.0 Hz, 6H), 1.26 (t, J = 6.9 Hz, 6H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 162.7, 155.3, 155.0, 150.0, 147.3, 129.0, 127.1, 115.0, 111.6, 108.5, 107.5, 106.7, 91.5, 41.1, 40.2, 16.4, 7.83, 7.79 ppm. HRMS (ESI⁺): m/z = 363.2448 (calcd for [M − ClO₄⁻]⁺, 363.2436).

6-(Diethylamino)-9-(4-(diethylamino)phenyl)-4-(2-(1,3,3-trimethylindolin-2-ylidene)ethylidene)-1,2,3,4-tetrahydroxanthylium perchlorate (1a). A solution of 5 (105.6 mg, 0.21 mmol) and Fischer aldehyde (43.0 mg, 0.21 mmol) in acetic anhydride (8.0 mL) was heated to 50 °C for 40 min. Then, water (8.0 mL) was added to the mixture to quench the reaction. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by column chromatography on silica gel eluting with dichloromethane and methanol (20 : 1, v/v) to afford the product as a dark brown solid (52.0 mg, 36.0%). Mp: > 250.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.52 (d, J = 14.0 Hz, 1H), 7.41 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 9.3 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.13 (br, 3H), 6.77 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 9.2 Hz, 1H), 6.58 (br, 1H), 5.99 (d, J = 13.9 Hz, 1H), 3.65 (s, 3H), 3.56 (q, J = 6.9 Hz, 4H), 3.45 (q, J = 6.8 Hz, 4H), 2.69 (t, J = 5.8 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 1.81–1.85 (m, 2H), 1.78 (s, 6H), 1.30 (t, J = 7.0 Hz, 6H), 1.25 (t, J = 7.0 Hz, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ 172.1, 163.7, 156.6, 153.5, 152.2, 148.7, 143.3, 140.9, 140.5, 131.0, 130.2, 128.9, 124.8, 122.4, 121.1, 119.6, 116.5, 113.8, 112.4, 111.0, 110.3, 98.6, 95.9, 49.0, 45.4, 44.6, 31.6, 28.7, 27.6, 24.6, 21.3, 12.8, 12.7 ppm. HRMS (ESI⁺): m/z = 586.3799 (calcd for [M − ClO₄⁻]⁺, 586.3797).

2-(3-(7-(Diethylamino)-4-(4-(diethylamino)phenyl)-2H-chromen-2-ylidene)prop-1-en-1-yl)-1,3,3-trimethyl-3H-indol-1-ium perchlorate (1b). A solution of 6 (35.0 mg, 0.10 mmol) and Fischer aldehyde (20.0 mg, 0.10 mmol) in acetic anhydride (4.0 mL) was heated to 50 °C for 40 min. Then, water (4.0 mL) was added to the mixture to quench the reaction. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by column chromatography on silica gel eluting with dichloromethane and methanol (20 : 1, v/v) to afford the product as a dark brown solid (15.0 mg, 23.0%). Mp > 250.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (t, J = 13.4 Hz, 1H), 7.74 (d, J = 9.1 Hz, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.33 (br, 2H), 7.15 (t, J = 7.3 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.80 (br, 3H), 6.70 (s, 1H), 6.68 (s, 1H), 6.26 (d, J = 13.5 Hz, 1H), 6.10 (d, J = 12.9 Hz, 1H), 3.58 (br, 7H), 3.48 (q, J = 6.6 Hz, 4H), 1.73 (s, 6H), 1.31 (t, J = 6.8 Hz, 6H), 1.25 (t, J = 6.8 Hz, 6H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 170.9, 165.2, 157.6, 155.1, 152.6, 150.0, 143.5, 143.2, 140.2, 131.4, 129.8, 128.5, 124.0, 122.0, 121.1, 112.3, 111.53, 111.48, 110.8, 109.6, 100.8, 96.8, 48.5, 45.2, 44.6, 31.0, 28.6, 12.57 ppm. HRMS (ESI⁺): m/z = 546.3453 (calcd for [M − ClO₄⁻]⁺, 546.3484).

7,7′-Bis(diethylamino)-4-(4-(diethylamino)phenyl)-2′-oxo-2′H-[2,3′-bichromen]-1-ylium perchlorate (1c). A solution of 4 (118.0 mg, 0.35 mmol) and 3-acetyl-7-diethylaminocoumarin (440.0 mg, 1.70 mmol) in concentrated H₂SO₄ (3.0 mL) was stirred at 90 °C for 12 h. After cooling to room temperature, the reaction mixture was poured into cold water (20.0 mL), and then 70% perchloric acid (400 μL) was added, and the crude product was obtained by filtration and washed with water. It was purified by column chromatography on silica gel eluting with dichloromethane and methanol (20 : 1, v/v) to afford the product as a dark brown solid (18.0 mg 8.0%). Mp > 250.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.35 (s, 1H), 8.42 (s, 1H), 8.10 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 9.4 Hz, 1H), 7.63 (d, J = 8.5 Hz, 2H), 7.52 (s, 1H), 6.99 (d, J = 9.2 Hz, 1H), 6.83 (br, 2H), 6.74 (d, J = 8.7 Hz, 1H), 6.45 (s, 1H), 3.67 (q, J = 5.9 Hz, 4H), 3.55–3.47 (m, 8H), 1.36 (t, J = 6.4 Hz, 6H), 1.27 (br, 12H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 160.2, 159.1, 158.9, 158.8, 158.1, 154.6, 154.1, 147.4, 134.2, 132.8, 130.6, 115.2, 113.2, 111.8, 111.0, 110.7, 110.1, 106.6, 98.1, 98.0, 96.2, 96.1, 45.9, 45.4, 44.9, 12.7, 12.6, 12.5 ppm. HRMS (ESI⁺): m/z = 564.3236 (calcd for [M − ClO₄⁻]⁺, 564.3226).

Absorption and fluorescence titration experiment

Stock solutions of dyes 1a–c (100 μM) were prepared in a volumetric flask (100 mL) with DMSO. Each test solution (10 μM) was prepared in a volumetric flask (10 mL) with 1.0 mL stock solution, 2.0 mL DMSO and the corresponding buffer solution to give a total volume of 10.0 mL. Absorption and fluorescence spectra were obtained with 1.0 cm × 1.0 cm quartz cells.

Selectivity experiment

Stock solutions of dyes 1a–c (100 μM) were prepared in a volumetric flask (100 mL) with DMSO. Stock solutions of various ions were prepared in volumetric flasks (10 mL) with concentrations of KCl (1.0 M), NaCl (1.0 M), CaCl₂ (5 mM), MgSO₄ (5 mM), CdCl₂ (3 mM), CuSO₄ (3 mM), CoCl₂ (3 mM), HgCl₂ (3 mM), MnCl₂ (3 mM), or NiCl₂ (3 mM) in doubly distilled water. Stock solutions of all kinds of amino acids were all prepared in volumetric flasks (10 mL) with concentrations of 1 mM in twice distilled water. Each test solution was prepared in a volumetric flask (10 mL) with 1 mL stock solution of dyes, 2 mL DMSO and 1.0 mL stock solutions of the corresponding ions or amino acids, diluted with neutral (pH 7.0 for dyes 1a–c) and acidic (pH 3.0, 2.0, and 1.6 for dyes 1a–c respectively) B–R buffer solution to give a total volume of 10.0 mL. Their fluorescence spectra were then recorded.

Determination of quantum yield

All the relative fluorescence quantum yields were determined and calculated with the following equation:

Φ_x/Φ_st = [A_st/A_x][n_x²/n_st²][D_x/D_st]

where st: standard; x: sample; Φ: quantum yield; A: absorbance at the excitation wavelength; D: area under the fluorescence spectra on an energy scale; and n: the refractive index of the solution calculated from the reported data (DMSO/water (v/v = 3.0/7.0)). The cyanine dye HPDITCP (Φ = 0.16 in ethanol) was used as standard.

Calculation details

The geometries of the dyes 1a⁺–c⁺ and their protonated forms in the ground state (S₀ state) were optimized by density functional theory (DFT) at the B3LYP/6-31+G(d) level, and the geometries at the lowest excited state (S₁ state) were optimized by TDDFT methods with B3LYP/6-31G(d) using the Gaussian program package.³⁵ In some cases, higher excited states were optimized. There are no imaginary frequencies in the frequency analysis of all the calculated structures; therefore, each calculated structure is in a local energy minimum. The energy gap between the S₀ state and the lowest excited state was calculated with the (TD)DFT//BP86/TZVP method based on the optimized S₀ state geometry (for absorption) and the lowest excited state geometry (for fluorescence), respectively. Water was used as the solvent for the (TD)DFT calculations (CPCM model).

Acknowledgements

The project is supported from the National Nature Science Foundation of China (51273136), Natural Science Fund of Jiangsu Province (BK20151262) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental section and related spectra. See DOI: 10.1039/c6ra19831g

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