Turn-off fluorescence sensing of benzenediols via guest-induced π-conjugation switching in bisimidazole-based hydrindacene allosteric receptors

Abstract

Fluorescent bisimidazole-based hydrindacene allosteric receptors 1a–c and 2 were synthesized for nonlinear ON/OFF sensing of benzenediols. By tuning their D–π–D conjugation through substituent modification, these receptors were designed to function as turn-off fluorescent sensors based on the cleavage of their π-conjugation accompanied by binding with benzenediols. The cooperative allosteric binding demonstrated selective and efficient nonlinear quenching toward orcinol.

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Introduction

Imidazole is a five-membered heteroaromatic ring with two electronically distinct nitrogen atoms, an electron donor and an electron acceptor.^1,2 This dual electronic nature allows the electronic behavior of imidazole to be tuned based on the substituents on the ring. Depending on whether an electron-withdrawing/neutral or electron-donating group is attached at the C2-position, imidazole can act as an electron-donating^3–5 or electron-accepting unit,^6–8 respectively. Due to these unique electronic characteristics, imidazole serves as a versatile building block for constructing chromophores,⁹ fluorescent materials with intramolecular charge transfer (ICT)^10,11 or excited state intramolecular proton transfer (ESIPT) properties,^12,13 and as ancillary ligands for phosphorescent materials.^14,15 Particularly, these luminescent properties have also been applied to the design of chemical sensors capable of selectively detecting specific metal^4,5,16–19 and non-metal ions,^20–24 and chemical species with particular functional groups.^25–30 Although such fluorescent sensors exhibit a linear response proportional to guest concentration and are suitable for quantitative analysis, they rely on an intensity change associated with 1 : 1 complexation as a threshold, which presents limitations in improving selectivity. To overcome this limitation and enhance selectivity, approaches based on positive homotropic allosteric binding have recently garnered attention.^31–34 The sigmoidal nonlinear response, in which binding of a guest molecule strengthens further binding of the same guest, is expected to enable improved sensitivity and selectivity.

Recently, we developed hydrindacene-based allosteric receptors with benzimidazole units that form 1 : 2 host–guest complexes with benzenediols (Scheme 1a).³⁵ These bisimidazole D–π–D systems function as highly π-conjugated chromophores.^36–38 In our system, guest binding induces a conformational twist in the benzimidazole units, which disrupts their π-conjugation and changes their spectroscopic properties. Unlike typical fluorescent sensors that show a linear response from 1 : 1 binding, our allosteric system exhibits the desired nonlinear response,³¹ enabling sharp ON/OFF switching once a concentration threshold is exceeded. This makes it a promising candidate for application in biosensing and environmental monitoring, where a clear threshold and enhanced guest selectivity are desired.^32–34

Scheme 1 (a) Allosteric binding of the bisimidazole-based hydrindacene receptor 1a with orcinol.³⁵ (b) Fluorescent receptors 1a–c (with benzimidazole units) and receptor 2 (with naphthoimidazole units), along with the proposed fluorescence quenching mechanism via D–π–D twisting (this work).

Herein, we investigate how substituents and π-conjugation extension influence the fluorescence sensing behavior of bisimidazole-based hydrindacene allosteric receptors through selective and cooperative guest binding (Scheme 1b). We synthesized a series of receptors, bisbenzimidazole 1a–c and bisnaphthoimidazole 2, whose emission colors were precisely tuned. Their fluorescence was efficiently quenched upon addition of benzenediols. Notably, orcinol induced nonlinear fluorescence quenching with increasing guest concentration due to allosteric 1 : 2 complexation. Our research demonstrates that this system functions as a turn-off supramolecular fluorescent sensor that exhibits enhanced guest selectivity and distinct emission changes in the visible-light region, unlike conventional benzenediol sensors.^39–41

Results and discussion

Preparation of bisimidazole-based hydrindacene receptors

Receptors 1a and 1b were prepared as shown in Scheme 2, following our previously reported procedure.³⁵ The diiode compound 3 was first converted to the dicyano compound by cyanation with CuCN. Subsequent hydrolysis with hydrogen peroxide afforded the diamide compound 4. The amide groups of 4 were then converted to the imidate intermediate 5 using Meerwein reagent, followed by condensation with 1,2-phenylenediamine or 4,5-dimethyl-1,2-phenylenediamine to give receptor 1a and receptor 1b, respectively.

Scheme 2 Synthesis of receptors 1a–1c and 2. Reagents and conditions: (i) CuCN, DMF, 150 °C, 16 h, 92%; (ii) H₂O₂ aq., K₂CO₃, acetone/DMSO, r.t., 3 d, 89%; (iii) Me₃O⁺·BF₄⁻, CH₂Cl₂, reflux, 2 d; (iv) 1,2-phenylenediamine, EtOH, reflux, 3 d, 2 steps 8%; (v) 4,5-dimethyl-1,2-phenylenediamine, EtOH, reflux, 3 d, 2 steps 7%; (vi) ⁱPrMgCl·LiCl, DMF, THF, −78 °C to r.t., 19 h, 78%; (vii) 4,5-dimethoxy-1,2-phenylenediamine, nitrobenzene/AcOH, 60 °C, 21 h, 38%; (viii) 2,3-diaminonaphthalene, nitrobenzene/AcOH, 60 °C, 2 d, 50%.

Receptors 1c and 2 were also prepared from 3. The diiodo compound 3 was converted to the diformyl compound 6 using the Bouveault aldehyde synthesis.⁴² The subsequent condensation of 6 with 4,5-dimethoxy-1,2-phenylenediamine or 2,3-diaminonaphthalene in nitrobenzene/AcOH⁴³ afforded receptor 1c and receptor 2, respectively.

X-ray structures of guest-free receptors

The structures of bisbenzimidazole receptor 1a³⁵ and bisnaphthoimidazole receptor 2 were elucidated by single-crystal X-ray structural analysis (Fig. 1). Both crystal structures of 1a and 2 include two molecules of methanol used as the recrystallization solvent with hydrogen bond formation between the amidine unit. The amidine units and phenylene ring of the hydrindacene framework were slightly tilted (34.0° in 1a and 28.0° in 2, respectively).

Fig. 1 X-ray crystal structures of receptors 1a and 2. Amidine N forms hydrogen bonds with the solvent methanol. Hydrogen atoms of C–H are omitted for clarity.

Allosteric binding of bisimidazole-based receptors toward benzenediols

First, to evaluate the guest-selective allosteric binding ability of the receptors—an essential step for demonstrating the nonlinear fluorescence quenching causing by D–π–D conjugation cleavage upon allosteric guest binding—we investigated the complexation behavior of receptor 1a with three representative guests (orcinol, catechol, and phenol) by ¹H NMR spectroscopy in CDCl₃ (Fig. 2). Upon addition of orcinol, the NH proton signal of the benzimidazole unit shifted downfield to 10.81 from 9.51 ppm (Δδ = +1.30 ppm), and the methylene proton signal of the five-membered ring (H_a) shifted upfield to 3.15 from 3.86 ppm (Δδ = −0.71 ppm). This suggests that 1a forms hydrogen bonds with orcinol, which is accompanied by shielding on the methylene protons by the benzene ring of the guest. The addition of catechol also caused a downfield shift of the NH proton (Δδ = +0.95 ppm) and an upfield shift of H_a (Δδ = −0.52 ppm), indicating a weaker guest binding than that with orcinol. In contrast, the addition of phenol induced only minor shifts in the NH proton (Δδ = +0.08 ppm) and H_a (Δδ = −0.04 ppm). These results indicate that, similar to the previously reported hydrindacenediamide receptor,⁴⁴ the bisimidazole receptors also selectively bind to benzenediols rather than phenol through cooperative hydrogen-bond formation based on allosteric binding.⁴⁵ The NMR titration profiles for orcinol and catechol were in good agreement with a 1 : 2 binding model as confirmed by curve fitting.⁴⁶ The macroscopic binding constants, K₁, K₂, and cooperative factor, α (= 4K₂/K₁), were estimated.⁴⁷ The values for orcinol were K₁ = 70 ± 9.1 M⁻¹, K₂ = 3560 ± 460 M⁻¹, and α = 210, while for catechol they were K₁ = 74 ± 14 M⁻¹, K₂ = 136 ± 23 M⁻¹, and α = 7.3. Both binding strength and cooperativity with orcinol were significantly greater than with catechol.

Fig. 2 (a) Complexation of receptor 1a with orcinol. (b) ¹H NMR spectra of (i) 1a only, (ii) 1a with 30 eq. of phenol, (iii) 1a with 30 eq. of catechol, (iv) 1a with 30 eq. of orcinol in CDCl₃.

Spectroscopic properties of bisimidazole-based receptors

Next, the spectroscopic properties of the receptors were examined in CH₂Cl₂ and DMSO (Fig. 3 and Table 1; additional spectra in acetone and MeCN are provided in the SI: Fig. S11, S13, S15 and S17). While benzimidazole (BZI) itself was non-emissive, all bisimidazole-based hydrindacene D–π–D receptors 1a–c and 2 exhibited clear fluorescence.

Fig. 3 UV/Vis absorption (solid lines) and fluorescence (dotted lines) spectra of receptors 1a (black), 1b (red), 1c (blue), and 2 (green) in (a) CH₂Cl₂ and (b) DMSO (10 µM).

Table 1 Spectroscopic properties of 1a–c and 2 in CH₂Cl₂ and DMSO (10 µM)

Receptor	Solvent	λ abs (nm)	λ em (nm)	Stokes shift (cm^−1)	φ F
a Fluorescence quantum yield (φF) was determined relative to quinine sulfate in 0.5 M H2SO4 (φF = 0.546).
BZI	CH2Cl2	274	—	—	—
	DMSO	275	—	—	—
1a	CH2Cl2	322	395	5740	0.67
	DMSO	321	395	5840	0.64
1b	CH2Cl2	337	412	5400	0.62
	DMSO	335	412	5580	0.59
1c	CH2Cl2	363	440	4820	0.72
	DMSO	359	444	5330	0.68
2	CH2Cl2	349	478	7730	0.24
	DMSO	349	501	8690	0.20

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The fluorescence spectra of 1a–c correspond to the electron-donating properties of the substituents,⁴⁸ showing progressive red-shifts in the order 1a < 1b < 1c (Fig. 3): receptor 1a exhibited deep blue emission (λ_em = 395 nm, φ_F = 0.67 in CH₂Cl₂; λ_em = 395 nm, φ_F = 0.64 in DMSO); 1b exhibited blue emission (λ_em = 412 nm, φ_F = 0.62 in CH₂Cl₂; λ_em = 412 nm, φ_F = 0.59 in DMSO); and 1c exhibited light blue emission (λ_em = 440 nm, φ_F = 0.72 in CH₂Cl₂; λ_em = 444 nm, φ_F = 0.68 in DMSO). The emission maxima (λ_em) remained nearly unchanged regardless of solvent polarity, which suggests that intramolecular charge transfer (ICT) is not a dominant feature in these systems. DFT calculations revealed that the electron-donating groups raised the HOMO energy levels while having a marginal effect on the LUMO energy levels, thereby narrowing the HOMO–LUMO energy gap (Fig. 4).

Fig. 4 HOMO and LUMO energy levels and Kohn–Sham orbitals of receptors 1a–c and 2, calculated at the r²SCAN-3c/def2-mTZVPP level.

The fluorescence spectrum of receptor 2 was further red-shifted, emitting a light green fluorescence. This result is consistent with DFT calculations, showing that the naphthoimidazole units narrow the HOMO–LUMO energy gap. This is attributed to the localization of the HOMO on the naphthoimidazole unit, which raises its energy level, whereas the LUMO is delocalized over both the naphthoimidazole and phenylene units, resulting in a lower energy level due to the extended π-conjugation. Furthermore, λ_em of 2 exhibited a large solvatochromic effect (λ_em = 478 nm, φ_F = 0.24 in CH₂Cl₂; λ_em = 501 nm, φ_F = 0.20 in DMSO). This large red-shift of 23 nm, with λ_em shifting from 478 to 501 nm, suggests the presence of intramolecular charge transfer (ICT) characteristics dependent on the solvent polarity.

Fluorescence intensity changes due to guest binding

To evaluate fluorescence changes induced by guest binding and protonation, the spectroscopic properties of each receptor in CH₂Cl₂ were examined (Fig. 5 and Table 2).

Fig. 5 UV-Vis absorption (solid lines) and fluorescence (dotted lines) spectra of receptor 1c (black), 1c with orcinol (red), 1c with catechol (blue), and 1c with phenol (green) in CH₂Cl₂ (1c: 10 µM; orcinol, catechol, and phenol: 30 mM).

Table 2 Spectroscopic properties of receptors 1a–c and 2 upon adding benzenediols and phenol in CH₂Cl₂ (receptors: 10 µM; orcinol, catechol, and phenol: 30 mM)

Receptor	λ abs (nm)	λ em (nm)	φ F	RFI
a Fluorescence quantum yield (φF) was determined relative to quinine sulfate in 0.5 M H2SO4 (φF = 0.546).
1a	322	395	0.67	1
1a + orcinol	—	395	0.047	0.020
1a + catechol	—	395	0.057	0.047
1a + phenol	318	395	0.38	0.58
1a + TFA	303	401	0.52	—
1b	337	412	0.62	1
1b + orcinol	—	412	0.049	0.030
1b + catechol	—	412	0.047	0.054
1b + phenol	331	412	0.38	0.75
1b + TFA	316	426	0.55	—
1c	363	440	0.72	1
1c + orcinol	308	440	0.16	0.060
1c + catechol	333	441	0.10	0.085
1c + phenol	357	439	0.60	0.78
1c + TFA	345	503	0.63	—
2	349	476	0.24	1
2 + orcinol	330	479	0.025	0.046
2 + catechol	335	482	0.027	0.081
2 + phenol	346	479	0.17	0.65
2 + TFA	337	580	0.006	—

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First, upon addition of phenol, only a slight blue-shift in the absorption maxima (λ_abs) was observed (1a: λ_abs = 322 to 318 nm; 1b: λ_abs = 337 to 331 nm; 1c: λ_abs = 363 to 357 nm; 2: λ_abs = 349 to 346 nm). Although a minor decrease in relative fluorescence intensity (RFI: 0.58–0.78) was noted, no significant quenching was observed. This result indicates that simple phenol does not cause either fluorescence quenching or a red-shift in the emission maxima (λ_em) that would be induced by protonation of the amidine units or ESIPT-like proton transfer in the excited state. The addition of TFA led to protonation of the imidazole units, resulting in a blue-shift in λ_abs and a red-shift in λ_em (1a: λ_abs = 322 to 303 nm, λ_em = 395 to 401 nm; 1b: λ_abs = 337 to 316 nm, λ_em = 412 to 426 nm; 1c: λ_abs = 363 to 345 nm, λ_em = 440 to 503 nm; 2: λ_abs = 349 to 337 nm, λ_em = 476 to 580 nm). In this case as well, no significant quenching was observed, except for receptor 2.⁴⁹

Upon addition of benzenediols, all receptors exhibited a blue-shift in λ_abs (1c: λ_abs = 363 to 308 nm; 2: λ_abs = 349 to 330 nm)⁵⁰ and a significant quenching of fluorescence (RFI: 0.020–0.085) (Fig. 6 and 7). We attribute these blue-shifts to the change in π-conjugation from an extended imidazole–phenylene–imidazole D–π–D system to imidazole units with reduced conjugation efficiency,³⁵ in which this D–π–D twisting raises the LUMO energy level and lowers HOMO energy level of the receptor (Fig. S29). In addition, this change promotes a shift of the HOMO toward the guest molecule, facilitating CT excited states from the guest to the twisted receptor (Fig. S30), resulting in the loss of the fluorescence property as a π-extended D–π–D chromophore in its initial state.⁵¹

Fig. 6 Relative fluorescence intensities of receptors (a) 1a, (b) 1b, (c) 1c, (d) 2 upon adding orcinol (O), catechol (C), and phenol (P) in CH₂Cl₂ (receptors: 10 µM; orcinol, catechol, and phenol: 30 mM).

Fig. 7 Photographs showing the fluorescence color of receptors 1a–c and 2 upon adding orcinol (O) and phenol (P) under UV light (365 nm) in CH₂Cl₂ (receptors: 10 µM; orcinol, catechol, and phenol: 30 mM).

Given that this quenching is induced by allosteric binding, it is expected to exhibit the desired nonlinear response. Therefore, the changes in fluorescence intensity of 1a were further investigated upon the sequential addition of benzenediols (Fig. 8). The Stern–Volmer plot, constructed by plotting I₀/I against guest concentration, exhibited a curved and nonlinearly increasing profile (Fig. 8b).⁵² This confirmed a nonlinear response based on allosteric binding, in which fluorescence quenching is accelerated with increasing guest concentration.

Fig. 8 (a) The decrease in relative fluorescence intensity with respect to guest concentration. (b) Stern–Volmer plots of receptor 1a in the presence of orcinol (black), catechol (red), and phenol (blue).

The limits of detection (LODs) for orcinol, catechol, and phenol were estimated 21, 34, and 362 µM, respectively, using the empirical equation LOD = 3σ/K_SV (Fig. S20).²⁷ In addition, the selectivity ratios for orcinol/catechol and orcinol/phenol were amplified to 3 and 50, respectively, at higher concentrations (Fig. S21 and S22). These remarkable results, which are attributed to the nonlinear behavior based on positive allosteric binding, demonstrate the successful integration of the allosteric effect for highly selective fluorescence quenching-based sensing.

Furthermore, investigation of the quenching behavior of the receptor based on the Stern–Volmer plot and fluorescence lifetime measurements (Fig. S23 and S24) revealed that the quenching process is predominantly static quenching, and that non-binding guest molecules have little influence on the emission of the receptors (Table S2).⁵³

These results collectively indicate that fluorescence quenching occurs specifically with benzenediols capable of allosteric binding, thereby demonstrating excellent guest selectivity. Furthermore, they suggest that these receptors have the potential to function as highly selective “turn-off” fluorescent sensors with nonlinear response characteristics, particularly toward 1,3-benzenediol derivatives.

Conclusions

In conclusion, we successfully synthesized a series of fluorescent bisimidazole-based hydrindacene allosteric receptors. These receptors demonstrated high fluorescence quantum yields and tunable emission colors, ranging from deep blue to light green, which can be modulated by the electron-donating nature of the substituents and the extent of π-conjugation. We found that these receptors underwent selective 1 : 2 allosteric binding with benzenediols, inducing disruption of the extended π-conjugation, resulting in a pronounced nonlinear fluorescence quenching response to increasing guest concentration. This allosteric “turn-off” sensing mechanism enabled clear discrimination between benzenediols and simple phenols, since the sensing behavior is governed by both the number and position of the hydroxy groups and the resulting differences in molecular structure. These findings demonstrate that bisimidazole-based hydrindacene receptors function as highly tunable π-conjugated chromophores and show great promise as supramolecular fluorescent sensors.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5cp04027b.

CCDC 2325723 (1a)³⁵ and 2490135 (2)⁵⁴ contain the supplementary crystallographic data for this paper.

Acknowledgements

This work was supported by the JSPS KAKENHI (Grant Number JP17K19129, JP20K05478 and JP24K08385 for H. K.). The authors thank the Cooperative Research Program of “NJRC Mater. & Dev.” from MEXT JAPAN.

Notes and references

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