Design and synthesis of a rhodol isomer and its derivatives with high selectivity and sensitivity for sensing Hg²⁺ and F⁻ in aqueous media

Abstract

Because of their high extinction coefficients, high quantum yields, and reasonable water solubility, xanthene dyes have played crucial roles in the field of molecular imaging as fluorescent tracers. In this work, a novel hydroxyl regioisomeric rhodol 1 has been designed and synthesized. Both absorption and fluorescence data in water revealed that the rhodol isomer 1 can generate stable optical signals in the pH range 4–10. The maximum absorbance wavelength (ca. 540 nm) of 1 meets perfectly the discrete excitation of the laser at 539 nm, and the emission maximum wavelength (ca. 590 nm) is largely red shifted compared to that of the rhodol fluorophore (ca. 540 nm). Meanwhile, the dye could be easily designed to form chemodosimeters or probes by modification of the carboxyl group or the hydroxyl group. The hydrazide derivative and the silyl ether derivative of the dye in aqueous media exhibited excellent selectivity and sensitivity toward Hg²⁺ and F⁻, respectively. Thus, the excellent optical properties and chemical properties of this dye allow it to be designed as a fluorescent tracer for biological applications.

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Introduction

The quest for fluorescent dyes is a continued attractive research theme with prime importance, as the signal detection via fluorescent methods relate to the fields of fluorescent sensing technology, fluorescent imaging technology, and photonic and electronic devices.¹ It is well known that xanthene dyes, such as fluorescein and rhodamine derivatives, have several interesting innate photophysical natures, e.g. high extinction coefficients, high quantum yields, and reasonable biocompatibilities, which are highly expected for their use as signal reporters for fluorescence tracers.^1,2 The merits for their use as fluorescent reporters in biological fields are that the intense absorption bands at ca. 490 nm for fluorescein derivatives and at ∼540 nm for rhodamine derivatives meet perfectly the discrete excitation of the laser at 488 nm and 539 nm, respectively, while also possessing reasonable quantum yields and excellent photostability in varieties of organic and aqueous media.³ To date, they have been widely used as fluorophore for the molecular design of chemosensors and probes.^3,4 However, a few of intrinsic deficiencies limited their applications as fluorescent labels and tracers, such as fluorescein-based dyes showed pH-dependent fluorescence properties,⁴ irreversible photobleaching under the intense illumination,^3,5 etc., and some rhodamine dyes exhibited strong tendency to form dimers.^6,7

As another member of xanthene family, rhodol fluorophore (Chart 1), a hybrid structure of fluorescein and rhodamine which also named “Rhodafluor”,⁵ has also been proved useful fluorophore for fluorescent sensors since it inherited all the photophysical properties from fluorescein and rhodamine, such as high extinction coefficients, quantum yields and photostability. It exhibited stable fluorescence emissions while the media pH is above 5.5. In the past few years, the rhodol fluorophore and its analogues have been successfully designed as a range of fluorescent probes, such as probes for all kinds of high reactive oxygen species,⁸ enzymes,⁹ and other chemical species.^10–13 To the best of our knowledge, the rhodol fluorophore derivatives are quite limited to date. Inspired by the relationships between the structures and the photophysical and photochemical properties of this type of fluorophores, we assumed that the change of the position of the electron donor substituent of the dyes will alter the “push–pull” properties of its internal charge transfer (ICT) excited state of the parent fluorophore, which will allow us to optimize the photophysical properties of the dyes for the desired applications. Based on this idea, we herein reported the synthesis, photophysical properties and chemical properties of a 2′-hydroxyl-6′-(diethylamino)fluoran 1, an unique xanthene based hydroxyl regio-isomeric rhodol dye (Chart 1). The rhodol isomer 1 exhibited the following advantages as a potential fluorophore for the molecular design of fluorescent chemodosimeters and probes: (1) it showed reasonable water solubility because it kept a ring-opened zwitterion structure in aqueous phase; (2) compared with the maximum absorbance wavelength (ca. 520 nm) of rhodol fluorophore, the maximum absorbance wavelength (ca. 540 nm) of 1 meet perfectly the discrete excitation of the laser at 539 nm, and the emission maximum wavelength (ca. 590 nm) are largely red shifted than that of rhodol fluorophore (ca. 550 nm); (3) the rhodol isomer 1 showed strong pH tolerance, and it can generate stable optical signals in the pH range 4–10; (4) the rhodol isomer 1 can be easily chemical modified to produce all kinds of chemodosimeters and probes in two ways, e.g. via modification of the carboxyl group and the hydroxyl group. At the same time, a Hg²⁺-selective fluorescent chemodosimeter 2 was prepared via modification of the carboxyl group by hydrazine, and a F⁻-selective silyl ether 3 was synthesized via silylation of the hydroxyl group of this fluorescent dye (Chart 1). We anticipated that the excellent innate nature, such as water-soluble, pH-independent optical properties, and the easily chemical modification properties of the dye will allow it to be widely used as fluorophore for the molecular design of chemodosimeters and probes.

Chart 1 Structure of rhodol fluorophore, rhodol isomer 1 and its derivatives 2–3.

Results and discussion

Synthesis of the rhodol isomer 1

As shown in Scheme 1, the rhodol isomer 1 was facilely synthesized in one step by the reaction of 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid and hydroquinone at 90 °C for 24 h in methanesulfonic acid. After crystallized from ethyl acetate, the rhodol isomer 1 was obtained as a pink powder in 85% yield, which is pure enough for analyses and for the next reactions. The structure of the rhodol isomer 1 was confirmed by NMR and high-resolution mass spectra (HRMS).

Scheme 1 Synthesis of rhodol isomer 1 and chemodosimeters 2 and 3. Reagents and conditions: (i) hydroquinone, CH₃SO₃H, 90 °C for 24 h; (ii) hydrazine, EtOH, refluxed for 24 h; (iii) tert-butyldiphenylchlorosilane, CH₂Cl₂, Et₃N, at room temperature overnight.

Modification of the rhodol isomer 1 as chemodosimeters

To get a general knowledge of its chemical properties, we then investigated the possibilities of the chemical modification of the rhodol isomer 1 as fluorescent chemodosimeters. It is well known that the hydrazide derivatives of xanthene dyes, such as rhodamine B and rhodamine 6G, showed excellent selectivity and sensitivity toward Cu²⁺ and Hg²⁺ cations,^14,15 respectively. Thus, we firstly tried the modification of the carboxyl group by the reaction of 1 with hydrazine monohydrate. A Hg²⁺-selective hydrazide derivative 2 was obtained in this way. Meanwhile, silyl ether derivative of some fluorescent dyes have been reported to be F⁻-selective chemodosimeters or probes.¹⁶ Therefore, we then tried the modification of the hydroxyl group by the reaction of 1 with tert-butyldiphenylchlorosilane. A F⁻-selective silyl ether 3 was obtained under such a modification of this dye. Structures of the hydrazide derivative 2 and the silyl ether 3 were confirmed by NMR and HRMS spectra.

Optical properties of the rhodol isomer 1

To understand the optical properties of the dye, we firstly explored the spectroscopic properties of the rhodol isomer 1 in solvents with different polarities in neutral and acidic media. As shown in Fig. 1a, the π–π* transitions of the ring-opened conjugate of 1 in water solutions showed two absorption bands at 506 nm (ε = 2.45 × 10⁴ M⁻¹ cm⁻¹) and 540 nm (ε = 2.4 × 10⁴ M⁻¹ cm⁻¹), respectively. It also showed a weak absorption at 546 nm (ε = 4 × 10³ M⁻¹ cm⁻¹) in ethanol solutions. No absorption bands over 400 nm were observed in other organic solvents (Fig. 1a). Upon addition of TFA (TFA/solvent, 1%, v/v) in most-organic solvents, however, it immediately induced prominent absorbance enhancements at ca. 505 nm and ca. 540 nm, respectively (inset in Fig. 1a). A very weak increase of the absorbance bands over 400 nm in acidic dioxane was observed. It is interesting to note that there is no obvious affect on the absorption of 1 in neutral and acidic water solutions. The results revealed that the rhodol isomer 1 kept a ring-closed spirolactone structure in neutral organic solvents, but the spirolactone structure is very sensitive to acid in most organic solvents. However, it is in a ring-opened π-conjugate in both neutral and acidic water media. Then, the fluorescence emission properties of 1 were investigated. As shown in Fig. 1b, the rhodol isomer 1 in acidic solvents with different polarities showed maxima fluorescence emission bands between 586 nm and 598 nm, whereas the maximum fluorescence emission band in acidic THF was largely red-shifted to 624 nm. Compared with the rhodol fluorophore, the maximum absorption band of the rhodol isomer 1 is red-shifted 20 nm, and the maximum fluorescence emission band is red-shifted ca. 40 nm in water at pH 7 (Fig. S1a and b, ESI†).

Fig. 1 (a) Absorption spectra of the rhodol isomer 1 (10 μM) in different solvents. Insets: 1 (10 μM) in different solvents in the presence of TFA (TFA/solvents, 1%, v/v). (b) Normalized fluorescence emission spectra of 1 (10 μM) in different solvents in the presence of TFA (TFA/solvents, 1%, v/v). λ_ex = 500 nm. Slit: 5 nm; 5 nm.

As described above, the rhodol isomer 1 kept in a ring-opened π-conjugate form in both neutral and acidic water media, which establishes the foundation to use this dye as a potential fluorophore for the molecular design of all kinds of fluorescent sensor. Thus, we next investigated the pH-dependent responses of the rhodol isomer 1 by absorption and fluorescence emission spectra in water solutions. 1 in water showed two strong absorption bands (at 505 nm and 540 nm, respectively) from pH 4 to 10.5 (Fig. S2a, ESI†). These two absorption bands are slightly red-shifted (ca. 5 nm) with the pH lower than 3, and they combined to a single band and red-shifted to 575 nm with the pH higher than 11. The pK_a data of 1 derived from the pH-dependent absorption changes at 540 nm are 2.7 and 10.9, correspond to the interconversions between cationic and zwitterion forms, and the interconversions between zwitterion and anionic forms, respectively (Scheme 2). A pH-dependent fluorescent emission change of 1 in water was also investigated (Fig. S2b, ESI†). It showed a strong fluorescent emission band centered at ca. 585 nm from pH 4 to 10.5, and it slightly red-shifted to 590 nm with the pH lower than 3. However, the fluorescence is fully quenched with the pH higher than 11, indicated that the anionic form of 1 is nonfluorescence. The pK_a derived from the pH-dependent fluorescence changes are 3.1 and 10.7, respectively. Both absorption and fluorescence data in water revealed that 1 kept a stable optical signals over a wide pH span from 4 to 10, which indicated that 1 established the foundation to be a pH-independent fluorophore for the molecular design of chemodosimeters.

Scheme 2 Representation of pH-dependent equilibrium of the rhodol isomer 1.

The molecular recognition properties of the hydrazide 2 toward Hg²⁺ ions

As described above, the excellent optical properties of the rhodol isomer 1 implied that, like classic rhodamines and fluorescein dyes, 1 could be employed as robust platforms for design of a range of fluorescent chemodosimeters. It has been well documented that many of the hydrazide derivatives of rhodamines had demonstrated excellent sensor properties towards Cu²⁺, Hg²⁺ or other chemical species.^14,15 Thus, we prepared the hydrazide derivative 2 from the rhodol isomer 1 and investigated the molecular recognition properties of 2 towards a range of cations in water phase by UV-Vis absorption and fluorescence measurements.

As shown in Fig. 2a and b, the hydrazide 2 showed no fluorescence and no absorption band over 400 nm in water, indicated that it kept a ring-closed circular spirolactam structure. However, addition of Hg²⁺ immediately elicited a dramatic change of the absorption bands over 400 nm and emission maximum at ca. 590 nm, which is similar to the spectroscopic features of the rhodol isomer 1. In addition, the obvious change of solution color in the presence of Hg²⁺ suggests that the hydrazide 2 would be a practical ‘naked-eye’ chemodosimeter of Hg²⁺ in water solutions (Fig. S3, ESI†). From the fluorescence titrations, the fluorescence turn-on constant (K_turn-on) was calculated to be (4 ± 0.088) × 10⁻⁵ M (with correlation coefficient R = 0.9976) (Fig. S4, ESI†).¹⁷ At the same time, the detection limit was estimated to be 3.31 × 10⁻⁹ M, which indicated that the limit of detection of the hydrazide 2 to Hg²⁺ met the limit for drinking water according to the China EPA standard (2 ppb, 10 nM) (Fig. S5, ESI†). Then, the response of the hydrazide 2 to other cations were investigated in water. There was no obvious effect on the UV-Vis absorption and fluorescence emission properties upon addition of 10 equivalents of a series of cations (Fig. S6a and b, ESI†). On the contrary, a prominent absorption enhancement over 400 nm and a significant enhancement of the emission intensity at 590 nm were observed upon addition of Hg²⁺. Further experiments for Hg²⁺-selective sensing were carried out with the hydrazide 2 in water with an excitation at 500 nm in the presence 10 equivalents of Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Na⁺, NH₄⁺, Ni²⁺, Pb²⁺ and Zn²⁺. Upon addition of 10 equivalents of Hg²⁺, the fluorescent emission spectra and the emission intensities at 590 nm were almost identical to that obtained in the presence of Hg²⁺ alone (Fig. 3). Stoichiometry for 2 and Hg²⁺ was measured on the basis of the Job's plot and was found to be 1 : 1 (Fig. S7†). Meanwhile, fluorescence changes of 2 as a function of pH were recorded in the presence and absence of Hg²⁺ in H₂O (Fig S8†). The chemodosimeter 2 exhibited very weak fluorescence emission over a pH range of 4–12. The fluorescence increased with the pH lower than 4 for the protonation of hydrazide moiety led to ring opening of spirolactam structure. Upon addition of Hg²⁺, the fluorescence exhibited prominent enhancement from pH 4 to 10. Thus, 2 was capable of detecting Hg²⁺ in the pH range 4–10. The results indicated that 2 can function as a highly selective and sensitive fluorescent probe for the Hg²⁺ cations in water.

Fig. 2 UV-Vis absorption (a) and fluorescence (b) spectra of 2 (10 μM) in the presence of different concentrations of Hg(NO₃)₂ in water. Inset in (a) and (b): the absorbance at 541 nm and the fluorescence changes at 590 nm as a function of the Hg²⁺ concentration. λ_ex = 500 nm. Slit: 5 nm; 5 nm.

Fig. 3 Change ratio (F_i − F₀)/(F_Hg²⁺ − F₀) of fluorescence intensity of 2 (10 μM) at 590 nm in various mixtures of metal ions (Hg(NO₃)₂ (50 μM) and one other metal ions (50 μM) in ethanol/water (v/v; 1 : 3)). (1) Hg²⁺ + Ag⁺; (2) Hg²⁺ + Al³⁺; (3) Hg²⁺ + Ca²⁺; (4) Hg²⁺ + Cd²⁺; (5) Hg²⁺ + Co²⁺; (6) Hg²⁺ + Cr³⁺; (7) Hg²⁺ + Cu²⁺; (8) Hg²⁺ + Fe²⁺; (9) Hg²⁺ + Fe³⁺; (10) Hg²⁺ alone; (11) Hg²⁺ + K⁺; (12) Hg²⁺ + Mg²⁺; (13) Hg²⁺ + Na⁺; (14) Hg²⁺ + NH₄⁺; (15) Hg²⁺ + Ni²⁺; (16) Hg²⁺ + Pb²⁺; (17) Hg²⁺ + Zn²⁺. λ_ex = 500 nm.

To elucidate the binding mechanism of the chemodosimeter 2 with Hg²⁺, the ¹H NMR titrations of 2 with Hg²⁺ were then carried out in DMSO-d₆ (Fig. 4). As shown in Fig. 4a, the aromatic protons of 2 were well split except that the protons 4–6 are overlapped at 6.36 ppm. On addition 1.0 equiv. of the D₂O Hg(ClO₄)₂ solutions, all the protons were slight low-field shifted (Fig. 4b). Meanwhile, the protons 4–6 were not only obviously low-field shifted but also well split at 6.49 ppm, 6.53 ppm and 6.75 ppm, respectively. Upon addition 2.0 equiv. of the Hg(ClO₄)₂, no obvious changes were observed for most of the protons except that the protons 4–6 were further low-field shifted (Fig. 4c). Further increase the Hg(ClO₄)₂ concentrations led to the ¹H NMR spectra became complex for the formation of new species (Fig. 4d). High-resolution mass spectra (HRMS) indicated that the chemodosimeter 2 was hydrolyzed to rhodol isomer 1 by Hg²⁺ (Fig. S9†). Based on the results of ¹H NMR titrations and HRMS, the recognition mechanism of the chemodosimeter 2 toward Hg²⁺ was proposed as Hg²⁺-promoted hydrolysis of the hydrazide moiety to the ring-opened rhodol isomer 1 as shown in Scheme 3.

Fig. 4 ¹H NMR titrations of 2 with Hg(ClO₄)₂ in DMSO-d₆. (a) 2 in DMSO-d₆; (b) 2 + 1 equiv. of Hg(ClO₄)₂; (c) 2 + 2 equiv. of Hg(ClO₄)₂; (d) 2 + 3 equiv. of Hg(ClO₄)₂.

Scheme 3 Representation of Hg²⁺ induced chemical reaction for the fluorescence turn on mechanism.

The recognition properties of the silyl ether 3 toward F⁻ ions

It is well known that F⁻ anions play a pivotal role in numerous health concerns, such as water depollution, metabolism disorders, dentistry.¹⁸ Whereas F⁻ have many beneficial impacts on human health, larger amounts of F⁻ exposure can lead to serious health problems, so that the World Health Organization recommends its content to be lower than 1.5 ppm in drinking water.¹⁹ However, a very large hydration enthalpy of F⁻ makes the selective sensing of this anion in aqueous solutions a real challenge. In recent years, it has been reported that the alkyl/arylsilyl ether derivatives of some fluorophores showed excellent F⁻ anion selectivities over other anions.¹⁶ Based on the excellent optical properties of the rhodol isomer 1, we prepared its tert-butyldiphenylsilyl ether derivative 3 to investigate F⁻-selective recognition behaviors in aqueous media.

The fluorescence titration experiment was firstly conducted to evaluate the sensing property of the tert-butyldiphenylsilyl ether 3 to F⁻ in DMSO–H₂O (v/v = 1 : 1) solution. Fig. 5 shows the spectroscopic changes of 3 at varied concentrations of NaF upon excitation at 514 nm. As expected, 3 displays a colorless solution and emits very weak fluorescence at ca. 590 nm in DMSO–H₂O (v/v = 1 : 1) solution over a 12 h period, indicated that the Si–O bond has a strong resistance to be hydrolyzed by water. Addition of NaF, however, it produced a light red purple color with emission maximum at ca. 586 nm (Fig. 5). The changes in fluorescence titration spectra terminated when the concentration of F⁻ reached 300 equivalents and over 10-fold fluorescence enhancement was reached under saturated conditions (ca. 300 equivalents). The fluorescence turn-on constant (K_turn-on) was calculated to be (9.1 ± 0.079) × 10⁻⁵ M (with correlation coefficient R = 0.9973) based on the fluorescence changes at 586 nm.¹⁷ In addition, a good linearity was obtained over the concentration range of 0–0.5 mM for F⁻. The regression equation was y = 71.22 + 0.76[F⁻] with a linear coefficient R of 0.998. From the F⁻-dependent fluorescence titrations (Fig. S10, ESI†), the detection limit was calculated to be 5.61 μM, indicating that the limit of detection of 3 to F⁻ met the World Health Organization recommended threshold limit for fluoride content in drinking water (1.5 ppm).²⁰

Fig. 5 Fluorescence spectra of 3 (5 μM) in the presence of different concentrations of NaF in DMSO–H₂O (v/v = 1 : 1) solution. λ_ex = 514 nm. Slit: 10 nm; 10 nm.

Subsequently, we investigated the response of the tert-butyldiphenylsilyl ether 3 (5.0 μM) to other anions in DMSO–H₂O (v/v = 1 : 1) solution. The addition of 40 equivalents of a series of anions, such as AcO⁻, Br⁻, Cl⁻, CO₃²⁻, H₂PO₄⁻, HCO₃⁻, HPO₄²⁻, HSO₄⁻, I⁻, NO₂⁻, NO₃⁻, PO₄³⁻, S²⁻ and SO₄²⁻, has no prominent effect on the fluorescence emission properties (Fig. 6). However, an obvious enhancement (ca. 2-fold) of the emission intensity at 586 nm was observed upon addition of F⁻. Then, the selectivity of 3 to F⁻ was further validated by competition experiments (Fig. 7). In the presence 40 equivalents of AcO⁻, Br⁻, Cl⁻, CO₃²⁻, H₂PO₄⁻, HCO₃⁻, HPO₄²⁻, HSO₄⁻, I⁻, NO₂⁻, NO₃⁻, PO₄³⁻, S²⁻, and SO₄²⁻, the addition of 40 equivalents of F⁻ resulted almost identical fluorescence enhancements of 3 at 586 nm to that obtained in the presence of F⁻ alone. Meanwhile, the fluorescence responses of 3 and 3 + F⁻ from pH 4 to 12 were examined, which showed that 3 could be used within a wide pH span of 4.0–9.0 (Fig. S11, ESI†). As described above, the unique fluorescence emission changes of 3 with F⁻ could be attributed to the cleavage of the Si–O bond by F⁻, which has been depicted in literatures (Fig. 8b).¹⁶ Free 3 is mainly in the non-fluorescent spirolactone structure in DMSO–H₂O (v/v = 1 : 1) solution. With the Si–O bond breakage by F⁻, the non-fluorescent 3 was converted to the ring-opened fluorescent rhodol isomer 1 and led an unique fluorescence enhancement. Thus, 3 can function as a selective fluorescent chemodosimeter for F⁻ anions via a F⁻ promoted Si–O bond breakage within a wide pH span of 4.0–9.0.

Fig. 6 The intensity of fluorescence of 3 (5.0 μM) in DMSO–H₂O (v/v = 1 : 1) solution, after the addition of various anions (40 equivalents). (1) Ac⁻; (2) Br⁻; (3) Cl⁻; (4) CO₃²⁻; (5) F⁻; (6) H₂PO₄⁻; (7) HCO₃⁻; (8) HPO₄²⁻; (9) HSO₄⁻; (10) I⁻; (11) NO₂⁻; (12) NO₃⁻; (13) PO₄³⁻; (14) S²⁻; (15) SO₄²⁻. λ_ex = 514 nm.

Fig. 7 Fluorescence change ratio ((F − F₀)/F₀) of 3 (5 μM) at 586 nm in various mixtures of NaF (40 equivalents) and one other anions (40 equivalents) in DMSO–H₂O (v/v = 1 : 1) solution. (1) F⁻ + Ac⁻; (2) F⁻ + Br⁻; (3) F⁻ + Cl⁻; (4) F⁻ + CO₃²⁻; (5) F⁻ alone; (6) F⁻ + H₂PO₄⁻; (7) F⁻ + HCO₃⁻; (8) F⁻ + HPO₄²⁻; (9) F⁻ + HSO₄⁻; (10) F⁻ + I⁻; (11) F⁻ + NO₂⁻; (12) F⁻ + NO₃⁻; (13) F⁻ + PO₄³⁻; (14) F⁻ + S²⁻; (15) F⁻ + SO₄²⁻. λ_ex = 514 nm.

Fig. 8 (a) ¹H NMR titrations of 3 with NaF in DMSO-d₆. (a) 3 in DMSO-d₆; (b) 3 + 1 equiv. of NaF; (c) 3 + 1.5 equiv. of NaF; (d) 3 + 2 equiv. of NaF; (e) 3 + 2 equiv. of NaF overnight. (b) Representation of fluoride induced chemical reaction for the fluorescence turn on mechanism and the numbering of protons of the rhodols for the ¹H NMR spectra.

Then, the ¹H NMR titrations of 3 with F⁻ were investigated in details as shown in Fig. 8a. The numbering of protons of the rhodols skeleton for assignment of the ¹H NMR spectra were illustrated in Fig. 8b. Before addition of NaF in DMSO-d₆, the aromatic protons of 3 are well split except that the protons 4–6 are overlapped at 6.42–6.37 ppm. When 1.0 equiv. of NaF in D₂O was added to the DMSO-d₆ solution of 3, most of the aromatic protons were low-field shifted except that the protons 2 and 3 were high field-shifted. Further increase of the NaF concentrations (1.5–2.0 equiv.) led no obvious change of the ¹H NMR spectra indicated the reaction was in a 1 : 1 stoichiometric. Meanwhile, there was no change can be observed from overnight.

Conclusion

In summary, a novel water-soluble and pH-independent fluorescent dye, the rhodol isomer 1 has been developed successfully from commercially available hydroquinone and 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid. Both absorption and fluorescence data in water revealed that 1 could generate stable optical signals in the pH range 4–10. Compared with those of rhodol fluorophore, the maximum absorption (ca. 540 nm) and fluorescent emission (ca. 585 nm) of 1 are obviously red-shifted. Meanwhile, the dye could be easily designed to chemodosimeters in two ways. A Hg²⁺-selective hydrazide derivative 2 was obtained via modification of the carboxyl group by the reaction of 1 with hydrazine monohydrate. At the same time, a F⁻-selective silyl ether 3 was obtained via silylation of the hydroxyl group by the reaction of 1 with tert-butyldiphenylchlorosilane. The molecular recognition properties of 2 and 3 were investigated in detail. 2 and 3 showed high selectivity and sensitivity toward Hg²⁺ and F⁻, respectively. An ongoing theme in our laboratory is the exploration of the dye as tracers in applications requiring pH tolerance.

Experimental

General methods

All solvents and reagents (analytical grade and spectroscopic grade) were obtained commercially and used as received unless otherwise mentioned. NMR spectra were recorded on a Varian Mercury Vx-300 at 300 (¹H NMR), Bruker spectrometer at 400 (¹H NMR) MHz and 100 (¹³C NMR) MHz. Chemical shifts (δ values) were reported in ppm down field from internal Me₄Si (¹H and ¹³C NMR). High-resolution mass spectra (HRMS) were acquired on an Agilent 6510 Q-TOF LC/MS instrument (Agilent Technologies, Palo Alto, CA) equipped with an electrospray ionization (ESI) source. Elemental analyses were performed on a Vanio-EL elemental analyzer (Analysensystem GmbH, Germany). UV absorption spectra were recorded on a UV-2550 UV-VIS spectrophotometer (Shimadzu, Japan). Fluorescence measurements were performed using an F-4600 fluorescence spectrophotometer (Hitachi, Japan) equipped with a quartz cell (1 cm × 1 cm). Melting points were recorded on a RY-2 Melting Point Analyzer (Analytical Instrument Factory, Tianjin) and are uncorrected.

Synthesis of 2′-hydroxyl-6′-(diethylamino)fluoran 1 (rhodol isomer 1)

To a 25 mL flask, was charged 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid (1.57 g, 5 mmol), hydroquinone (0.88 g, 8 mmol), methanesulfonic acid (5 mL). The reaction mixture was stirred at 90 °C for 24 h. After being cooled to room temperature, the reaction mixture was poured into ice water (50 mL). The pH of the mixture was adjusted to ∼7 by the addition of NaHCO₃. The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate. After filtration, the filtrate was condensed to dryness to afford the crude product. The crude product was recrystallized from ethyl acetate to afforded pure 1 as pink powder in 85% yield; mp: 188–190 °C. HRMS: m/z [M + H]⁺ = 388.1549; calcd: 388.1471; ¹H NMR (400 MHz, DMSO-d₆, ppm): 9.32 (s, 1H), 8.02 (d, J = 8 Hz, 1H), 7.81 (t, J = 8 Hz, 1H), 7.73 (t, J = 8 Hz, 1H), 7.30 (d, J = 8 Hz, 1H), 7.20 (d, J = 8 Hz, 1H), 6.89 (q, J = 4 Hz, 1H), 6.50–6.42 (m, 3H), 6.06 (d, J = 4 Hz, 1H), 3.34 (t, J = 8 Hz, 7H), 1.09 (t, J = 8 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆, ppm): 169.2, 153.5, 153.0, 149.7, 144.6, 136.0, 129.1, 126.7, 125.0, 124.6, 119.1, 116.1, 112.4, 108.9, 104.3, 97.3, 84.2, 44.2, 12.8.

Synthesis of the hydrazide 2

To a 25 mL flask, was charged 1 (3 g, 7.8 mmol), hydrazine (5 mL), ethanol (30 mL). The mixture was heated to reflux for 24 h under nitrogen atmosphere. After being cooled down to room temperature, the solvent was filtered. The filtrate was evaporated to dryness. The residue was recrystallized from methanol to afforded pure 2 as white powder in 61% yield; mp: 220–222 °C. HRMS: m/z [M + H]⁺ = 402.1819; calcd: 402.1739; ¹H NMR (400 MHz, DMSO-d₆, ppm): 8.76 (s, 1H), 7.59 (d, J = 8 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 7.24 (t, J = 8 Hz, 1H), 7.18 (d, J = 8 Hz, 1H), 7.09 (d, J = 8 Hz, 1H), 6.67 (q, J = 8 Hz, 1H), 6.44 (t, J = 8 Hz, 2H), 6.32 (d, J = 8 Hz, 1H), 6.28 (d, J = 8 Hz, 1H), 3.76–3.49 (m, 2H), 3.36 (q, J = 8 Hz, 4H), 1.19 (t, J = 8 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆, ppm): 167.00, 153.61, 153.26, 151.67, 145.86, 133.01, 128.55, 128.27, 127.61, 123.78, 122.88, 118.46, 118.04, 111.42, 77.36, 77.04, 76.73, 66.82, 44.42, 12.56.

Synthesis of the tert-butyldiphenylsilyl ether 3

To a 50 mL flask, was added the rhodol isomer 1 (387.4 mg, 1 mmol), tert-butyldiphenylchlorosilane (549.7 mg, 2 mmol), anhydrous dichloromethane (10 mL). To this solution, triethyl-amine (303.6 mg, 3 mmol) was added dropwise. After the addition of triethylamine, the reaction mixture was stirred overnight at room temperature. Then, the reaction mixture was poured in water (50 mL). The organic layer was separated, and dried over sodium sulfate. After filtration, the filtrate was condensed to dryness. The residue was purified by crystallization from dichloromethane to afford 3 (417 mg) as a white powder in 67% yield; mp: 84–86 °C. HRMS: m/z [M + H]⁺ = 626.2784; calcd: 626.2727. ¹H NMR (400 MHz, DMSO-d₆, ppm): 0.92 (s, 9H), 1.04 (t, J = 7.0 Hz, 6H), 3.30 (q, J = 6.8 Hz, 4H), 5.83 (d, J = 2.8 Hz, 1H), 6.37 (q, J = 9.6 Hz, 3H), 6.97–7.03 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.28–7.35 (m, 6H), 7.38–7.45 (m, 4H), 7.58–7.64 (m, 2H), 7.78–7.80 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 12.7, 19.2, 26.8, 44.2, 83.7, 97.2, 104.1, 107.0, 117.1, 118.5, 119.9, 123.1, 123.9, 125.0, 126.3, 126.4, 126.9, 130.3, 130.6, 130.7, 131.8, 132.1, 135.2, 135.8, 146.0, 149.8, 150.9, 151.8, 152.9, 168.7.

Acknowledgements

We gratefully acknowledge the Natural Science Foundation of China (NNSFC 21272172; 11432016), and the Natural Science Foundation of Tianjin (12JCZDJC21000).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details; spectra or other electronic format. See DOI: 10.1039/c6ra14287g

‡ Graduate students Jun Qin, Huirong Yao make same contribution for this work.

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