Synthesis of a new highly sensitive near-infrared fluorescent iridium(III) probe and its application for the highly selective detection of glutathione

Abstract

A new near-infrared fluorescent probe 1 based on the IR 780 skeleton was designed, synthesized and structurally characterized for the development of a chemosensor. UV-vis absorption and fluorescence spectroscopic studies show that probe 1 exhibits a high selectivity and sensitivity towards iridium(III) in an EtOH/H₂O (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM) solution by forming a 1 : 1 complex with iridium(III). Experimental results further demonstrate that the probe 1–iridium(III) complex also has excellent application for the highly selective detection of glutathione.

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Introduction

Glutathione (GSH) is a peptide that is synthesized naturally in the human body and is widely distributed in various organs. GSH helps to maintain the normal function of the immune system¹ and shows anti-oxidation and integration detoxification activity.² The main physiological role of GSH is the ability to rid the body of free radicals by acting as an important antioxidant, protecting the thiol groups of many proteins and enzyme molecules.³ To date, many investigations have shown that some diseases are more closely related to the levels of GSH, including hepatic, cardiac and renal failure,⁴ leucocyte loss,⁵ Parkinson's disease,⁶ and cancer.⁷ Therefore, accessing the level of GSH in a biological system is helpful in the early diagnosis of certain diseases, and designing a new probe to detect GSH with high selectivity, sensitivity and simplicity is extremely necessary for diagnosing those diseases.

Over the past several years, some new fluorescent probes (including fluoresceins, rhodamine, BODIPY, stilbene, naphthalimide and phthalocyanine dyes) have been designed to distinguish thiol from other amino acids in living systems.⁸ For example, phthalocyanine dyes with a wide spectrum, a molar extinction coefficient, sensitivity, non-fluorescent background fluorescence quantum yield are gradually replacing the traditional fluorescent marker dyes to become a new generation of marker dyes.⁹ Moreover, the indocyanine green (ICG) of the phthalocyanine dyes has been certified by the FDA.¹⁰ In the relevant papers, some probes composed of phthalocyanine dyes have also been developed for identification of GSH.¹¹ In these papers, GSH is detected to achieve the change in fluorescence by the method of bond cleavage, but most of researchers suffer from the problem which has low thiol selectivity for GSH,¹² due to GSH, Cys and Hcy often interfered with each other during the process of detection.^13,14 Because the emission wavelength of phthalocyanine dyes is so long (650–900 nm), it is quite difficult to quench the fluorescence by the photo introduced electron transfer (PET) approach.¹⁵ In addition, some metal ions are well known to play an important role in quenching the fluorescence of the probe.¹⁶ Several research groups have prepared a series of fluorescence probes that can be used for fluorescence turn-on sensors to detect cell receptors.¹⁷ In those articles we can find that those ‘on–off’ chemical sensor achieved the purpose of changing fluorescence by complexation of metal with probe.

Therefore, a design probe of phthalocyanine is a good method to detect GSH efficiently by metal complexation. In this paper, we designed a new composite probe containing metal ions to achieve the target of identification of GSH. The 3,5-bis(2,5-dioxocyclopent-3-en-1-yl)benzoyl chloride, containing the IR780 skeleton, was used to design a novel near-infrared fluorescent probe. After the Michael addition reaction of GSH with a group from dimaleimide, the GSH was selectively distinguished with the aid of the metal ions.¹⁸ The results showed that the iridium(III) was the best metal ion suitable for quenching the fluorescence of probe 1 in EtOH/H₂O (1 : 4, v/v, pH 7.40, HEPES buffer, 1 mM) solution. The iridium(III) exhibited a high sensitivity and strong complexation capacity. At the same time, the composite iridium(III) probe exhibited a high selectivity and sensitivity toward GSH.

Results and discussion

Synthesis of probe 1

Target probe 1 was synthesized by multi-step reactions in accordance with the route as shown in Scheme 1. Reaction of piperazine with IR-780 iodine under the condition of nucleophilic substitution reaction afforded compound 1 according to the reported procedure.¹² Subsequently, for synthesis of reaction unit of GSH-selective, we employed 3,5-diaminobenzoic acid and maleic anhydride as starting materials to prepare compound 2, which was then under the condition of acylation reaction to produce the key intermediate 3.¹⁹ Finally, treatment of compound 1 with compound 3 in the presence of Et₃N efficiently provided probe 1 in 80% yield. The chemical structures from compound 1 to probe 1 were all confirmed by ¹H-NMR, ¹³C-NMR and HRMS (Fig. S1–7, ESI†).

Scheme 1 Reagents and conditions: (a) CH₃CN, N₂, 82 °C, 4 h, 90%; (b) (i) CHCl₃, reflux 20 h, (ii) Ac₂O, NaOAc, 100 °C, 2 h, 69% for two steps; CH₂Cl₂/Et₃N, 75–82%. (c) SOCl₂, reflux, 10 h, 90%; (d) Et₃N, DCM, N₂, rt, 5 h, 60%.

Effect of pH values

The pH-dependence of probe 1 (10 μM) was first evaluated in an optimized EtOH/H₂O (1 : 4, v/v) solution (Fig. S8, ESI†), exhibiting a suitable pH range for probe 1 in biological applications. The fluorescence intensities of probe 1 were recorded at 30 min after the addition of probe 1 into solutions of different pH values (the pH values included 2.7, 3.4, 4.5, 5.6, 6.3, 7.0, 7.3, 7.5, 8.3, 9.4) at λ_ex/em = 764/800 nm. As shown in Fig. 1, the results demonstrated that the fluorescence of probe 1 have a good stability in the pH range of 2.7–9.4 and the fluorescence intensity of probe 1–Ir³⁺ complex can be influenced in the pH range of 5.6–9.4. Hence, the pH range of 5.6–9.4 was a reasonable value for detection of the fluorescence performance of probe 1. Based on the above range of reasonable pH values, further fluorescence and UV-vis studies were detected in a EtOH/H₂O (1 : 4, v/v, pH, 7.4, HEPES buffer, 1 mM) solution.

Fig. 1 The spectra of probe 1 (10 μM) in solutions of different pH values containing 20% EtOH. Fluorescence spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). Each spectrum was recorded 30 min after the addition of probe 1.

Response time of probe 1 for iridium(III)

To evaluate the sensitivity of probe 1 toward Ir³⁺, the response time of probe 1 with iridium(III) was detected in HEPES (1 mM, pH = 7.4) containing 20% EtOH at λ_ex/em = 764/800 nm. The concentration of probe 1 and iridium(III) was 10 μm and 50 μm. As shown in Fig. 2, the fluorescence intensity of probe 1 at 764 nm decreased rapidly after Ir³⁺ (50 μM) was added into probe 1 (10 μM) solution, and stabilized after 20 min after the addition of iridium(III). These results indicated that probe 1 was a sensitive sensor for Ir³⁺ in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH. Therefore, the reaction time of 20 min was used in subsequent experiments to ensure enough time for metal ions to complex with the probe 1.

Fig. 2 The spectra of probe 1 (10 μM) with iridium(III) (50 μM) in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH. Fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). The spectra were recorded at 2 min intervals after the addition of iridium(III). Inset: trend of fluorescence intensity, with probe 1 recorded at different times.

Selectivity studies for metal ions using UV-vis spectra

The selectivity of probe 1 (10 μM) in a HEPES (1 mM, pH = 7.4) containing 20% EtOH was examined with various metal ions (including Na⁺, K⁺, Ca²⁺, Cu⁺, Mg²⁺, Fe²⁺, Fe³⁺, Cu²⁺, Al³⁺, Sn²⁺, Ag⁺, Hg²⁺, Ni²⁺, Co²⁺, Cd²⁺, Cr³⁺, Pb²⁺, Ir³⁺, Rh³⁺, Mn²⁺, Li⁺, and Ba²⁺, at 50 μM). As shown in Fig. 3 after iridium(III) ions complexed with the probe 1, the maximum UV absorption wavelength (λ_max = 691) had a slight blue shift, and the absorption intensity (ε_max = 0.531) was significantly weaker than all the other metal ions. The results indicate that probe 1 exhibits excellent selectivity for iridium(III) in a EtOH/H₂O (4 : 1, v/v, pH 7.40, HEPES buffer, 1 mM) solution.

Fig. 3 The spectra of probe 1 (10 μM) with different metal ions (50 μM) in HEPES (1 mM, pH = 7.4) containing 20% EtOH. Each spectrum was recorded 20 min after the addition of the metal ions.

To gain better insight into the effect of the relationship between the probe 1 and iridium(III), the probe 1 (10 μM) was titrated with iridium(III) in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH and analyzed using UV-vis absorbance spectra. As shown in Fig. 4 with the increase in the concentration of iridium(III), UV absorption of the probe 1 decreased continuously at 775 nm. When the concentration of iridium(III) was increased by a factor of 3, the UV absorbance intensity of probe 1 reached its lowest value.

Fig. 4 The UV-vis absorbance spectra of probe 1 (10 μM) for different concentrations of iridium(III) in HEPES (1 mM, pH = 7.40) containing 20% EtOH. Each spectrum was recorded 20 min after the addition of iridium(III).

Selectivity studies for metal ions by fluorescence spectra

To achieve further in-depth analysis of the selectivity of probe 1 for metal ions, the fluorescence intensity of probe 1 was also further investigated by fluorescence spectra with various metal ions in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH at λ_ex/em = 764/800 nm under the same conditions. As shown in Fig. 5, when Ir³⁺ (50 μM) was introduced to the solution of probe 1, a remarkable fluorescence quenching was observed. All other metal ions showed no obvious fluorescence quench under the same conditions. This result indicated that the probe 1 had better selectivity for iridium(III) due to the quenching of its fluorescence. Therefore, the experiments of fluorescence titration further demonstrated that probe 1 functions as a highly selective and sensitive fluorescent chemosensor for iridium(III).

Fig. 5 The spectra of probe 1 (10 μM) with different metal ions (50 μM) in HEPES (1 mM, pH = 7.4) containing 20% EtOH. Fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). Each spectrum was recorded 20 min after the addition of the metal ions.

Fluorescence titration of probe 1 with Ir³⁺

To gain further insight into the binding of probe 1 (10 μM) with Ir³⁺, Ir³⁺ titration against probe 1 in an EtOH/H₂O (4 : 1, v/v, pH 7.4, HEPES buffer, 1 mM, λ_ex/em = 764/800 nm) was monitored by using fluorescence spectra. As shown in Fig. 6, when no Ir³⁺ ions were added to the solutions of probe 1, the free probe 1 (10 μM) exhibited very strong fluorescence (λ_ex = 764 nm) at 800 nm. However, upon the addition of the Ir³⁺ (0–5 eq.), the titration of Ir³⁺ ions with probe 1 led to a significant decrease in the emission intensity at 800 nm. The fluorescence spectra also had been slightly blue shifted from 800 nm to 790 nm with the increase concentrations of iridium(III). The emission intensity reached its lowest value after the addition of 5 equivalent of Ir³⁺.

Fig. 6 The spectra of probe 1 (10 μM) for different concentrations of iridium(III) in HEPES (1 mM, pH = 7.4) containing 20% EtOH. Fluorescent spectra: (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). Each spectrum was recorded 20 min after the addition of iridium(III). Inset: plots of fluorescence intensity where probe 1 was recorded with different concentrations of iridium(III).

Next, the complexation ratio of probe 1 with iridium(III) was also investigated in the EtOH/H₂O mixed solution (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM, λ_ex/em = 764/800 nm) by a Job's plot. The molar concentration of iridium(III) ranged from 0 to 1 in a solution of [Ir³⁺] + [probe 1]; the total concentration of probe 1 with iridium(III) was 40 μM. The results revealed that when the mole fraction of iridium(III) was 0.5, the fluorescence intensity of probe 1 reached the maximum value (Fig. 7), indicating that the ratio by which probe 1 complexed with iridium(III) is 1 : 1.

Fig. 7 Job's plot of probe 1 in an EtOH/H₂O solution (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM) with a total concentration of [probe 1] + Ir³⁺ = 40 μM. Fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm).

Detection limit of probe 1 with iridium(III)

The detection limit of probe 1 for iridium(III) was further calculated based on an experiment with the fluorescence titration. The fluorescence spectrum of probe 1 was collected 10 times. Each spectrum was recorded at 10 min intervals at λ_ex/em = 764/800 nm. As shown in Fig. 8, the iridium(III) concentration changed over the range of 2–7 μM. A good linear regression equation was obtained as y = −20 507x + 76 135 (R = 0.9940). The detection limit of iridium(III) was calculated as 2.0 × 10⁻⁸ M by (K = 3), showing a high detection sensitivity for iridium(III).²⁰

Fig. 8 Detection limit of probe 1 with iridium(III). The spectra of probe 1 (10 μM) with different concentrations of iridium(III) in HEPES (1 mM, pH = 7.4) containing 20% EtOH. Fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm).

Calculations for the binding association constant

The association constant for probe 1 with iridium(III) ion was calculated using a Benesi–Hildebrand plot.²¹ The result is shown in Fig. 9. A good linear regression equation was obtained as y = −2 × 10⁻¹¹x + 1× 10⁻⁵ based on the fluorescence titration curves of probe 1 with iridium(III) ion. Thus, the result of K_a (the binding association constant) for iridium(III) was 5.0 × 10⁵ M⁻¹ in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH at λ_ex/em = 764/800 nm, indicating that iridium(III) with probe have a strong binding capacity.

Fig. 9 The binding association constant of probe 1 with iridium(III) was based on a Benesi–Hildebrand plot, fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm).

Fluorescence spectroscopic studies of probe 1and probe 1–Ir³⁺ complex with various amino acid

We have concluded that probe 1 could strongly and selectively bind with Ir³⁺ to form a probe 1–Ir³⁺ complex, causing a considerable change in the spectral properties of probe 1. We further studied the effects of different amino acids on the effect of probe 1–Ir³⁺ complex. First, we performed the UV-vis absorption (Fig. 10A) and fluorescence spectroscopic (Fig. 10B) experiments of the probe 1 (10 μM) in the presence of different amino acids such as Ala, Arg, Gly, Glu, Lys, Met, Pro, Cys, GSH, Thr, Trp, Val, and Hcy (20 μM) in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH. Every spectrum was recorded 60 min after the addition of the amino acids (20 μM) into probe 1 (10 μM).^22,23 Both UV-vis absorption and fluorescence spectroscopic results showed that all amino acids failed to produce any discernible spectral change under the same conditions.

Fig. 10 The spectra of probe 1 (10 μM) relative to different amino acids (20 μM) in HEPES (1 mM, pH = 7.4) containing 20% EtOH: (A) UV-vis absorption spectra and (B) fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). Fluorescent spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). Each spectrum was recorded 60 min after the addition of amino acids.

Next, to better understand the property of probe 1–Ir³⁺ complex on sensing of amino acids in a HEPES (1 mM, pH = 7.4) solution containing 20% EtOH. We further performed UV-vis absorption and fluorescence titration experiments of probe 1–Ir³⁺ complex with various amino acids. Every spectrum was recorded after the addition of iridium(III) (30 μM) into probe 1 (10 μM) for 30 min followed by further addition of the amino acids (20 μM) for another 30 min. As shown in Fig. 11, after the addition of GSH (20 μM, 2 eq.) to the solution, a significant absorption enhancement at 775 nm was observed (Fig. 11A). No obvious response was observed at 775 nm after other amino acids were added under the same conditions. The sensoring behavior of probe 1–Ir³⁺ complex towards different amino acids was also investigated by fluorescence spectroscopy to further evaluate the selectivity of probe 1–Ir³⁺ complex towards GSH. The change in fluorescence intensity upon addition of various amino acids under the same conditions was recorded. As shown in Fig. 11B, the fluorescence spectra of probe 1–Ir³⁺ complex exhibit a weak fluorescence at 800 nm (λ_ex = 764 nm). When GSH (20 μM, 2 eq.) was added to the solution containing probe 1–Ir³⁺ complex, a strong emission band centered at 580 nm appeared, and a greater than 8-fold fluorescence enhancement was observed. Compared with probe 1, 24.29 per fluorescence intensity was recovered upon the addition of GSH to probe 1–Ir³⁺ complex. However, no significant variation in the fluorescence spectra was observed with any other amino acids under identical conditions. Therefore, both the UV-vis absorption and the fluorescence titration experiments demonstrated that probe 1–Ir³⁺ complex functions as a highly sensitive and selective fluorescent chemosensor for GSH.

Fig. 11 The spectra of the probe 1–Ir³⁺ ensemble (probe 1 10 μM, iridium(III) 30 μM) with different amino acids (20 μM) in HEPES (1 mM, pH = 7.4) containing 20% EtOH. (A) UV-vis absorption spectra and (B) fluorescence spectra (λ_ex = 764 nm, λ_em = 800 nm, slit: 5/5 nm). Probe 1 complexed with iridium(III) for 20 minutes. Then, each spectrum was recorded 60 min after the addition of amino acids.

Competitive selectivity of probe 1–Ir³⁺ complex for GSH with other amino acids and anions

To further evaluate the selectivity of the probe 1–Ir³⁺ complex for GSH, the competitive selectivity of the probe 1–Ir³⁺ complex for GSH under the same conditions was also examined in the presence of other amino acids. As shown in Fig. 12, no significant variation in the emission of the probe 1–Ir³⁺ GSH complex was observed when comparing the results with or without other amino acids. These results clearly indicated that the detection of GSH is not interfered with by other amino acids and that probe 1–Ir³⁺ complex could be used as a selective GSH fluorescent chemosensor.

Fig. 12 The competitive selectivity of probe 1–Ir³⁺ for GSH with other amino acids was examined with various amino acids (including Ala, Arg, Gly, Glu, Lys, Met, Pro, Cys, GSH, Thr, Trp, Val, and Hcy, 20 μM). The selectivity of these amino acids was detected in HEPES (1 mM, pH = 7.4) containing 20% EtOH at λ_ex/em = 764/800 nm. Every spectrum was recorded at 20 min after the addition of iridium(III) (30 μM) and 60 min after the addition of the amino acids.

Additionally, the competitive selectivity of the probe 1–Ir³⁺ complex for GSH under the identical conditions was also examined in the presence of other anions. As shown in Fig. 13, the emission of the probe 1–Ir³⁺ GSH complex shows no significant change when introducing various anions including: F⁻, Cl⁻, Br⁻, I⁻, HCO₃⁻, SO₄²⁻, H₂PO₄⁻, S₂²⁻, HPO₄²⁻, PO₄³⁻, NO₃⁻ and CO₃²⁻. These results further demonstrate that the detection of GSH is not remarkable interfered by other anions and thus the probe 1–Ir³⁺ complex could be used as a highly selective GSH fluorescent chemosensor.

Fig. 13 The competitive selectivity of probe 1–Ir³⁺ for GSH was examined with various anions (including: F⁻, Cl⁻, Br⁻, I⁻, HCO₃⁻, SO₄²⁻, H₂PO₄⁻, S₂²⁻, HPO₄²⁻, PO₄³⁻, NO₃⁻ and CO₃²⁻, 20 μM). The selectivity of these anions was detected in HEPES (1 mM, pH = 7.4) containing 20% EtOH at λ_ex/em = 764/800 nm. Every spectrum was recorded at 20 min after the addition of iridium(III) (30 μM) and 60 min after the addition of the amino acids.

Mechanism studies

NMR spectral analysis of probe 1 with iridium(III): to determine the reaction mechanism of probe 1 with iridium(III), the ¹H-NMR spectral analysis of probe 1 was studied between the absence and presence of iridium(III) (Fig. 14). Obviously, the probe 1 (5 mg) can be quite uniformly dispersed in the CD₃OD : D₂O (2 : 1, v/v, 500 μL) (I). Then iridium(III) (1 eq.) was added into the CD₃OD : D₂O (2 : 1, v/v) containing 5 mg probe 1 (II), suggesting that the chemical shift of the resulting product was identical to the free probe 1, except its intensity of the resulting product was significantly reduced upon addition of iridium(III). Next, when iridium(III) (2 eq.) was added, the NMR's relative intensity of the final product had been almost completely disappeared upon introducing iridium(III), and the final product of probe 1 with iridium(III) can rapidly precipitated. The above results means that complexation of probe 1 with iridium(III) can generated between the probe 1 and the iridium(III), meanwhile, since iridium(III) complexed with probe 1, it also led to reduce the solubility of the probe 1 and further precipitate from solvent system.

Fig. 14 The ¹H-NMR (500 MHz) spectra of probe 1 (I), probe 1 + iridium(III) (1 eq.) (II), and probe 1 + iridium(III) (2 eq.) (III) in the CD₃OD : D₂O (2 : 1, v/v).

Reaction mechanism of probe 1 with GSH: from the above experimental results with UV-vis and fluorescence spectral data, the reaction mechanism for probe 1 (10 μM) with GSH (20 μM) was studied in 3 mL HEPES (1 mM, pH = 7.4) containing 20% EtOH at room temperature by MS. The MS were recorded after the addition of GSH for 60 min. The intense peak at m/z 1497.6211 was present in the HRMS spectrum (Fig. 15A).

Fig. 15 HRMS spectrum of probe 1 with GSH (A) and probe 1 (B).

In contrast to that probe 1's the original molecular weight of 883.4566 (Fig. 15B), revealing that the two molecules GSH just reacted with probe 1. That is to say, the probe 1 reaction occurred by the Michael addition reaction with GSH (2 eq.).

To further explain the interaction relationship between probe 1 with iridium trichloride and GSH, the energy-optimized structures of probe 1, iridium trichloride and GSH were obtained by DFT calculations with the B3LYP method using a site of Gaussian 09 programs. As shown in Fig. 16, spatial distributions showed that the conjugated structure of IR780 was changed after the chlorine atom of IR780 were replaced by nitrogen atom of the piperazine (Fig. 16a). After iridium(III) complexed with probe 1, the conjugate structure of the probe 1 was obviously destroyed by iridium(III) (Fig. 16b). Then, with the addition of GSH, the conjugate structure of IR780 was restored duo to move up the iridium(III) (Fig. 16c). As a consequence, the orbital energies of the HOMO and LUMO of probe 1, probe 1–Ir³⁺, probe 1–Ir³⁺–GSH were generated by DFT calculations too (Fig. 17). Indicating that the LUMO of probe 1 was distributed on one of the two maleimide group moiety, the HOMO was spread over the group of IR780 (Fig. 17a). However, after the probe 1 complexed with iridium(III) (Fig. 17b), the π electrons on the LUMO of probe 1 was distributed on the right of the group of IR780 moiety, the HOMO was spread over the two maleimide group moieties. Subsequently, adding the GSH (Fig. 17c), the electron cloud of new product also occurred significant changes, the π electrons on the LUMO of probe 1 was distributed on the IR780 group, benzene and two molecules of GSH, and the HOMO was spread over the IR780 group and the right of GSH. The energy gaps between the LUMO and HOMO in the probe 1, probe 1–Ir³⁺, probe 1–Ir³⁺–GSH were calculated to be 54.15, 39.97 and 4.36 kcal mol⁻¹ respectively. The results show that the binding of GSH to probe 1–Ir³⁺ reduced the energy gap of HOMO–LUMO of the new product and stabilized the system.

Fig. 16 Energy-minimized structures of probe 1 (a), probe 1–Ir³⁺ (b) and probe 1–Ir³⁺–GSH (c).

Fig. 17 HOMO and LUMO orbitals of probe 1 (a), probe 1–Ir³⁺ (b) and probe 1–Ir³⁺–GSH (c).

Base on the above points, we try to deduce the following inference about the mechanism: probe 1–Ir³⁺ ensemble shows a high selectivity for GSH. The probe 1 fluorescence is quenched owing to leading to the destruction the conjugate structure of the probe 1 by iridium(III) and its poor solubility in the EtOH/H₂O (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM) solution after the complexation of probe 1 with iridium(III). However, with the addition of GSH, the conjugate structure of IR780 was restored owing to shifting the iridium(III). In addition, GSH is a hydrophilic compound, it is easy to improve the solubility of the complexation of iridium(III) with probe 1, when GSH reacts with probe 1. Based on the above mechanism, probe 1 illustrate a excellent selectivity for GSH in presence of iridium(III).

Conclusions

In summary, a new near-infrared composite probe (probe 1 complexed with iridium(III)) was prepared for the determination of GSH based on the Michael addition reaction and metal complexation. Chemical structure of the probe 1 was characterized by ¹H-NMR, ¹³C-NMR and HRMS. The results of performance testing (UV-vis and fluorescence response) show that the chemosensor of probe 1 had excellent selectivity and sensitivity for iridium(III) in EtOH/H₂O (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM) solution. Their fluorescence intensities were basically stable in the pH range of 5.6–9.4. The 1 : 1 complexation mode was achieved based on a Job's plot. The K_a was calculated to be 5.0 × 10⁵ M⁻¹. Moreover probe 1–Ir³⁺ complex had excellent selectivity and sensitivity for GSH in aEtOH/H₂O (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM) solution. Fluorescence of probe 1 was not only destruction of the conjugate structure of the probe 1, but also precipitation from the solvent after the complexation of probe 1 with iridium(III), once the hydrophilic GSH reacted with the probe 1, probe 1 again tended to hydrophilic and resumed its partial fluorescent.

Experimental

All chemicals were purchased from J&K Scientific (Shanghai, China) and used without further purification. Acetonitrile was HPLC grade, other solvents were of analytical grade, and double-distilled water was used throughout all experiments.

The salts used in stock aqueous solutions of ions were NaCl, KCl, CaCl₂, CuCl, MgCl₂·6H₂O, FeCl₂·4H₂O, FeCl₃·6H₂O, CuCl₂·2H₂O, AlCl₃, SnCl₂·2H₂O, AgNO₃, HgCl₂, NiCl₂·6H₂O, CoCl₂·6H₂O, CdCl₂·5H₂O, CrCl₃·6H₂O, PbCl₂, IrCl₃·H₂O, RhCl₃·H₂O, MnCl₂·4H₂O, Li₂CO₃, Ba(NO₃)_2, KF·2H₂O, KBr, KI, Na₂S·6H₂O, NaHCO₃, NaSO₄, KH₂PO₄, K₂HPO₄, K₃PO₄·2H₂O, NaNO₃, and K₂CO₃. Column chromatography was performed on Haiyang silica gel (type: 200–300 mesh ZCX-2). TLC was performed on Haiyang silica gel F254 plate.

The ¹H- (500 MHz) and ¹³C-NMR (126 MHz) spectra were recorded on a Bruker Avance 500 spectrometer with CDCl₃ and DMSO-d₆ as solvents and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a double-focusing high-resolution instrument (Autospec; Micromass Inc.) under electron ionization conditions. All pH measurements were measured by PHS-25C Precision pH/mV Meter (Aolilong, Hangzhou, China) at room temperature (approximately 298 K). UV-vis spectra were recorded on a UV-3600 spectrophotometer (Shimadzu, Japan). Fluorescence measurements were recorded on an Edinburgh FLS920 fluorescence spectrophotometer (Livingston, UK).

Stock solution preparation for spectral detection

The stock solutions (50 μM) of the metal ions of Cd²⁺, Co²⁺, Cr³⁺, Ir³⁺, Ag⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mg²⁺, Mn²⁺, Na⁺, Al³⁺, Ba²⁺, Ca²⁺, Cu⁺, Cu²⁺, Ni²⁺, Pb²⁺, Sn²⁺, K⁺, Li⁺ and Zn²⁺, and amino acids of Ala (alanine), Arg (arginine), Gly (glycine), Glu (glutamine), Lys (lysine), Met (methionine), Pro (proline), Cys (cysteine), GSH, Thr (threonine), Trp (tryptophane), Val (valine), and Hcy (homocysteine) were prepared in EtOH/H₂O (1 : 4, v/v, pH 7.40). The working solution of probe 1 (10 μM) was prepared in EtOH/H₂O (1 : 4, v/v, pH 7.4). Detection solutions of probe 1 were freshly prepared for spectroscopic measurements.

UV-vis and fluorescence spectral studies

All experiments were performed in EtOH/H₂O solution (1 : 4, v/v, pH 7.4, HEPES buffer, 1 mM). In the selectivity experiments, the test samples were prepared by adding an appropriate amount of the cation stock or amino acid solution (50 μM) to a 3 mL solution of the probe 1 (10 μM). In the titration experiment, a 10 μM solution of the probe 1 was prepared at room temperature with an appropriate amount of the ion stock solution or GSH solution, which was added to the quartz optical cell using a micropipette. Spectral data were recorded 30 min after the addition of the amino acids or metal ions. In the fluorescence measurements (slit: 5/5 nm), excitation was provided at 764 nm and emission was 800 nm, collected from 784 to 860 nm.

Synthesis of compound 1 (ref. 12)

Under a N₂ atmosphere, piperazine (34.5 mg, 0.4 mm) was added to a 5 mL solution of IR-780 iodine (67 mg, 0.1 mmol) in anhydrous acetonitrile. After stirring for 4 h at 82 °C, the solvent was removed in vacuo. The resulting residue was purified by column chromatography (silica gel; DCM/MeOH, 100/2, v/v) to give compound 1 (68 mg, 90%) as a blue solid. ¹H-NMR (500 MHz, CDCl₃, 298 K, TMS) δ (ppm): 7.69 (d, J = 13.0 Hz, 2H), 7.29–7.36 (m, 4H), 7.15 (s, 2H), 6.97 (d, J = 6.0 Hz, 2H), 5.79 (d, J = 12.0 Hz, 2H), 3.97 (d, 8H), 3.32 (s, 4H), 2.50 (s, 4H), 1.87 (s, 6H), 1.74 (s, 12H), 1.08 (s, 6H). ¹³C-NMR (126 MHz, CDCl₃, 298 K, TMS) δ (ppm): 172.7, 169.5, 142.8, 141.2, 140.5, 128.3, 123.7, 123.4, 122.3, 109.4, 96.2, 55.2, 48.4, 46.8, 45.3, 29.6, 25.1, 21.7, 20.4, 11.7.

Synthesis of compound 2 (ref. 19)

Maleic anhydride (193 mg, 1.97 mmol) was added to a 6 mL solution of 3,5-diaminobenzoic acid (100 mg, 0.66 mmol) in CHCl₃. After refluxing for 20 h, the residue of yellow precipitate was filtered, and sodium acetate (17.6 mg, 0.26 mmol) and acetic anhydride (5 mL) were added. The reacting mixture was heated for 2 h at 100 °C. The resulting reaction mixture was poured on ice, and cold water (15 mL) was added. The mixture was stirred vigorously for an addition hour. The precipitate was filtered and washed with cold water (50 mL) to afford compound 2 (144.38 mg, 69%) as a beige solid. ¹H-NMR (500 MHz, DMSO-d₆, 298 K, TMS) δ (ppm): 7.97 (s, 2H), 7.64 (s, 1H), 7.22 (s, 4H). ¹³C-NMR (126 MHz, DMSO-d₆, 298 K, TMS) δ (ppm): 170.0, 166.4, 135.3, 132.8, 132.5, 129.0, 126.8.

Synthesis of compound 3 (ref. 19)

Compound 2 (200 mg, 0.65 mmol) was added to thionyl chloride (10 mL). The mixture was reflux at 85 °C until solid particles disappeared completely. The excess SOCl₂ was evaporated in vacuo, then the resulting solid was dried under vacuum to afford compound 3 (191.49 mg, 90%) as a beige solid that was used without further purification to add directly into the next reaction.

Synthesis of probe 1

Compound 3 (29 mg, 0.75 mmol) and Et₃N (12.14 mg, 0.11 mmol) in anhydrous DCM (2 mL) were added dropwise to 3 mL anhydrous DCM solution containing compound 1 (60 mg, 0.08 mmol) at 0 °C. The reaction mixture was allowed to stir for an additional 5 h under a N₂ atmosphere at room temperature. After completion of the reaction, the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica gel; DCM/MeOH, 100/1, v/v) to give probe 1 (56.53 mg, 80%) as a classical blue solid. ¹H-NMR (500 MHz, CDCl₃, 298 K, TMS) δ (ppm): 7.86 (d, J = 13.7 Hz, 2H), 7.73 (t, J = 1.9 Hz, 1H), 7.67 (d, J = 2.0 Hz, 2H), 7.407–7.32 (m, 4H), 7.20 (t, J = 7.5, 0.9 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 6.93 (s, 4H), 6.01 (d, J = 13.7 Hz, 2H), 4.03 (t, J = 7.3 Hz, 4H), 3.65 (s, 4H), 2.56 (t, J = 6.4 Hz, 4H), 1.89 (p, J = 7.7 Hz, 6H), 1.73 (s, 16H), 1.07 (t, J = 7.40 Hz, 6H). ¹³C-NMR (126 MHz, CDCl₃, 298 K, TMS) δ (ppm): 170.2, 169.7, 168.7, 168.6, 142.6, 141.6, 140.5, 135.9, 134.5, 132.5, 128.6, 126.8, 124.3, 123.4, 123.2, 122.1, 110.1, 98.5, 54.0, 48.6, 45.8, 28.8, 25.4, 21.5, 20.6, 11.7. HRMS (M − I⁻)⁺ found, 883.4566; calcd for C₅₅H₅₉N₆O₅⁺, 883.4521.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (81371616), Specially-appointed Professor Grant by JiangSu province (2015, Prof. Jing Zhu), and the Research Fund for the Doctoral Program of Higher Education of China (20133219120020). We thank the sensor Lab of NUST for supporting facilities for UV-vis studies.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H, ¹³C-NMR and HRMS spectra. See DOI: 10.1039/c5ra14847b

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