A smart “off–on” gate for the in situ detection of hydrogen sulphide with Cu(ii)-assisted europium emission

A novel responsive europium-based luminescence “off–on” gate for the in situ detection of H₂S in water was developed.

Abstract

A water-soluble and emissive Eu-complex (EuL1) bearing a DO3A(Eu³⁺)–pyridine–aza-crown motif has been prepared and its Cu²⁺ complex has been demonstrated to be a smart luminescence “off–on” gate for H₂S detection in water with a nano-molar detection limit (60 nM). EuL1 binds to Cu²⁺ ions selectively (K_B = 1.2 × 10⁵ M^–1) inducing 17-fold luminescence quenching and forming a 1 : 1 stoichiometric complex (EuL1–Cu²⁺), which responds to H₂S selectively with restoration of the original Eu emission of EuL1 followed by a further 40-fold luminescence enhancement, forming a 1 : 1 stoichiometric complex (EuL1–Na₂S, K_B = 1.5 × 10⁴ M^–1). Without Cu²⁺ ions, EuL1 showed non-specific binding towards H₂S with only a 5-fold luminescence enhancement.

Introduction

Hydrogen sulphide (H₂S) is the smallest bioactive thiol that may act as a gaseous signalling agent,1 and its production in different tissue types is associated with a wide range of physiological responses such as vascular smooth muscle relaxation,2 mitochondrial ATP production,3 insulin-signalling inhibition,4 regulation of inflammation response5 and mediation of neurotransmission.6 Moreover, recent investigations show that abnormal levels of H₂S are associated with a variety of diseases, such as neurodegenerative diseases,7 diabetes8 and cancer.9 However, the biological targets of H₂S and the mechanisms of these H₂S-related physiological phenomena remain unclear. Therefore the development of responsive and reversible luminescence probes for non-invasive real time monitoring of H₂S may be useful for understanding its biological modes of action.

One of the major approaches for developing luminescence H₂S detection10 is based on sulphide-specific chemical reactions, such as reduction of an azide11 and nucleophilic addition of a sulphide ion.12 This type of luminescence probe is generally irreversible and usually requires a considerably long incubation time. An alternative approach is based on CuS precipitation13 due to the low-solubility of CuS (K_sp = 6.3 × 10^–36). These luminescence probes are generally reversible with low detection limits. We are particularly interested in developing H₂S luminescence sensors based on organo-lanthanide complexes due to their water-solubility and unique photophysical properties, including line-like emission spectra and long luminescence lifetimes (micro to milli second scale) that can effectively separate the observing signal from biological autofluorescence noise and are suitable for time-gated detection. Recently, a few studies have been found in the literature with irreversible H₂S lanthanide probes.12a Herein, we report the development of a novel responsive europium-based luminescence “off–on” gate for the in situ detection of H₂S in water.

As illustrated in Fig. 1, EuL1 contains a DO3A–Eu³⁺ complex and an aza-18-crown-6 moiety, which are linked to the 2- and 6-positions of a pyridine-containing chromophore constituting a switch-like structure. In the ground state, EuL1 should be emissive due to the coordination of the pyridine chromophore to a Eu³⁺ ion, which favours energy transfer from the organic chromophore to the Eu³⁺ ion. Upon binding of the aza-18-crown-6 moiety with a Cu²⁺ ion, pyridine is expected to coordinate with the Cu²⁺ ion, resulting in luminescence quenching. The europium emission should be recovered after the displacement of the Cu²⁺ ion upon copper sulphide precipitation.

Fig. 1. The structure of EuL1 and the illustration of the design of a reversible Eu-based luminescence probe (EuL1–Cu²⁺) for H₂S detection.

Results and discussion

Synthesis and photophysical properties of L1 and EuL1

Ligand L1 was readily prepared from (4-iodopyridine-2,6-diyl)dimethanol (1)14via a desymmetrization synthetic strategy. As shown in Scheme 1, a pyridine-containing chromophore (based on a D–π–A motif) was established via a Sonogashira cross-coupling reaction between 1 and 1-ethynyl-4-propoxybenzene (2).15 After converting both hydroxyl groups of 3 into the corresponding bromide, the aza-18-crown-6 and DO3A moieties were incorporated into 4 sequentially under basic conditions and afforded L1 in good yields. L1 was fully characterized using ¹H and ¹³C NMR spectroscopy and HRMS. Finally, acid hydrolysis of the t-butyl esters followed by Eu complex formation provided EuL1, which was characterized unambiguously using HRMS and HPLC (Table S1 and Fig. S1†).

Scheme 1. Synthesis of L1 and EuL1.

In the UV-vis absorption spectrum, L1 showed strong absorption bands at 235 and 310 nm in methanol which are attributed to the π to π* transitions. The absorption bands were broadened and red-shifted in EuL1 (245 and 333 nm, ε_{333 nm} = 7560 M^–1 cm^–1) in water (Fig. S2†). The excitation spectrum of EuL1 at 615 nm showed maxima at 240 and 340 nm (Fig. S2†), evidencing an antenna effect due to energy transfer from the ligand to the Eu³⁺ ion. The ⁵D₀ → ⁷F_J transitions of EuL1 (λ_ex = 325 nm) were found at 578 (J = 0), 585–603 (J = 1), 604–637 (J = 2), 646–658 (J = 3), and 673–712 nm (J = 4) in the emission spectrum (Fig. 2). The quantum yield of EuL1 corresponding to the ⁵D₀ → ⁷F₂ transitions of Eu³⁺ ions in water is 0.5% (Table S2†).

Fig. 2. Emission spectrum of EuL1 (H₂O, λ_ex = 325 nm, 10 μM).

Fluorimetric titration studies of EuL1

With EuL1 in hand, its binding properties towards Cu²⁺ ions were investigated. Upon the addition of 1 equiv. of Cu²⁺ ions (CuCl₂ as the source of Cu²⁺ ions), the absorption maximum of EuL1 showed a slight red shift and the absorption ability slightly decreased due to the effect of the copper metal. In a titration study, EuL1 exhibited a 17-fold quenching of the europium emission with an excess of Cu²⁺ ions and the Benesi–Hildebrand plot showed a 1 : 1 binding stoichiometry with K_B = 1.2 × 10⁵ M^–1 (inset of Fig. 3a).16 The Job's plot also supported the formation of a EuL1–Cu²⁺ complex in a 1 : 1 ratio (Fig. S3†). In a competitive study, the addition of a large excess of various metal ions, such as Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Co²⁺, Zn²⁺, Ni²⁺, Fe²⁺, Mn²⁺, Cu⁺ and Li⁺ ions, to EuL1 resulted in only slight luminescence changes (red columns in Fig. 3b). The subsequent addition of excess Cu²⁺ ions caused significant luminescence quenching (blue columns in Fig. 3b). These results indicate the high selectivity of EuL1 towards Cu²⁺ ions and that the binding between EuL1 and Cu²⁺ ions is not interfered by other metal ions. In a pH study, EuL1 remains highly emissive and was quenched by Cu²⁺ ions in the pH range 6 to 8 (Fig. S4†), indicating that EuL1 is stable and can bind to Cu²⁺ ions under physiological conditions.

Fig. 3. (a) Fluorimetric titration of EuL1 (10 μM) towards Cu²⁺. The inset shows the plot of I₀/(I – I₀) vs. [Cu²⁺] (0–20 μM). I and I₀ stand for intensity of europium emission ⁵D₀ → ⁷F₂. (b) Effects of various metal ions on the luminescence intensity of EuL1 (10 μM). 1: EuL1 only; 2: Na⁺; 3: K⁺; 4: Ca²⁺; 5: Mg²⁺; 6: Ba²⁺; 7: Co²⁺; 8: Zn²⁺; 9: Ni²⁺; 10: Fe²⁺; 11: Mn²⁺; 12: Cu⁺; 13: Li⁺; 14: Cu²⁺; 15: all of the above metal ions except Cu²⁺. All spectra were acquired in water with excitation at 325 nm.

To study the reversibility of the binding between EuL1 and Cu²⁺ ions, a small amount of H₂S (Na₂S as the source of H₂S) was added. The EuL1–Cu²⁺ complex responded instantaneously (requiring only 40 s to reach saturation without stirring or shaking) (Fig. S5†), and Eu emission resumed with a similar profile for the emission spectrum to that of EuL1 (Fig. 4). This result indicated that the DO3A–Eu³⁺ complex was not displaced by a Cu²⁺ ion, forming the EuL1–Cu²⁺ complex in the previous step. More interestingly, Eu emission was further enhanced (40-fold) with an excess of H₂S and the Eu³⁺ emission profile showed significant changes, suggesting binding between EuL1 and H₂S (Fig. 5a). The Benesi–Hildebrand plot showed a 1 : 1 binding stoichiometry with K_B = 1.5 × 10⁴ M^–1 (inset of Fig. 5a).16 The detection limit of EuL1 towards H₂S was calculated according to the 3S_D/slope as low as 60 nM. Surprisingly, direct titration of EuL1 against H₂S resulted in only about a 5-fold luminescence enhancement with a non-linear relationship in the 1 : 1 Benesi–Hildebrand plot (Fig. 6). These results indicated that the Cu²⁺ ion facilitates the specific 1 : 1 binding of EuL1 and H₂S, presumably via pre-organizing the conformation of EuL1. On the other hand, non-specific binding (possibly a mixture of 1 : 1 and 2 : 1 binding) between EuL1 and H₂S resulted without the favourable conformation that is induced by the pre-complexation of a Cu²⁺ ion. This proposal was further supported by the dramatic luminescence drop of the EuL1–Na₂S complex upon heating (>70 °C) (Fig. S6†). This type of Cu²⁺-assisted luminescence enhancement of Eu emission is unprecedented. In a competitive study, EuL1–Cu²⁺ showed insignificant changes in luminescence with a large excess of anions, including Cl^–, SO₄^2–, HSO₄^–, I^–, CO₃^2–, HPO₄^2–, Br^– and HCO₃^–, and only small changes for GSH and cysteine (red columns in Fig. 5b). Upon the addition of H₂S, the Eu emissions were recovered in all the above cases, indicating a high selectivity of EuL1–Cu²⁺ towards H₂S.

Fig. 4. The emission spectra of EuL1 (10 μM) (red), with 1 equiv. of Cu²⁺ ions (green), and with 1 equiv. of Cu²⁺ ions and 1 equiv. of H₂S (black). All spectra were acquired in water with λ_ex at 325 nm.

Fig. 5. (a) Fluorimetric titration of EuL1–Cu²⁺ (10 μM, generated in situ with 2 equiv. of Cu²⁺) towards H₂S (0–100 μM). The inset shows the plot of I₀/(I – I₀) vs. [Na₂S] (0–100 μM). I and I₀ stand for intensity of europium emission ⁵D₀ → ⁷F₂. (b) Effects of various anions on the luminescence intensity of EuL1 (10 μM). 1: EuL1 only; 2: Cl^–; 3: SO₄^2–; 4: HSO₄^–; 5: I^–; 6: CO₃^2–; 7: HPO₄^2–; 8: Br^–; 9: HCO₃^–; 10: S^2–; 11: GSH; 12: cysteine. All spectra were acquired in water with excitation at 325 nm.

Fig. 6. Fluorimetric titration of EuL1 (10 μM) towards H₂S (0–300 μM). The inset shows the plot of I₀/(I – I₀) vs. [H₂S] (0–300 μM). I and I₀ stand for intensity of europium emission ⁵D₀ → ⁷F₂. All spectra were acquired in water with λ_ex at 325 nm.

Mechanistic studies

The binding mechanisms of EuL1 towards Cu²⁺ ions and the EuL1–Cu²⁺ complex towards H₂S were studied using a comparative analysis of the emission spectra of the Eu complexes and the ¹H NMR spectra of La complexes.17 As shown in Fig. 7, the profile of the emission spectrum of EuL1 did not change significantly upon the addition of Cu²⁺ ions. Comparing [EuL1], [EuL1 + Cu²⁺] and [EuL1 + Cu²⁺ + H₂S], measured under the same solution conditions, similar spectra were observed for [EuL1] and [EuL1 + Cu²⁺] (⁵D₀ → ⁷F₁ : ⁷F₂ : ⁷F₄ of [EuL1] = 1 : 1.122 : 0.55 and ⁵D₀ → ⁷F₁ : ⁷F₂ : ⁷F₄ [EuL1 + Cu²⁺] = 1 : 1.186 : 0.91, Table 1). This is correlated with the NMR data and shows that the Cu²⁺ ion is coordinated in the aza-crown. However, signal broadening was observed in the ¹H NMR spectrum of LaL1, indicating rapid metal–ligand exchange. These results suggested that the pyridine moiety of the organic chromophore is rapidly switching between the DO3A–Eu³⁺ and aza-18-crown-6–Cu²⁺ complexes, causing significant luminescence quenching. Moreover, the binding of Cu²⁺ would also provide a favourable conformation for forming a new 1 : 1 complex with H₂S. Upon the addition of H₂S, the emission profile of EuL1 changed significantly, ΔJ = 2/ΔJ = 1 for [EuL1 + Cu²⁺ + H₂S],18 and the intensity ratio was about >200% higher for [EuL1] and [EuL1 + Cu²⁺]. This increase can be attributed to the lower symmetry of the complexes with the addition of sulphide ions (Fig. 7) and the ¹H NMR signals of LaL1 were sharpened. These results suggested new complex formation after the displacement of the Cu²⁺ ion via CuS precipitation. This proposal is further supported by the HRMS spectrum of the EuL1–Na₂S complex (Fig. S7†) and the change in the quantum yields (Table S2†). The EuL1–Na₂S complex is highly emissive probably due to its rigid structure.

Fig. 7. Top: proposed binding mechanism of EuL1 towards Cu²⁺ and H₂S (Na₂S as the source of H₂S). Bottom left: emission spectra of the Eu complexes (λ_ex = 325 nm). Bottom right: ¹H NMR spectra of the La complexes (6.5–8.5 ppm).

Table 1. The ratio of ⁵D₀ → ⁷F_J (J = 0 to 4) emission bands of EuL1, EuL1 + Cu²⁺ and EuL1 + Cu²⁺ + H₂S ^a .

5D0→	7F0	7F1	7F2	7F3	7F4
EuL1	0.01	1	1.22	0.08	0.55
EuL1 + Cu2+	0.08	1	1.86	0.15	0.91
EuL1 + Cu2+ + H2S	0.48	1	3.98	0.15	1.95

^aAll spectra were acquired in water with excitation at 325 nm.

The proposed binding mechanism was also examined using a series of negative control compounds (Fig. 8).19EuL2 showed no luminescence quenching upon the addition of Cu²⁺ ions (Fig. 9a). This result indicated that the carbonyl linker of aza-18-crown-6 may be too rigid for coordination between Cu²⁺ and pyridine, which could be essential for Eu emission quenching. Without the aza-crown moiety, EuL3 also showed no luminescence quenching towards Cu²⁺ (Fig. 9b), suggesting DO3A–Eu³⁺ is stable with Cu²⁺ and the aza-crown motif is important for the Cu²⁺ binding. L4 bearing the pyridine-chromophore showed profound luminescence quenching, but its phenyl analogue (L5) showed no significant change in luminescence upon the addition of Cu²⁺ ions (Fig. 9c and d). These results indicated that the pyridine moiety of the chromophore is essential for the binding of Cu²⁺ to the aza-crown moiety. The results of this series of negative control compounds are in full agreement with the proposed mechanism in Fig. 7.

Fig. 8. The structures of the negative control compounds EuL2, EuL3, L4 and L5.

Fig. 9. The emission spectra of negative control compounds (10 μM) with various concentration of Cu²⁺ ions. (a): EuL2; (b): EuL3; (c): L4; (d): L5. All spectra were acquired in water with λ_ex at 325 nm.

Conclusions

In summary, we have prepared a water-soluble and emissive Eu-complex (EuL1) based on a DO3A(Eu³⁺)–pyridine–aza-crown motif, and studied its consecutive binding properties towards Cu²⁺ and H₂S extensively. EuL1 binds to Cu²⁺ ions selectively (K_B = 1.2 × 10⁵ M^–1) inducing 17-fold luminescence quenching and forming a 1 : 1 stoichiometric complex (EuL1–Cu²⁺), which responds to H₂S selectively with restoration of the original EuL1 emission followed by a further 40-fold luminescence enhancement and a nano-molar detection limit (60 nM). Mass spectroscopic analysis showed the formation of a 1 : 1 stoichiometric complex (EuL1–Na₂S) with K_B = 1.5 × 10⁴ M^–1. Without Cu²⁺ ions, EuL1 shows non-specific binding towards H₂S with only a 5-fold luminescence enhancement. These results indicate that the Cu²⁺ ion may pre-organize the conformation of EuL1 and facilitate the formation of the EuL1–Na₂S complex. The studies on this unprecedented Cu²⁺-assisted luminescence enhancement of Eu emission are still ongoing. With long-lived Eu emission, reversible binding properties, an instantaneous response and high selectivity towards H₂S, this Eu-based luminescence “off–on” gate could find suitable applications for H₂S imaging in biological systems.