A smart “off–on” gate for the in situ detection of hydrogen sulphide with Cu(ii)-assisted europium emission† †Electronic supplementary information (ESI) available: Detailed experimental procedures, characterization of compounds, NMR spectra and supplementary fluorometric titration studies. See DOI: 

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


Introduction
Hydrogen sulphide (H 2 S) 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 inammation response 5 and mediation of neurotransmission. 6 Moreover, recent investigations show that abnormal levels of H 2 S are associated with a variety of diseases, such as neurodegenerative diseases, 7 diabetes 8 and cancer. 9 However, the biological targets of H 2 S and the mechanisms of these H 2 S-related physiological phenomena remain unclear. Therefore the development of responsive and reversible luminescence probes for non-invasive real time monitoring of H 2 S may be useful for understanding its biological modes of action.
One of the major approaches for developing luminescence H 2 S detection 10 is based on sulphide-specic chemical reactions, such as reduction of an azide 11 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 precipitation 13 due to the low-solubility of CuS (K sp ¼ 6.3 Â 10 À36 ). These luminescence probes are generally reversible with low detection limits. We are particularly interested in developing H 2 S 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 autouorescence noise and are suitable for time-gated detection. Recently, a few studies have been found in the literature with irreversible H 2 S lanthanide probes. 12a Herein, we report the development of a novel responsive europium-based luminescence "off-on" gate for the in situ detection of H 2 S in water.
As illustrated in Fig. 1, EuL1 contains a DO3A-Eu 3+ complex and an aza-18-crown-6 moiety, which are linked to the 2-and 6positions 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 Fig. 1 The structure of EuL1 and the illustration of the design of a reversible Eu-based luminescence probe (EuL1-Cu 2+ ) for H 2 S detection.
to a Eu 3+ ion, which favours energy transfer from the organic chromophore to the Eu 3+ ion. Upon binding of the aza-18crown-6 moiety with a Cu 2+ ion, pyridine is expected to coordinate with the Cu 2+ ion, resulting in luminescence quenching. The europium emission should be recovered aer the displacement of the Cu 2+ ion upon copper sulphide precipitation.

Results and discussion
Synthesis and photophysical properties of L1 and EuL1 Ligand L1 was readily prepared from (4-iodopyridine-2,6-diyl) dimethanol (1) 14 via a desymmetrization synthetic strategy. As shown in Scheme 1, a pyridine-containing chromophore (based on a D-p-A motif) was established via a Sonogashira crosscoupling reaction between 1 and 1-ethynyl-4-propoxybenzene (2). 15 Aer 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 1 H and 13 C 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 †).

Fluorimetric titration studies of EuL1
With EuL1 in hand, its binding properties towards Cu 2+ ions were investigated. Upon the addition of 1 equiv. of Cu 2+ ions (CuCl 2 as the source of Cu 2+ ions), the absorption maximum of EuL1 showed a slight red shi 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 Scheme 1 Synthesis of L1 and EuL1.  europium emission with an excess of Cu 2+ ions and the Benesi-Hildebrand plot showed a 1 : 1 binding stoichiometry with K B ¼ 1.2 Â 10 5 M À1 (inset of Fig. 3a). 16 The Job's plot also supported the formation of a EuL1-Cu 2+ 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 + , Ca 2+ , Mg 2+ , Ba 2+ , Co 2+ , Zn 2+ , Ni 2+ , Fe 2+ , Mn 2+ , Cu + and Li + ions, to EuL1 resulted in only slight luminescence changes (red columns in Fig. 3b). The subsequent addition of excess Cu 2+ ions caused signicant luminescence quenching (blue columns in Fig. 3b). These results indicate the high selectivity of EuL1 towards Cu 2+ ions and that the binding between EuL1 and Cu 2+ ions is not interfered by other metal ions. In a pH study, EuL1 remains highly emissive and was quenched by Cu 2+ ions in the pH range 6 to 8 (Fig. S4 †), indicating that EuL1 is stable and can bind to Cu 2+ ions under physiological conditions.
To study the reversibility of the binding between EuL1 and Cu 2+ ions, a small amount of H 2 S (Na 2 S as the source of H 2 S) was added. The EuL1-Cu 2+ complex responded instantaneously (requiring only 40 s to reach saturation without stirring or shaking) (Fig. S5 †), and Eu emission resumed with a similar prole for the emission spectrum to that of EuL1 (Fig. 4). This result indicated that the DO3A-Eu 3+ complex was not displaced by a Cu 2+ ion, forming the EuL1-Cu 2+ complex in the previous step. More interestingly, Eu emission was further enhanced (40-fold) with an excess of H 2 S and the Eu 3+ emission prole showed signicant changes, suggesting binding between EuL1 and H 2 S (Fig. 5a). The Benesi-Hildebrand plot showed a 1 : 1 binding stoichiometry with K B ¼ 1.5 Â 10 4 M À1 (inset of Fig. 5a). 16 The detection limit of EuL1 towards H 2 S was calculated according to the 3S D /slope as low as 60 nM. Surprisingly, direct titration of EuL1 against H 2 S resulted in only about a 5fold luminescence enhancement with a non-linear relationship in the 1 : 1 Benesi-Hildebrand plot (Fig. 6). These results indicated that the Cu 2+ ion facilitates the specic 1 : 1 binding of EuL1 and H 2 S, presumably via pre-organizing the conformation of EuL1. On the other hand, non-specic binding (possibly a mixture of 1 : 1 and 2 : 1 binding) between EuL1 and H 2 S resulted without the favourable conformation that is induced by Fig. 4 The emission spectra of EuL1 (10 mM) (red), with 1 equiv. of Cu 2+ ions (green), and with 1 equiv. of Cu 2+ ions and 1 equiv. of H 2 S (black). All spectra were acquired in water with l ex at 325 nm. ; 8: Br À ; 9: HCO 3 À ; 10: S 2À ; 11: GSH; 12: cysteine. All spectra were acquired in water with excitation at 325 nm. the pre-complexation of a Cu 2+ ion. This proposal was further supported by the dramatic luminescence drop of the EuL1-Na 2 S complex upon heating (>70 C) (Fig. S6 †). This type of Cu 2+assisted luminescence enhancement of Eu emission is unprecedented. In a competitive study, EuL1-Cu 2+ showed insignicant changes in luminescence with a large excess of anions, including Cl À , SO 4 2À , HSO 4 À , I À , CO 3 2À , HPO 4 2À , Br À and HCO 3 À , and only small changes for GSH and cysteine (red columns in Fig. 5b). Upon the addition of H 2 S, the Eu emissions were recovered in all the above cases, indicating a high selectivity of EuL1-Cu 2+ towards H 2 S.

Mechanistic studies
The binding mechanisms of EuL1 towards Cu 2+ ions and the EuL1-Cu 2+ complex towards H 2 S were studied using a comparative analysis of the emission spectra of the Eu complexes and the 1 H NMR spectra of La complexes. 17 As shown in Fig. 7 Table 1). This is correlated with the NMR data and shows that the Cu 2+ ion is coordinated in the aza-crown. However, signal broadening was observed in the 1 H 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-Eu 3+ and Fig. 7 Top: proposed binding mechanism of EuL1 towards Cu 2+ and H 2 S (Na 2 S as the source of H 2 S). Bottom left: emission spectra of the Eu complexes (l ex ¼ 325 nm). Bottom right: 1 H NMR spectra of the La complexes (6.5-8.5 ppm). ]. This increase can be attributed to the lower symmetry of the complexes with the addition of sulphide ions (Fig. 7) and the 1 H NMR signals of LaL1 were sharpened. These results suggested new complex formation aer the displacement of the Cu 2+ ion via CuS precipitation. This proposal is further supported by the HRMS spectrum of the EuL1-Na 2 S complex (Fig. S7 †) and the change in the quantum yields (Table S2 †). The EuL1-Na 2 S complex is highly emissive probably due to its rigid structure. The proposed binding mechanism was also examined using a series of negative control compounds (Fig. 8). 19 EuL2 showed no luminescence quenching upon the addition of Cu 2+ ions (Fig. 9a). This result indicated that the carbonyl linker of aza-18crown-6 may be too rigid for coordination between Cu 2+ and pyridine, which could be essential for Eu emission quenching. Without the aza-crown moiety, EuL3 also showed no luminescence quenching towards Cu 2+ (Fig. 9b), suggesting DO3A-Eu 3+ is stable with Cu 2+ and the aza-crown motif is important for the Cu 2+ binding. L4 bearing the pyridine-chromophore showed profound luminescence quenching, but its phenyl analogue (L5) showed no signicant change in luminescence upon the addition of Cu 2+ ions ( Fig. 9c and d). These results indicated that the pyridine moiety of the chromophore is essential for the binding of Cu 2+ 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.

Conclusions
In summary, we have prepared a water-soluble and emissive Eucomplex (EuL1) based on a DO3A(Eu 3+ )-pyridine-aza-crown motif, and studied its consecutive binding properties towards Cu 2+ and H 2 S extensively. EuL1 binds to Cu 2+ ions selectively (K B ¼ 1.2 Â 10 5 M À1 ) inducing 17-fold luminescence quenching and forming a 1 : 1 stoichiometric complex (EuL1-Cu 2+ ), which responds to H 2 S 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-Na 2 S) with K B ¼ 1.5 Â 10 4 M À1 . Without Cu 2+ ions, EuL1 shows non-specic binding towards H 2 S with only a 5-fold luminescence enhancement. These results indicate that the Cu 2+ ion may pre-organize the conformation of EuL1 and facilitate the formation of the EuL1-Na 2 S complex. The studies This journal is © The Royal Society of Chemistry 2016 on this unprecedented Cu 2+ -assisted luminescence enhancement of Eu emission are still ongoing. With long-lived Eu emission, reversible binding properties, an instantaneous response and high selectivity towards H 2 S, this Eu-based luminescence "off-on" gate could nd suitable applications for H 2 S imaging in biological systems.