During the recent two decades,the design and synthesis of compounds for sensing relevant environmentally and biologically important ionic species,particularly for heavy metal and transition metal cations,are currently of great interest ^[1]. Numerous excellent works have focused on the selective and sensitive detection of transition metal ions (such as,Cu²⁺,Pb²⁺,Zn²⁺and Fe³⁺) ^[2]. Governmental restrictions on the levels of residual heavy metals in end products are also very strict. Therefore,detection of Hg²⁺ levels has attracted tremendous attention. Many probes for Hg²⁺ have been proposed thus far ^[3]. However,most of the probes act only in organic media. Consequently,there is an urgent need to develop chemical sensors capable of detecting the presence of Hg²⁺ in aqueous media. As everyone knows,rhodamine derivatives are non-fluorescent and colorless,whereas ring-opening of the corresponding spirolactam gives rise to strong fluorescence emission and a pink color ^[4]. Recently,some rhodamine-based fluorescent probes were reported for the detection of Hg²⁺ metal ions ^[5]. In this paper,we report a new rhodamine-based derivative,turn-on fluorescent sensor 1 (Scheme 1) for Hg²⁺ , which shows very high sensitivity and selectivity toward Hg²⁺ over other metal ions. 2. Experimental

All the materials for synthesis were purchased from commercial suppliers and used without further purification. The solutions of metal ions were prepared from their nitrate salts,except for FeCl₂, CrCl₃and MnCl₂. The use of Fe(NO₃)₃and FeCl₃yielded nearly the same results. Rhodamine B derivative (2) was synthesized according to the literature ^[6]. A Hitachi F-4500 spectrofluorimeter was used for fluorescence measurements. The absorption spectra were recorded with a Techcomp UV-8500 spectrophotometer (Shanghai,China). The NMR spectra were measured on a Bruker AMX-400 spectrometer at 400 MHz in CDCl₃. Elemental analyses were carried out with a Flash EA 1112 instrument. Mass spectra were acquired in positive ion mode using a Bruker ESQUIRE 3000 ion trap spectrometer equipped with a gas nebulizer probe, capable of analyzing ions up tom/z6000.

Synthesis of 1: Benzoyl chloride (0.16 g,1.1 mmol) was slowly added to the mixture of 2 (0.5 g,1.0 mmol) and triethylamine (1 mL) in 10 mL CH₂Cl₂at 0°C. After the addition,the mixture was stirred at room temperature for 4 h. The solvent was removed and the residue was purified by silica gel column chromatography with CH₂Cl₂/MeOH (15/1,v/v) as eluent to afford 1,yield 85%. Mp 116- 118°C; ¹H NMR (400 MHz,CDCl₃): δ8.17-8.19 (m,1H),7.92 (s, 1H),7.84 (d,2H,J= 7.2 Hz),7.40-7.52 (m,5H),7.10-7.13 (m,1H), 6.34-6.41 (m,2H),6.24 (d,2H,J= 8.8 Hz),3.95(s,2H),3.32-3.37 (m,8H),1.17-1.20 (m,12H); ESI-MS: m/z 605.6 [M+H]⁺; Anal. calcd. for C₃₇H₄₀N₄O₂S: C 73.48,H 6.67,N 9.26,found: C 73.42,H 6.69,N 9.30. 3. Results and discussion

Compound 1 was facilely synthesized from rhodamine B according to procedures in the literature and obtained as light orange crystals. As shown in Scheme 1,compound 1 was prepared in high yield by reacting 2 with benzoyl chloride in the presence of triethylamine. Its structure has been confirmed using ¹H NMR,ESI mass spectrometry,and elemental analysis. Although 1 is a derivative of rhodamine B,it forms a nearly colorless solution in either water or absolute ethanol,indicating that the spirocyclic form exists predominantly. Besides,neither the color nor the fluorescence (excited at 530 nm) characteristics of rhodamine could be observed for1in ethanol-water (4:1,v/v),suggesting that the spirocyclic form was still preferred. Addition of mercuric ion to the solution of1causes instantaneous development of a pink color and a strong fluorescence (Fig. 1). This observation shows that the mercury-induced,ring-opening reaction takes place rapidly at room temperature.

Scheme 1

	Download: JPG larger image
Scheme 1. The synthesis of title compound.

Fig. 1

	Download: JPG larger image
Fig. 1. Color change of the neutral buffer solution of1in the presence of metal ions.

To further investigate the interaction of Hg²⁺ and 1,an ultraviolet photometric titration experiment was carried out. An increasing,linear absorption intensity of 1 could be observed with increasing Hg²⁺ concentration accompanied by color changes from colorless to pink. To determine the stoichiometry of the Hg-ligand complex,Job’s method for absorbance measurement was applied. Keeping the sum of the initial concentration of Hg²⁺ and 1 at 100mmol/L,the molar ratio of Hg²⁺ was varied from 0 to 2. A plot of [Hg²⁺ ]/{[Hg²⁺ ]+[1]}versusthe molar fraction of Hg²⁺ was provided in Fig. 2. It showed that the [Hg²⁺ ]/{[Hg²⁺ ]+[1]} value went through a maximum at a molar fraction of 0.5,indicating a 1:1 stoichiometry of the Hg²⁺ to 1 in the complex.

Fig. 2

	Download: JPG larger image
Fig. 2. Plots according to the method for continuous variations,indicating the 1:1 stoichiometry for1-Hg2+ (the total concentration of1and Hg2+ is 100mmol/L).

Fig. 3 shows the absorption spectra of 1 in the presence of various metal ions in ethanol-water. When no metal ion was added to the solution of 1(10mmol/L),almost no absorption above 562 nm could be observed,whereas a significant enhancement of the characteristic absorption of rhodamine B emerged soon after Hg²⁺ was injected into the solution. There was a large enhancement factor (34-fold) of absorbance at λ_max= 562 nm upon the addition of 20 equiv. of Hg²⁺ . Other cations of interest gave no response (Fig. 4).

Fig. 3

	Download: JPG larger image
Fig. 3. Changes in the absorption spectra of1in the presence of different metal ions in ethanol-water (4:1,v/v).

Fig. 4

	Download: JPG larger image
Fig. 4. Change in the absorbance at 562 nm of1(10mmol/L) in presence of 20 equiv. of various different metal ions in ethanol-water (4:1,v/v).

Changes in the fluorescence properties of 1 caused by other metal ions,including Mg²⁺ ,K⁺,Co²⁺,Cu²⁺,Mn²⁺,Ba²⁺,Pb²⁺,Na⁺,Ca²⁺,Cr³⁺,Ag⁺,Ni²⁺,Cd²⁺,Zn²⁺,Fe²⁺,Li⁺and Fe³⁺were also measured. Fluorescence spectra of solutions of 1(10 umol/L), recorded within 5 min after the addition of 1 equiv. of each of these metal ions,are displayed in Fig. 5. Only Hg²⁺ ion promote significant fluorescence intensity changes,while other metal ions did not cause any changes under identical conditions. In addition, the enhancement in fluorescence intensity resulting from addition of Hg²⁺ is not influenced by subsequent addition of other metal ions. Finally,while the colorless to pink color change associated with the reaction of1with Hg²⁺ is readily detected visually,no significant color changes are promoted by other metal ions. This interesting feature reveals that 1can serve as a selective ‘‘nakedeye’’ chemosensor for Hg²⁺ .

Fig. 5

	Download: JPG larger image
Fig. 5. Fluorescence spectra of1(10mmol/L) in ethanol-water (4:1,v/v) in the presence of 1 equiv. of Mg2+,K+,Co2+,Cu2+,Mn2+,Ba2+,Pb2+,Na+,Ca2+,Hg2+ ,Zn2+, Ni2+,Cd2+,Ag+,Fe2+,Li+,Cr3+and Fe3+Ex: 560 nm,Em: 580 nm,slit: 5.

Generally,one of the most important and useful applications for a fluorescent chemosensor is the detection of metal ions,especially for heavy metals. The limit of the chemosensor for Hg²⁺ ion has been tested. When 1 was employed at 0.1mmol/L in EtOH/H₂O (4:1,v/v),the fluorescent intensity of 1 was proportional to the added concentration of Hg²⁺ (Fig. 6). The detection limit was measured to be 1.0 ppb,establishing 1 was capable of distinguishing safe and toxic levels of Hg²⁺ in drinking water according to the U.S. (EPA) standard (2 ppb).

Fig. 6

	Download: JPG larger image
Fig. 6. The fluorescence intensity at 580 nm of compound 1(0.1umol/L) as a function of the Hg2+ concentration (0-20 ppb) in ethanol-water (4:1,v/v).

Both UV-vis and fluorescence data lead to a significant OFF-ON signal. From the molecular structure and spectral results of 1 ,itis concluded that the reaction mechanism should involve two steps. First,the addition of the Hg²⁺ ion induced the N atom of amide to attack the C atom of C55S bond,and thus a ring opening of the spirolactam of rhodamine took place. Second,a water-promoted hydrolysis followed. After the removal of HgS and phthalic acid,an intramolecular guanylation took place. Finally,a cyclic product 5 was formed through an irreversible hydrolysis,desulfurization reaction,as depicted in Scheme 2. In order to prove this reaction mechanism of the present system,the reaction products of 1 with Hg²⁺ were subjected to electrospray ionization mass spectral analyses. In ESI-MS,a major ion peaks were detected atm/z471.4 (Fig. 7). It was characterized as the ring opened product of rhodamine derivative,indicating the generation of 5 as a final product.

Scheme 2

	Download: JPG larger image
Scheme 2. Proposed mechanism.

Fig. 7

	Download: JPG larger image
Fig. 7. ESI-MS of 1 +Hg2+ .

In conclusion,we synthesized a new rhodamine-based fluorescent probe for Hg²⁺ . The colorimetric and fluorescent response to Hg²⁺ can be conveniently detected by the naked eye,which provides a facile method for visual detection of Hg²⁺ . The selectivity of this system for Hg²⁺ over other metal ions is excellent. Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos. 20972143,21375113) and Program for New Century Excellent Talents in University (No. NCET-11-0950).