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Synthesis and evaluation of a water-solubility glycosyl-rhodamine fluorescent probe detecting Hg2+
Accepted Manuscript Title: Synthesis and evaluation of a water-solubility glycosyl-rhodamine fluorescent probe detecting Hg2+ Author: Zhengjun Chen, Wei Hu, Mian Wang, Lisheng Wang, Guifa Su, Jianyi Wang PII: DOI: Reference:
S0008-6215(16)30065-9 http://dx.doi.org/doi: 10.1016/j.carres.2016.03.018 CAR 7153
To appear in:
Carbohydrate Research
Received date: Revised date: Accepted date:
5-2-2016 17-3-2016 20-3-2016
Please cite this article as: Zhengjun Chen, Wei Hu, Mian Wang, Lisheng Wang, Guifa Su, Jianyi Wang, Synthesis and evaluation of a water-solubility glycosyl-rhodamine fluorescent probe detecting Hg2+, Carbohydrate Research (2016), http://dx.doi.org/doi: 10.1016/j.carres.2016.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and evaluation of a water-solubility glycosyl-rhodamine fluorescent probe detecting Hg2+ Zhengjun Chena, Wei Hua, Mian Wanga, Lisheng Wanga, Guifa Suc, Jianyi Wang*a, b a
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, People’s
Republic of China b
Guangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology and
Resource Development, Nanning 530004, People’s Republic of China c
Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi
Normal University), Ministry of Education of China
*Corresponding author. [email protected] Highlights A water-solubility fuorescent probe (RBGlc-3) was designed and synthesized. RBGlc-3 can selectively recognize Hg2+ in water environment. 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 against Hg2+. Graphical Abstract
Abstract A glycosyl-rhodamine fluorescent probe with good water-solubility has been designed and synthesized through click reaction. Compared with control compound 1, the obtained target compound (RBGlc-3) could be independently applied for the detection of Hg2+ in water medium, and not disturbed by Ce3+, Eu3+, Ca2+, Cd2+, Fe2+, Ba2+, Co2+, Cu2+, Zn2+, Pb2+, Mg2+, Ni2+, K+, Ag+, Na+, NH4+, CH3COO-, S2O42-, SO42-, SO32- and Cl-. 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 toward Hg2+ ion, and the OFF-ON fluorescent mechanism of RBGlc-3 is proposed. Key words: Rhodamine, water-solubility, Hg2+, glucose, click reaction 1
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1. Introduction Mercury is always considered as major heavy metal pollution. Regardless of the element form or ion form, mercury could be converted into methyl mercury by bacteria and bioaccumulation in the food chain1-6, inducing a variety of serious damages, such as central nervous and cardiovascular systems7,8, permanent deterioration of the brain, kidneys, and developing fetus9. Therefore, it is very significant to develop a convenient and effective chemosensor for the detection of mercury. Up to date, many fluorescent chemosensors such as calixarenes, quinolines, fluorescein, rhodamine, coumarin, pyrene, naphthalimide, boron dipyrromethene difluoride (BODIPY), squaraine and nitrobenzofurazan have been developed10-12, and rhodamine is still the most popular chemosensor for the detection of Hg2+ because of its excellent quantum yields, photostability, and relatively long emission wavelengths10. However, most of the rhodamine-based fluorescent probes had to be employed in the mixture of organic solvents and water, such as CH3CN/H2O medium13,14, CH3CN/HEPES buffer15, DMF/H2O medium16, CH3OH/H2O medium17, CH3OH/HEPES buffer18,19,20, CH3OH/Tris-HCl buffer21, CH3CH2OH/H2O medium22, CH3CH2OH/HEPES buffer medium23. Clearly, poor water-solubility of rhodamine-based fluorescent probe is the largest bottleneck to limit its application for detection of mercury ion in the water-solubility environment and biological systems. Therefore, the improvement of water-solubility of rhodamine-based fluorescent probe becomes very necessary. As is commonly known, glucose is a highly hydrophilic nutrient in the biological and pathological processes24, and an environmentally friendly molecule. The glucose molecule had been even used to improve the water-solubility of fluorescent probe via incorporating D-glucosamine group and quinoline group25. Inspired by these, in this work we introduced three glucose units into rhodamine framework via click reaction technology, which has been widely used to construct complex architectures in carbohydrate chemistry26, to increase the water-solubility of rhodamine molecule, and broaden its application for the detection of Hg2+ ion in the water-solubility environment and biological systems. The obtained target compound (RBGlc-3) to detect Hg2+ ion was correspondingly characterized and evaluated in the water-solubility environment. 2. Results and Discussion 2.1 Synthesis of RBGlc-3 As was shown in Scheme 1, a strong fluorescent rhodamine B, which was starting material, first undergo a cyclization reaction with 2-aminoethanol to give a non-fluorescent compound 127. Under the promotion of NaH (60% dispersion in mineral oil) in anhydrous THF, OH group of compound 1 then nucleophilically substituted Br group of compound 2 to provide compound 3, where compound 2 was in advance prepared by tris(hydroxymethyl)aminomethane and propargyl bromide28. Under the catalysis of copper(I), compound 3 and per-O-acetyl glycosyl azide 4 then went through a click reaction to generate compound 5, where compound 4 was in 2
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advance prepared according to the reference29,30. Compound 5 was finally deacetylated to afford the target compound (RBGlc-3).
2.2 Water-solubility evaluation of RBGlc-3 In 1.5 mL plastic centrifuge, RBGlc-3 (10 mg, 0.0073 mmol) and the control compound 1 (3.5 mg, 0.0072 mmol) were dispersed in 0.5 mL pure water, respectively. The control compound 1 was very difficult to dissolve in pure water even if it was treated by ultrasound for one hour. However, RBGlc-3 was easily dissolved in pure water when three glucose units were introduced into rhodamine framework. Under the irradiation of ultraviolet lamp (365 nm), both the RBGlc-3 solution and the compound 1 dispersion system did not emit fluorescence, shown in Fig. 1a. When Hg2+ ion (5 equiv) was separately added into the RBGlc-3 solution and the compound 1 dispersion system with the ultrasound of 2 minutes, the RBGlc-3 solution indicated a significant color switch from light red to pink and the fluorescence was gradually enhanced. While the compound 1 dispersion system were hardly observed fluorescence change (Fig. 1b). However, when 1 mL organic solvent methanol was added to the compound 1 dispersion system, the compound 1 was dissolved into a homogeneous system (solution), and the compound 1 system also presented a similar color change and the enhancement effect of fluorescence (Fig. 1c). Clearly, the fluorescence effect of compound is closely related to its solubility in the environment. Therefore, the introduce of three glucose units into rhodamine framework would greatly improve water-solubility of rhodamine derivative RBGlc-3, and broaden its application for detection of Hg2+ ion in the water-solubility environment and biological systems. 2.3 Selectivity and competition of RBGlc-3 for Hg2+ ion Seen from Fig. 2a and 2b, RBGlc-3 (10 M) in pure water medium exhibited a significant fluorescent enhancement at 583 nm when Hg2+ ion (5 equiv) was added. However, when Hg2+ ion was replaced by other cations or anions, such as Ce3+, Eu3+, Ca2+, Cd2+, Fe2+, Ba2+, Co2+, Cu2+, Zn2+, Pb2+, Mg2+, Ni2+, K+, Ag+, Na+, NH4+, CH3COO-, S2O42-, SO42-, SO32- and Cl-, no obvious fluorescent changes were observed. These results suggested that RBGlc-3 in pure water medium could be selectively recognized by Hg2+. With gradual addition of Hg2+ ion (0-5 equiv)，the fluorescent intensity and UV absorption also gradually enhanced for the solution of RBGlc-3 (10 M) in pure water, shown in Fig. 3a and Fig. 3b. Toward better understanding of the fluorescent enhancement effect of the RBGlc-3 solution induced by Hg2+, a reversible experiment was performed. When 50 M S2- ion was added to the RBGlc-3 solution system with 50 M Hg2+ ion, the fluorescence of RBGlc-3 solution system dramatically disappeared (Fig. 4). A probable reason is that Hg2+ ion formed HgS precipitation with S2- ion and decreased the binding with RBGlc-3, causing that the ring-opening fluorescent RBGlc-3 re-cyclized to give a non-fluorescent spirocyclic RBGlc-3. To explore the practical application of RBGlc-3 for the detection of Hg2+ in water, 3
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the competitive experiments were also carried out for RBGlc-3 in pure water. As shown in Fig. 5, when other metal ions (50 M) was added into the test solution with Hg2+ ion, the RBGlc-3 solution (10 M) did not show obvious fluorescent enhancement. Conversely, when the same amount of Hg2+ ion was added into the test solution with other metal ion, the fluorescent intensity of test solutions were dramatically enhanced. Therefore, RBGlc-3 can be independently used to detect Hg2+ in water environment through judging the change of color, absorption and fluorescence, and be not disturbed by the ions listed above. 2.4 Stoichiometry and recognition mode of RBGlc-3 toward Hg2+ In Fig. 6, total concentration of RBGlc-3 and Hg2+ was kept constant (10 M) and [Hg2+]/[RBGlc-3+Hg2+] value was varied from 0 to 0.9. When [Hg2+]/[RBGlc-3+Hg2+] value was 0.5 ([Hg2+]/[RBGlc-3] ratio was 1:1), the solution of RBGlc-3 and Hg2+ would emit the strongest fluorescence. A reasonable explanation for this phenomenon is that 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 against Hg2+ ion. Moreover, the RBGlc-3 and Hg2+ complex with a ratio of 1:1 could be also confirmed by spectra (m/z 1576.56799 for [RBGlc-3 + Hg2+]), shown in Fig. 7. To better understand the recognition mode of RBGlc-3 toward Hg2+ ion, H1 NMR of RBGlc-3 and Hg2+ mixed in 1:1 ratio was recorded in CD3OD-d4 solvent (Fig. 8). The signals of H5, H6, H7 of RBGlc-3 separately move to low-field of 0.2, 0.06, 0.08 ppm, indicating that N atom in the triazole rings participated in the binding of RBGlc-3 against Hg2+ ion, leading to their deshielding effects. Based on the findings above, OFF-ON fluorescent mechanism of RBGlc-3 was correspondingly proposed, shown in Scheme 2. 3. Conclusions In summary, to improve water-solubility of rhodamine fluorescent probe, three glucose units were introduced into rhodamine framework via click reaction technology. The synthesized RBGlc-3 showed good water-solubility compared with the control compound 1. The competition experiments revealed that RBGlc-3 could be independently applied for the detection of Hg2+ in pure water medium, and not disturbed by Ce3+, Eu3+, Ca2+, Cd2+, Fe2+, Ba2+, Co2+, Cu2+, Zn2+, Pb2+, Mg2+, Ni2+, K+, Ag+, Na+, NH4+, CH3COO-, S2O42-, SO42-, SO32- and Cl-. The Job’s plot, HRMS data and NMR analysis indicated that 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 toward Hg2+ ion, and the corresponding OFF-ON fluorescent mechanism of RBGlc-3 is proposed. 4. Materials and methods All regents were commercially available and used without further purification, except THF was dried through sodium. 1H NMR and 13C NMR spectra were recorded on Brucker 600 MHz at 25 oC, TMS as the internal standard in CDCl3, and solvent peak as the internal reference in CD3OD. HRMS analysis was performed by Thermo Fisher Scientific LTQ FT Ultra. The UV-vis absorption and the fluorescence emission 4
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spectra were carried out by Shanghai Jing Hua 7600 UV visible spectrophotometer (dual beam) and Shimadzu RF-5301PC at room temperature, respectively. 4.1. Synthesis of compound 1 The synthesis of compound 1 was performed according to the literature27. The solution of rhodamine B (10 g), 2-aminoethanol (25 mL) and methanol (15 mL) was stirred at 75 oC until the red in the solution disappeared. H2O (200 mL) was added to the mixture. The resulting mixture was extracted with CH2Cl2, washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (petroleum ether:EtOAc 4:1) to afford compound 1, which showed the same spectroscopic properties as the literature27. 1H NMR (600 MHz, CDCl3) δ 7.92 (dd, J = 5.6, 3.0 Hz, 1H), 7.46 (dd, J = 5.6, 3.1 Hz, 2H), 7.09 (dd, J = 5.2, 3.2 Hz, 1H), 6.51 (d, J = 8.9 Hz, 2H), 6.40 (d, J = 2.4 Hz, 2H), 6.31 (dd, J = 8.9, 2.4 Hz, 2H), 3.51 – 3.47 (m, 2H), 3.36 (q, J = 7.0 Hz, 8H), 3.32 – 3.29 (m, 2H), 1.19 (t, J = 7.1 Hz,12H). 13C NMR (151 MHz, CDCl3) δ 170.10, 153.92, 153.28, 148.90, 132.69, 130.45, 128.51, 128.14, 123.81, 122.90, 108.24, 104.80, 97.80, 65.87, 62.68, 62.66, 44.65, 44.37, 12.61. 4.2. Synthesis of compound 2 Compound 2 was synthesized according to the reference28, as shown in the Scheme 3. NMR data were the same as the literature reported28. 1H NMR (600 MHz, CDCl3) δ 6.70 (s, 1H), 4.18 (d, J = 2.4 Hz, 6H), 3.87 (s, 6H), 3.82 (s, 2H), 2.47 (t, J = 2.3 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ165.39, 79.40, 74.81, 68.14, 59.76, 58.70, 29.47. 4.3. Synthesis of compound 3 A solution of NaH (0.291 g, 7.28 mmol, 60%) in anhydrous THF (6 mL) was cooled to 0 oC under argon. Compound 1 (0.9183 g, 1.88 mmol) in anhydrous THF (6 mL) was added dropwise. After stirred for 15 min, a solution of 2 in anhydrous THF (6 mL) was added dropwise. The reaction was allowed to warm up to room temperature and was stirred for overnight. The reaction mixture was poured into water (100 mL). The aqueous solution was extracted with CH2Cl2 (3×40 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, concentrated, and then purified by silica gel chromatography (petroleum ether:EtOAc 4:1) to give 3 an off-white solid (1.106 g, 62%). 1H NMR (600 MHz, CDCl3) δ 7.94 – 7.91 (m, 1H), 7.47 – 7.42 (m, 2H), 7.11 – 7.06 (m, 1H), 6.82 (s, 1H), 6.46 (d, J = 8.9 Hz, 2H), 6.39 (d, J = 2.3 Hz, 2H), 6.29 (dd, J = 8.9, 2.5 Hz, 2H), 4.11 (d, J = 2.4 Hz, 6H), 3.85 (s, 6H), 3.55 (s, 2H), 3.42 (t, J = 6.6 Hz, 2H), 3.35 (q, J = 7.1 Hz, 8H), 3.06 (t, J = 6.6 Hz, 2H), 2.40 (t, J = 2.3 Hz, 3H), 1.18 (t, J = 7.1 Hz, 12H). 13C NMR (151 MHz, CDCl3) δ 169.47, 168.35, 153.67, 153.20, 148.83, 132.41, 130.94, 128.88, 128.01, 123.76, 122.89, 108.13, 105.44, 97.69, 79.74, 74.54, 70.39, 68.27, 68.12, 64.62, 59.17, 58.58, 44.38, 39.06, 12.61. ESI-MS (m/z) 761.4. 4.4. Synthesis of compound 4 5
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Synthesis of compound 4 is routine, which was prepared according to the literature29,30. NMR data were the same as the literature reported. 1H NMR (600 MHz, CDCl3) δ 5.23 (t, J = 9.5 Hz, 1H), 5.12 (t, J = 9.8 Hz, 1H), 4.99 – 4.95 (m, 1H), 4.66 (d, J = 8.9 Hz, 1H), 4.29 (dd, J = 12.5, 4.8 Hz, 1H), 4.18 (dd, J = 12.5, 2.2 Hz, 1H), 3.81 (ddd, J = 10.1, 4.8, 2.3 Hz, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 170.60, 170.11, 169.30, 169.20, 87.91, 74.02, 72.60, 70.64, 67.89, 61.66, 20.69, 20.56, 20.54. 4.5. Synthesis of compound 5 To a solution of 3 (0.2052 g, 0.2697 mmol) and 4 (0.315 g, 0.8438 mmol) in THF (4 mL) was added a solution of sodium ascorbate (62.3 mg, 0.3145 mmol) and CuSO4·5H2O (40.2 mg, 0.161 mmol) in distilled H2O (4 mL). The reaction mixture was stirred for overnight at r.t. under argon. TCL indicated the reaction completion (petroleum ether: EtOAc 1:6). Deionized H2O was added and the aqueous solution was extracted with CH2Cl2 (3×15 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated to afford a crude solid residue, which was purified by silica gel chromatography (petroleum ether:EtOAc 1:4) to get 5 as a light red solid (0.4268 g, 84.2%).1H NMR (600 MHz, CDCl3) δ 8.01 (s, 3H), 7.93 (dd, J = 5.9, 2.6 Hz, 1H), 7.45 (dd, J = 5.3, 3.2 Hz, 2H), 7.10 (dd, J = 5.8, 2.5 Hz, 1H), 6.84 (s, 1H), 6.47 (t, J = 8.4 Hz, 2H), 6.34 (d, J = 60.0 Hz, 4H), 6.01 (d, J = 9.4 Hz, 3H), 5.61 (t, J = 9.5 Hz, 3H), 5.46 (t, J = 9.5 Hz, 3H), 5.36 (t, J = 9.8 Hz, 3H), 4.61 (dd, J = 13.3, 3.9 Hz, 6H), 4.32 (dd, J = 12.6, 4.8 Hz, 3H), 4.20 – 4.15 (m, 3H), 4.13 – 4.08 (m, 3H), 3.82 (d, J = 9.2 Hz, 3H), 3.75 (d, J = 9.2 Hz, 3H), 3.52 (q, J = 14.7 Hz, 2H), 3.46 – 3.39 (m, 2H), 3.34 (dd, J = 13.5, 6.5 Hz, 8H), 3.08 – 2.98 (m, 2H), 2.09 (s, 9H), 2.04 (s, 18H), 1.79 (s, 9H), 1.17 (t, J = 6.9 Hz, 12H). 13C NMR (151 MHz, CDCl3) δ 170.64, 170.10, 169.67, 169.47, 168.81, 168.48, 153.67, 153.19, 148.88, 145.65, 132.56, 130.84, 128.84, 128.13, 123.84, 122.86, 121.89, 120.93, 108.18, 97.69, 85.45, 74.92, 72.94, 70.32, 68.79, 68.25, 67.84, 64.70, 61.68, 59.63, 56.84, 44.38, 39.12, 21.81, 20.66, 20.61, 20.57, 20.06, 12.58. 4.6. Synthesis of RBGlc-3 To a solution of 5 (0.42 g, 0.2233 mmol) in dry MeOH (6 mL) was added sodium methoxide until pH 9-10. The reaction mixture was stirred for overnight at r.t. under argon. The mixture was then neutralized by addition of ion-exchange resin (Amberlite IR 120 H+) until pH 7, filtered and the solvent was evaporated to afford a crude solid residue. The residue was then purified by silica gel chromatography (CH3OH:CH2Cl2 10 - 50%) to give a light red solid RBGlc-3 (0.2725 g, 88.7%).1H NMR (600 MHz, MeOD) δ 8.16 (s, 3H), 7.92 – 7.89 (m, 1H), 7.52 (dd, J = 5.5, 3.1 Hz, 2H), 7.06 – 7.03 (m, 1H), 6.46 – 6.41 (m, 4H), 6.39 – 6.35 (m, 2H), 5.67 (d, J = 9.2 Hz, 3H), 4.60 (s, 1H), 4.53 (s, 6H), 3.98 (t, J = 9.1 Hz, 3H), 3.89 (d, J = 10.9 Hz, 3H), 3.74 (t, J = 8.8 Hz, 9H), 3.65 – 3.61 (m, 6H), 3.59 – 3.55 (m, 3H), 3.44 (s, 2H), 3.37 (dd, J = 13.6, 6.5 Hz, 10H), 3.01 (t, J = 5.7 Hz, 2H), 1.16 (t, J = 7.0 Hz, 12H). 13C NMR (151 MHz, CD3OD) δ 170.41, 169.08, 153.82, 153.30, 149.06, 144.45, 132.81, 130.50, 128.42, 128.22, 123.62, 123.13, 122.51, 108.24, 104.78, 97.63, 88.14, 79.68, 77.05, 72.63, 6
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69.61, 69.51, 67.89, 67.62, 65.32, 63.76, 61.03, 59.68, 44.02, 38.97, 11.58. HRMS calcd for C63H86N13O22: [M + H+] 1376.59, found 1376.6. 4.7. Spectroscopic measurements The stock solutions of 10 mM of HgCl2, Cu(NO3)2·3H2O, Pb(NO3)2, Eu(NO3)3·6H2O, Co(NO3)2·6H2O, Ce(NO3)3·6H2O, Cd(NO3)2·4H2O, AgNO3, NH4NO3, Zn(NO3)2·6H2O, Ni(NO3)2·6H2O, Ca(NO3)2·4H2O, NaNO3, KNO3, Mg(NO3)2, NH4NO3, FeSO4·7H2O, CH3COONa, Na2S2O4, Na2SO4, Na2SO3, NaCl and Na2S were prepared in pure water. The stock solution of RBGlc-3 (2 mM) was prepared in methanol. Ions selective spectrum of RBGlc-3 (10 M) solution was measured by adding 20 L of RBGlc-3 stock solution and 20 L metal ion into cuvette, and diluting to 4 mL with pure water. The changes of UV absorption and fluorescence spectra of RBGlc-3 were obtained by adding 2 L metal ion stock solution every time to the solution of RBGlc-3 (10 M) in 4 mL pure water. For fluorescence measurements, emission was acquired by scanning the spectra between 545 nm and 700 nm at 530 nm of excitation (excitation slit=3.0 nm, emission slit=3.0 nm). 4.8.Measurements of Job’s plot The stock solutions of 2 mM of HgCl2 and RBGlc-3 were prepared in pure water and methanol, respectively. 20, 18, 16, 14, 12, 10, 8, 6, 4 and 2 L of RBGlc-3 solution were put in fluorescent pools. 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 L of HgCl2 solution were then added to each fluorescent pool containing RBGlc-3 solution. Each fluorescent pool was then diluted with pure water to 4 mL. After stirring for a period of time, the spectra were scanned between 545 nm and 700 nm at 530 nm of excitation (excitation slit=3.0 nm, emission slit=3.0 nm). Acknowledgments This work is supported by the National Natural Science Foundation of China (No. 21262004), the Project of Guangxi Natural Science Foundation (No. 2013GXNSFBA019152), the high level innovation team and outstanding scholar project of Guangxi institutions of higher education (guijiaoren [2014] 49 hao), and State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (No. CMEMR2013-B03). Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/. These data of the most important compounds described in this article.
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Fig. 1 The influence of water-solubility of RBGlc-3 on fluorescence. (a) RBGlc-3 was completely dissolved in pure water and compound 1 was not dissolved in pure water; (b) Addition of Hg2+ ion, the RBGlc-3 solution emitted fluorescence and the compound 1 solution hardly showed fluorescence; (c) Addition of methanol, the compound 1 solution also emitted fluorescence.
Fig. 2 Fluorescence spectra of RBGlc-3 (10 μM) in pure water at 583 nm (ex = 530nm). (a) Addition of 5.0 equiv cations; (b) Addition of 5.0 equiv anions.
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Fig. 3 The influence of Hg2+ concentration on spectra of RBGlc-3 (10 μM) i pure water. (a) Fluorescence change with 0-5 equiv Hg2+ ion; (b) Absorption change with 0-5 equiv Hg2+ ion.
Fig. 4 Fluorescence spectra of RBGlc-3 (10 μM) i pure w ter with 5.0 equiv of H and followed by addition of 5.0 equiv. of S2- (ex = 530nm).
2+
Fig. 5 Fluorescence response of RBGlc-3 (10 μM) i pure w ter to v rious c tio s and those with additional Hg2+. 1, Fe2+; 2, NH4+; 3, Ba2+; 4, Ca2+; 5, Cd2+; 6, Co2+; 7, K+; 8, Mg2+; 9, Na+; 10, Ni2+;11, Pb2+; 12, Ce3+; 13, Cu2+; 14, Zn2+; 15, Ag+; 16, Eu3+.
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Fig. 6 Job’s plot for RBGlc-3 and Hg2+complexation (the total concentration of RBGlc-3 and Hg2+ was 10 M).
Fig. 7 HRMS spectra of [RBGlc-3 + Hg2+].
Fig. 8 1H NMR spectra of RBGlc-3 and [RBGlc-3 + Hg2+] in CD3OD.
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Scheme 1 Synthetic route of RBGlc-3. Reagents and conditions: (a) 2-aminoethanol, methanol, 75 oC; (b) NaH(60%), anhydrous THF, 0o C to r.t., overnight, 62%; (c) CuSO4·5H2O, sodium ascorbate, THF/H2O (1:1), r.t., overnight, 84.2%; (d) CH3ONa, CH3OH, r.t., overnight, 88.7%.
Scheme 2 The proposed OFF-ON fluorescent mechanism of RBGlc-3.
Scheme 3 The synthesis of compound 2.
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S0008-6215(16)30065-9 http://dx.doi.org/doi: 10.1016/j.carres.2016.03.018 CAR 7153
To appear in:
Carbohydrate Research
Received date: Revised date: Accepted date:
5-2-2016 17-3-2016 20-3-2016
Please cite this article as: Zhengjun Chen, Wei Hu, Mian Wang, Lisheng Wang, Guifa Su, Jianyi Wang, Synthesis and evaluation of a water-solubility glycosyl-rhodamine fluorescent probe detecting Hg2+, Carbohydrate Research (2016), http://dx.doi.org/doi: 10.1016/j.carres.2016.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and evaluation of a water-solubility glycosyl-rhodamine fluorescent probe detecting Hg2+ Zhengjun Chena, Wei Hua, Mian Wanga, Lisheng Wanga, Guifa Suc, Jianyi Wang*a, b a
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, People’s
Republic of China b
Guangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology and
Resource Development, Nanning 530004, People’s Republic of China c
Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi
Normal University), Ministry of Education of China
*Corresponding author. [email protected] Highlights A water-solubility fuorescent probe (RBGlc-3) was designed and synthesized. RBGlc-3 can selectively recognize Hg2+ in water environment. 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 against Hg2+. Graphical Abstract
Abstract A glycosyl-rhodamine fluorescent probe with good water-solubility has been designed and synthesized through click reaction. Compared with control compound 1, the obtained target compound (RBGlc-3) could be independently applied for the detection of Hg2+ in water medium, and not disturbed by Ce3+, Eu3+, Ca2+, Cd2+, Fe2+, Ba2+, Co2+, Cu2+, Zn2+, Pb2+, Mg2+, Ni2+, K+, Ag+, Na+, NH4+, CH3COO-, S2O42-, SO42-, SO32- and Cl-. 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 toward Hg2+ ion, and the OFF-ON fluorescent mechanism of RBGlc-3 is proposed. Key words: Rhodamine, water-solubility, Hg2+, glucose, click reaction 1
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1. Introduction Mercury is always considered as major heavy metal pollution. Regardless of the element form or ion form, mercury could be converted into methyl mercury by bacteria and bioaccumulation in the food chain1-6, inducing a variety of serious damages, such as central nervous and cardiovascular systems7,8, permanent deterioration of the brain, kidneys, and developing fetus9. Therefore, it is very significant to develop a convenient and effective chemosensor for the detection of mercury. Up to date, many fluorescent chemosensors such as calixarenes, quinolines, fluorescein, rhodamine, coumarin, pyrene, naphthalimide, boron dipyrromethene difluoride (BODIPY), squaraine and nitrobenzofurazan have been developed10-12, and rhodamine is still the most popular chemosensor for the detection of Hg2+ because of its excellent quantum yields, photostability, and relatively long emission wavelengths10. However, most of the rhodamine-based fluorescent probes had to be employed in the mixture of organic solvents and water, such as CH3CN/H2O medium13,14, CH3CN/HEPES buffer15, DMF/H2O medium16, CH3OH/H2O medium17, CH3OH/HEPES buffer18,19,20, CH3OH/Tris-HCl buffer21, CH3CH2OH/H2O medium22, CH3CH2OH/HEPES buffer medium23. Clearly, poor water-solubility of rhodamine-based fluorescent probe is the largest bottleneck to limit its application for detection of mercury ion in the water-solubility environment and biological systems. Therefore, the improvement of water-solubility of rhodamine-based fluorescent probe becomes very necessary. As is commonly known, glucose is a highly hydrophilic nutrient in the biological and pathological processes24, and an environmentally friendly molecule. The glucose molecule had been even used to improve the water-solubility of fluorescent probe via incorporating D-glucosamine group and quinoline group25. Inspired by these, in this work we introduced three glucose units into rhodamine framework via click reaction technology, which has been widely used to construct complex architectures in carbohydrate chemistry26, to increase the water-solubility of rhodamine molecule, and broaden its application for the detection of Hg2+ ion in the water-solubility environment and biological systems. The obtained target compound (RBGlc-3) to detect Hg2+ ion was correspondingly characterized and evaluated in the water-solubility environment. 2. Results and Discussion 2.1 Synthesis of RBGlc-3 As was shown in Scheme 1, a strong fluorescent rhodamine B, which was starting material, first undergo a cyclization reaction with 2-aminoethanol to give a non-fluorescent compound 127. Under the promotion of NaH (60% dispersion in mineral oil) in anhydrous THF, OH group of compound 1 then nucleophilically substituted Br group of compound 2 to provide compound 3, where compound 2 was in advance prepared by tris(hydroxymethyl)aminomethane and propargyl bromide28. Under the catalysis of copper(I), compound 3 and per-O-acetyl glycosyl azide 4 then went through a click reaction to generate compound 5, where compound 4 was in 2
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advance prepared according to the reference29,30. Compound 5 was finally deacetylated to afford the target compound (RBGlc-3).
2.2 Water-solubility evaluation of RBGlc-3 In 1.5 mL plastic centrifuge, RBGlc-3 (10 mg, 0.0073 mmol) and the control compound 1 (3.5 mg, 0.0072 mmol) were dispersed in 0.5 mL pure water, respectively. The control compound 1 was very difficult to dissolve in pure water even if it was treated by ultrasound for one hour. However, RBGlc-3 was easily dissolved in pure water when three glucose units were introduced into rhodamine framework. Under the irradiation of ultraviolet lamp (365 nm), both the RBGlc-3 solution and the compound 1 dispersion system did not emit fluorescence, shown in Fig. 1a. When Hg2+ ion (5 equiv) was separately added into the RBGlc-3 solution and the compound 1 dispersion system with the ultrasound of 2 minutes, the RBGlc-3 solution indicated a significant color switch from light red to pink and the fluorescence was gradually enhanced. While the compound 1 dispersion system were hardly observed fluorescence change (Fig. 1b). However, when 1 mL organic solvent methanol was added to the compound 1 dispersion system, the compound 1 was dissolved into a homogeneous system (solution), and the compound 1 system also presented a similar color change and the enhancement effect of fluorescence (Fig. 1c). Clearly, the fluorescence effect of compound is closely related to its solubility in the environment. Therefore, the introduce of three glucose units into rhodamine framework would greatly improve water-solubility of rhodamine derivative RBGlc-3, and broaden its application for detection of Hg2+ ion in the water-solubility environment and biological systems. 2.3 Selectivity and competition of RBGlc-3 for Hg2+ ion Seen from Fig. 2a and 2b, RBGlc-3 (10 M) in pure water medium exhibited a significant fluorescent enhancement at 583 nm when Hg2+ ion (5 equiv) was added. However, when Hg2+ ion was replaced by other cations or anions, such as Ce3+, Eu3+, Ca2+, Cd2+, Fe2+, Ba2+, Co2+, Cu2+, Zn2+, Pb2+, Mg2+, Ni2+, K+, Ag+, Na+, NH4+, CH3COO-, S2O42-, SO42-, SO32- and Cl-, no obvious fluorescent changes were observed. These results suggested that RBGlc-3 in pure water medium could be selectively recognized by Hg2+. With gradual addition of Hg2+ ion (0-5 equiv)，the fluorescent intensity and UV absorption also gradually enhanced for the solution of RBGlc-3 (10 M) in pure water, shown in Fig. 3a and Fig. 3b. Toward better understanding of the fluorescent enhancement effect of the RBGlc-3 solution induced by Hg2+, a reversible experiment was performed. When 50 M S2- ion was added to the RBGlc-3 solution system with 50 M Hg2+ ion, the fluorescence of RBGlc-3 solution system dramatically disappeared (Fig. 4). A probable reason is that Hg2+ ion formed HgS precipitation with S2- ion and decreased the binding with RBGlc-3, causing that the ring-opening fluorescent RBGlc-3 re-cyclized to give a non-fluorescent spirocyclic RBGlc-3. To explore the practical application of RBGlc-3 for the detection of Hg2+ in water, 3
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the competitive experiments were also carried out for RBGlc-3 in pure water. As shown in Fig. 5, when other metal ions (50 M) was added into the test solution with Hg2+ ion, the RBGlc-3 solution (10 M) did not show obvious fluorescent enhancement. Conversely, when the same amount of Hg2+ ion was added into the test solution with other metal ion, the fluorescent intensity of test solutions were dramatically enhanced. Therefore, RBGlc-3 can be independently used to detect Hg2+ in water environment through judging the change of color, absorption and fluorescence, and be not disturbed by the ions listed above. 2.4 Stoichiometry and recognition mode of RBGlc-3 toward Hg2+ In Fig. 6, total concentration of RBGlc-3 and Hg2+ was kept constant (10 M) and [Hg2+]/[RBGlc-3+Hg2+] value was varied from 0 to 0.9. When [Hg2+]/[RBGlc-3+Hg2+] value was 0.5 ([Hg2+]/[RBGlc-3] ratio was 1:1), the solution of RBGlc-3 and Hg2+ would emit the strongest fluorescence. A reasonable explanation for this phenomenon is that 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 against Hg2+ ion. Moreover, the RBGlc-3 and Hg2+ complex with a ratio of 1:1 could be also confirmed by spectra (m/z 1576.56799 for [RBGlc-3 + Hg2+]), shown in Fig. 7. To better understand the recognition mode of RBGlc-3 toward Hg2+ ion, H1 NMR of RBGlc-3 and Hg2+ mixed in 1:1 ratio was recorded in CD3OD-d4 solvent (Fig. 8). The signals of H5, H6, H7 of RBGlc-3 separately move to low-field of 0.2, 0.06, 0.08 ppm, indicating that N atom in the triazole rings participated in the binding of RBGlc-3 against Hg2+ ion, leading to their deshielding effects. Based on the findings above, OFF-ON fluorescent mechanism of RBGlc-3 was correspondingly proposed, shown in Scheme 2. 3. Conclusions In summary, to improve water-solubility of rhodamine fluorescent probe, three glucose units were introduced into rhodamine framework via click reaction technology. The synthesized RBGlc-3 showed good water-solubility compared with the control compound 1. The competition experiments revealed that RBGlc-3 could be independently applied for the detection of Hg2+ in pure water medium, and not disturbed by Ce3+, Eu3+, Ca2+, Cd2+, Fe2+, Ba2+, Co2+, Cu2+, Zn2+, Pb2+, Mg2+, Ni2+, K+, Ag+, Na+, NH4+, CH3COO-, S2O42-, SO42-, SO32- and Cl-. The Job’s plot, HRMS data and NMR analysis indicated that 1:1 stoichiometry is the most likely recognition mode of RBGlc-3 toward Hg2+ ion, and the corresponding OFF-ON fluorescent mechanism of RBGlc-3 is proposed. 4. Materials and methods All regents were commercially available and used without further purification, except THF was dried through sodium. 1H NMR and 13C NMR spectra were recorded on Brucker 600 MHz at 25 oC, TMS as the internal standard in CDCl3, and solvent peak as the internal reference in CD3OD. HRMS analysis was performed by Thermo Fisher Scientific LTQ FT Ultra. The UV-vis absorption and the fluorescence emission 4
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spectra were carried out by Shanghai Jing Hua 7600 UV visible spectrophotometer (dual beam) and Shimadzu RF-5301PC at room temperature, respectively. 4.1. Synthesis of compound 1 The synthesis of compound 1 was performed according to the literature27. The solution of rhodamine B (10 g), 2-aminoethanol (25 mL) and methanol (15 mL) was stirred at 75 oC until the red in the solution disappeared. H2O (200 mL) was added to the mixture. The resulting mixture was extracted with CH2Cl2, washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (petroleum ether:EtOAc 4:1) to afford compound 1, which showed the same spectroscopic properties as the literature27. 1H NMR (600 MHz, CDCl3) δ 7.92 (dd, J = 5.6, 3.0 Hz, 1H), 7.46 (dd, J = 5.6, 3.1 Hz, 2H), 7.09 (dd, J = 5.2, 3.2 Hz, 1H), 6.51 (d, J = 8.9 Hz, 2H), 6.40 (d, J = 2.4 Hz, 2H), 6.31 (dd, J = 8.9, 2.4 Hz, 2H), 3.51 – 3.47 (m, 2H), 3.36 (q, J = 7.0 Hz, 8H), 3.32 – 3.29 (m, 2H), 1.19 (t, J = 7.1 Hz,12H). 13C NMR (151 MHz, CDCl3) δ 170.10, 153.92, 153.28, 148.90, 132.69, 130.45, 128.51, 128.14, 123.81, 122.90, 108.24, 104.80, 97.80, 65.87, 62.68, 62.66, 44.65, 44.37, 12.61. 4.2. Synthesis of compound 2 Compound 2 was synthesized according to the reference28, as shown in the Scheme 3. NMR data were the same as the literature reported28. 1H NMR (600 MHz, CDCl3) δ 6.70 (s, 1H), 4.18 (d, J = 2.4 Hz, 6H), 3.87 (s, 6H), 3.82 (s, 2H), 2.47 (t, J = 2.3 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ165.39, 79.40, 74.81, 68.14, 59.76, 58.70, 29.47. 4.3. Synthesis of compound 3 A solution of NaH (0.291 g, 7.28 mmol, 60%) in anhydrous THF (6 mL) was cooled to 0 oC under argon. Compound 1 (0.9183 g, 1.88 mmol) in anhydrous THF (6 mL) was added dropwise. After stirred for 15 min, a solution of 2 in anhydrous THF (6 mL) was added dropwise. The reaction was allowed to warm up to room temperature and was stirred for overnight. The reaction mixture was poured into water (100 mL). The aqueous solution was extracted with CH2Cl2 (3×40 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, concentrated, and then purified by silica gel chromatography (petroleum ether:EtOAc 4:1) to give 3 an off-white solid (1.106 g, 62%). 1H NMR (600 MHz, CDCl3) δ 7.94 – 7.91 (m, 1H), 7.47 – 7.42 (m, 2H), 7.11 – 7.06 (m, 1H), 6.82 (s, 1H), 6.46 (d, J = 8.9 Hz, 2H), 6.39 (d, J = 2.3 Hz, 2H), 6.29 (dd, J = 8.9, 2.5 Hz, 2H), 4.11 (d, J = 2.4 Hz, 6H), 3.85 (s, 6H), 3.55 (s, 2H), 3.42 (t, J = 6.6 Hz, 2H), 3.35 (q, J = 7.1 Hz, 8H), 3.06 (t, J = 6.6 Hz, 2H), 2.40 (t, J = 2.3 Hz, 3H), 1.18 (t, J = 7.1 Hz, 12H). 13C NMR (151 MHz, CDCl3) δ 169.47, 168.35, 153.67, 153.20, 148.83, 132.41, 130.94, 128.88, 128.01, 123.76, 122.89, 108.13, 105.44, 97.69, 79.74, 74.54, 70.39, 68.27, 68.12, 64.62, 59.17, 58.58, 44.38, 39.06, 12.61. ESI-MS (m/z) 761.4. 4.4. Synthesis of compound 4 5
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Synthesis of compound 4 is routine, which was prepared according to the literature29,30. NMR data were the same as the literature reported. 1H NMR (600 MHz, CDCl3) δ 5.23 (t, J = 9.5 Hz, 1H), 5.12 (t, J = 9.8 Hz, 1H), 4.99 – 4.95 (m, 1H), 4.66 (d, J = 8.9 Hz, 1H), 4.29 (dd, J = 12.5, 4.8 Hz, 1H), 4.18 (dd, J = 12.5, 2.2 Hz, 1H), 3.81 (ddd, J = 10.1, 4.8, 2.3 Hz, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 170.60, 170.11, 169.30, 169.20, 87.91, 74.02, 72.60, 70.64, 67.89, 61.66, 20.69, 20.56, 20.54. 4.5. Synthesis of compound 5 To a solution of 3 (0.2052 g, 0.2697 mmol) and 4 (0.315 g, 0.8438 mmol) in THF (4 mL) was added a solution of sodium ascorbate (62.3 mg, 0.3145 mmol) and CuSO4·5H2O (40.2 mg, 0.161 mmol) in distilled H2O (4 mL). The reaction mixture was stirred for overnight at r.t. under argon. TCL indicated the reaction completion (petroleum ether: EtOAc 1:6). Deionized H2O was added and the aqueous solution was extracted with CH2Cl2 (3×15 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated to afford a crude solid residue, which was purified by silica gel chromatography (petroleum ether:EtOAc 1:4) to get 5 as a light red solid (0.4268 g, 84.2%).1H NMR (600 MHz, CDCl3) δ 8.01 (s, 3H), 7.93 (dd, J = 5.9, 2.6 Hz, 1H), 7.45 (dd, J = 5.3, 3.2 Hz, 2H), 7.10 (dd, J = 5.8, 2.5 Hz, 1H), 6.84 (s, 1H), 6.47 (t, J = 8.4 Hz, 2H), 6.34 (d, J = 60.0 Hz, 4H), 6.01 (d, J = 9.4 Hz, 3H), 5.61 (t, J = 9.5 Hz, 3H), 5.46 (t, J = 9.5 Hz, 3H), 5.36 (t, J = 9.8 Hz, 3H), 4.61 (dd, J = 13.3, 3.9 Hz, 6H), 4.32 (dd, J = 12.6, 4.8 Hz, 3H), 4.20 – 4.15 (m, 3H), 4.13 – 4.08 (m, 3H), 3.82 (d, J = 9.2 Hz, 3H), 3.75 (d, J = 9.2 Hz, 3H), 3.52 (q, J = 14.7 Hz, 2H), 3.46 – 3.39 (m, 2H), 3.34 (dd, J = 13.5, 6.5 Hz, 8H), 3.08 – 2.98 (m, 2H), 2.09 (s, 9H), 2.04 (s, 18H), 1.79 (s, 9H), 1.17 (t, J = 6.9 Hz, 12H). 13C NMR (151 MHz, CDCl3) δ 170.64, 170.10, 169.67, 169.47, 168.81, 168.48, 153.67, 153.19, 148.88, 145.65, 132.56, 130.84, 128.84, 128.13, 123.84, 122.86, 121.89, 120.93, 108.18, 97.69, 85.45, 74.92, 72.94, 70.32, 68.79, 68.25, 67.84, 64.70, 61.68, 59.63, 56.84, 44.38, 39.12, 21.81, 20.66, 20.61, 20.57, 20.06, 12.58. 4.6. Synthesis of RBGlc-3 To a solution of 5 (0.42 g, 0.2233 mmol) in dry MeOH (6 mL) was added sodium methoxide until pH 9-10. The reaction mixture was stirred for overnight at r.t. under argon. The mixture was then neutralized by addition of ion-exchange resin (Amberlite IR 120 H+) until pH 7, filtered and the solvent was evaporated to afford a crude solid residue. The residue was then purified by silica gel chromatography (CH3OH:CH2Cl2 10 - 50%) to give a light red solid RBGlc-3 (0.2725 g, 88.7%).1H NMR (600 MHz, MeOD) δ 8.16 (s, 3H), 7.92 – 7.89 (m, 1H), 7.52 (dd, J = 5.5, 3.1 Hz, 2H), 7.06 – 7.03 (m, 1H), 6.46 – 6.41 (m, 4H), 6.39 – 6.35 (m, 2H), 5.67 (d, J = 9.2 Hz, 3H), 4.60 (s, 1H), 4.53 (s, 6H), 3.98 (t, J = 9.1 Hz, 3H), 3.89 (d, J = 10.9 Hz, 3H), 3.74 (t, J = 8.8 Hz, 9H), 3.65 – 3.61 (m, 6H), 3.59 – 3.55 (m, 3H), 3.44 (s, 2H), 3.37 (dd, J = 13.6, 6.5 Hz, 10H), 3.01 (t, J = 5.7 Hz, 2H), 1.16 (t, J = 7.0 Hz, 12H). 13C NMR (151 MHz, CD3OD) δ 170.41, 169.08, 153.82, 153.30, 149.06, 144.45, 132.81, 130.50, 128.42, 128.22, 123.62, 123.13, 122.51, 108.24, 104.78, 97.63, 88.14, 79.68, 77.05, 72.63, 6
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69.61, 69.51, 67.89, 67.62, 65.32, 63.76, 61.03, 59.68, 44.02, 38.97, 11.58. HRMS calcd for C63H86N13O22: [M + H+] 1376.59, found 1376.6. 4.7. Spectroscopic measurements The stock solutions of 10 mM of HgCl2, Cu(NO3)2·3H2O, Pb(NO3)2, Eu(NO3)3·6H2O, Co(NO3)2·6H2O, Ce(NO3)3·6H2O, Cd(NO3)2·4H2O, AgNO3, NH4NO3, Zn(NO3)2·6H2O, Ni(NO3)2·6H2O, Ca(NO3)2·4H2O, NaNO3, KNO3, Mg(NO3)2, NH4NO3, FeSO4·7H2O, CH3COONa, Na2S2O4, Na2SO4, Na2SO3, NaCl and Na2S were prepared in pure water. The stock solution of RBGlc-3 (2 mM) was prepared in methanol. Ions selective spectrum of RBGlc-3 (10 M) solution was measured by adding 20 L of RBGlc-3 stock solution and 20 L metal ion into cuvette, and diluting to 4 mL with pure water. The changes of UV absorption and fluorescence spectra of RBGlc-3 were obtained by adding 2 L metal ion stock solution every time to the solution of RBGlc-3 (10 M) in 4 mL pure water. For fluorescence measurements, emission was acquired by scanning the spectra between 545 nm and 700 nm at 530 nm of excitation (excitation slit=3.0 nm, emission slit=3.0 nm). 4.8.Measurements of Job’s plot The stock solutions of 2 mM of HgCl2 and RBGlc-3 were prepared in pure water and methanol, respectively. 20, 18, 16, 14, 12, 10, 8, 6, 4 and 2 L of RBGlc-3 solution were put in fluorescent pools. 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 L of HgCl2 solution were then added to each fluorescent pool containing RBGlc-3 solution. Each fluorescent pool was then diluted with pure water to 4 mL. After stirring for a period of time, the spectra were scanned between 545 nm and 700 nm at 530 nm of excitation (excitation slit=3.0 nm, emission slit=3.0 nm). Acknowledgments This work is supported by the National Natural Science Foundation of China (No. 21262004), the Project of Guangxi Natural Science Foundation (No. 2013GXNSFBA019152), the high level innovation team and outstanding scholar project of Guangxi institutions of higher education (guijiaoren [2014] 49 hao), and State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (No. CMEMR2013-B03). Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/. These data of the most important compounds described in this article.
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Fig. 1 The influence of water-solubility of RBGlc-3 on fluorescence. (a) RBGlc-3 was completely dissolved in pure water and compound 1 was not dissolved in pure water; (b) Addition of Hg2+ ion, the RBGlc-3 solution emitted fluorescence and the compound 1 solution hardly showed fluorescence; (c) Addition of methanol, the compound 1 solution also emitted fluorescence.
Fig. 2 Fluorescence spectra of RBGlc-3 (10 μM) in pure water at 583 nm (ex = 530nm). (a) Addition of 5.0 equiv cations; (b) Addition of 5.0 equiv anions.
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Fig. 3 The influence of Hg2+ concentration on spectra of RBGlc-3 (10 μM) i pure water. (a) Fluorescence change with 0-5 equiv Hg2+ ion; (b) Absorption change with 0-5 equiv Hg2+ ion.
Fig. 4 Fluorescence spectra of RBGlc-3 (10 μM) i pure w ter with 5.0 equiv of H and followed by addition of 5.0 equiv. of S2- (ex = 530nm).
2+
Fig. 5 Fluorescence response of RBGlc-3 (10 μM) i pure w ter to v rious c tio s and those with additional Hg2+. 1, Fe2+; 2, NH4+; 3, Ba2+; 4, Ca2+; 5, Cd2+; 6, Co2+; 7, K+; 8, Mg2+; 9, Na+; 10, Ni2+;11, Pb2+; 12, Ce3+; 13, Cu2+; 14, Zn2+; 15, Ag+; 16, Eu3+.
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Fig. 6 Job’s plot for RBGlc-3 and Hg2+complexation (the total concentration of RBGlc-3 and Hg2+ was 10 M).
Fig. 7 HRMS spectra of [RBGlc-3 + Hg2+].
Fig. 8 1H NMR spectra of RBGlc-3 and [RBGlc-3 + Hg2+] in CD3OD.
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Scheme 1 Synthetic route of RBGlc-3. Reagents and conditions: (a) 2-aminoethanol, methanol, 75 oC; (b) NaH(60%), anhydrous THF, 0o C to r.t., overnight, 62%; (c) CuSO4·5H2O, sodium ascorbate, THF/H2O (1:1), r.t., overnight, 84.2%; (d) CH3ONa, CH3OH, r.t., overnight, 88.7%.
Scheme 2 The proposed OFF-ON fluorescent mechanism of RBGlc-3.
Scheme 3 The synthesis of compound 2.
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