A Novel Fluorescence “Turn-Off” Sensor Base On A Triazole-Linked BINOL-Glucose Derivative For The Sensitive Detection of Copper Ion

A novel sensitive chiral uorescent “turn-off” sensor based on 3,3′-positions modied triazole-linked BINOL-Glucose derivative has been synthesized via “click” reaction. The uorescence emission intensity of (S, β-D)-1 was almost completely quenched along with obvious color change from yellow to green upon the coordination with a Cu(II) ion while other metal ions had no obvious change. The detection limit of the sensor (S, β-D)-1 toward copper ion was calculated to be 0.31 μmol L -1 . The stoichiometry ratio of (S, β-D)-1 - Cu 2+ complex was proved to be 1:1 by the analysis of NMR spectroscopic, ESI-MS data and the job’s plot. HNMR spectroscopic and IR were also used to study the mechanism, demonstrated copper ion was coordinated with (S, β-D)-1 by 1+1complex formation.


Introduction
Followed by zinc and iron, as the third most abundant essential microelement in human body, Cu 2+ also plays vital roles in the human body and plant tissues during the fundamental physiological processes of organisms [1][2][3][4][5][6][7] . However, at higher concentration levels, copper produces toxicity and could result in a wide variety of potential health risks 8-16 , such as Hashimoto's disease, pelvic in ammatory disease (PID), Alzheimer's, neurodegenerative disorders, stomach cramps, Wilson's diseases, brocystic breast disease and prion diseases. Furthermore, the development of modern industry and agriculture has caused serious transition metals pollutions, such as Cu 2+ , have resulted in severe environmental pollution and destructed natural ecosystems, particularly the serious drinking water pollution. [17][18][19][20] Hence, it is highly meaningful and still a challenging to protect human health and environment and nd or design a new method to monitor and selective detect Cu 2+ .
A large amount of analytical methods, for example, electrochemical method, atomic absorption spectroscopy (AAS), ICP-MS and colorimetric method had been applied to detect Cu 2+ , in consideration of the importance of copper ion. 3,19−23 But these analytical methods have not been achieved applications for online monitoring of Cu 2+ feasibly and effectively owing to their expensive instruments, wellcontrolled experimental conditions, tedious time-consuming sample preparation and delayed responses.
Compared to other classical techniques, uorescent probes have attracted enormous attention because of theirs rapid response, real-time analysis, facile operation and high selectivity and sensitivity. [21][22][23][24] The uorescence method with innate high sensitivity has attracted increasing attention to investigate molecule-based uorescent sensors, which could offer multiple signaling modes, for example enhancement, quenching, lifetime, anisotropy, and excimer/ exciplex formation of substrate analysis. Fluorescent sensors applied or tagged to detect anions, metal cations, neutral molecules, protons and chiral organic compounds have received detailed investigated. At present, monitoring of copper ion was focused on the design and synthesis of uorescent probes with high sensitivity and selectivity. Over the past few years, different uorescent sensors based on rhodamine 25,26 , naphthalimides 27 , coumarin 28,29 , Bodipy 30,31 and qulioline 32 , using the detection of Cu 2+ have been reported. Most of the probes were interfered by other transition metals, especially Hg 2+ in the discrimination of Cu 2+ with the emission quenching intensity 33 . However, it still remains rather challenging to design effective speci c uorescent sensors for copper ion with high sensitivity and selectivity.
As a uorescent chemical sensor, rapid identi cation of metal ions and chiral isomers with high selectivity and sensitivity are the remarkable advantage of chiral BINOL derivatives. As the versatile backbone of BINOL, it can be easily modi ed from space effects and electronic effects, it and its derivatives [34][35][36][37][38] have caused considerable focus on the eld of uorescent chemical sensors [39][40][41][42][43][44][45][46][47][48][49] and asymmetric catalysis. Chiral BINOL derivatives have the distinct characteristics: 50-54 easily modi ed by functional groups especially tuned to the 2-, 3-, 4-, 5-and 6-positions of the chiral optical pure BINOL and commercially available with both enantioselective enantiomers, (R)-and (S)BINOL. Moreover, glucose has attracted more attention because of its good biocompatibility, variety structural modi cation, natural existence and without toxic during the recent years. Furthermore, the best water solubility of glucose was the most ideal advantage as composing uorescent chemical sensors. In the past few years, 1,2,3-triazole modi ed sugar derivatives have attracted continuous focus on the chemistry research 55-57 and sugarbased uorescent chemical sensor 58-65 . We have investigated the application of a BINOL-Glucose derivative 66 uorescent sensor to discriminate Ag + companied with high enantioselectivity and sensitivity without interference of Hg 2+ . Based on the 3,3′-positions structural modi cation versatile backbone of (S)-BINOL and considered (S)-BINOL and two 1,2,3-triazole units as uorophore and recognition group, respectively. Detailed investigation on the application of the Glucose modi ed triazole-based sensor in the molecular recognition of metal ions was processed in this study. At the same time, 3,3′-position modi ed triazole-linked 1,1′-bi-2-naphthol (BINOL) derivative displayed highly uorescent quenching by the combination with Cu 2+ in the presence of large amounts of competing ions as we expected.

Fluorescence response to Cu 2+
The uorescence response of (S, β-D)-1(20 µM) in acetonitrile solution upon irradiation with UV/vis lights were also investigated by a uorescence spectroscopy. As shown in Fig. 1, (S, β-D)-1 displayed moderate uorescence at 376 nm with the excitation light at 287 nm and 333 nm. The absolute uorescence quantum yield of (S, β-D)-1 was valued to be 0.032. The uorescence responses to various metal ions including Zn 2+ , Co 2+ , K + , Ag + , Ba 2+ , Mg 2+ , Ca 2+ , Cr 3+ , Ni 2+ , Cd 2+ , Cu 2+ , Al 3+ , Hg 2+ , Mn 2+ , Sr 2+ and Pb 2+ in acetonitrile solution were recorded by the same uorescence spectroscopy. The uorescence measurement of (S, β-D)-1 was performed in CH 3 CN ([(S, β-D)-1] = 20 µM). As shown in Fig. 1, it was clearly observed that none of other metal ions but Cu 2+ exhibited obvious quenching uorescence emission intensity under excited at 287 nm with the color changed from pale yellow to green. Upon the addition of copper, the characteristic color change of the detected solution implied the detection of copper ions over naked eye was feasible. The uorescence emission intensity of (S, β-D)-1 at λ = 376 nm (λex = 287 nm) was almost entirely quenched by the coordination with cooper ion to form 1 + 1 complex. The uorescence intensity remained no variation when other different metal ions were added. All of these results were meant that (S, β-D)-1 could be potentially act as a Cu 2+ uorescence sensor in acetonitrile with high selectivity and sensitivity. The quenching uorescence intensity upon the addition of Cu 2+ ion to (S, β-D)-1 may be attributed to the intramolecular proton transfer through six-member ring transition state from phenolic hydroxyl groups to the adjacent OCH 2 group in excited-state and owing to metal ion chelation which probably due to the photo-induced electron transfer (PET effect). It was indicated that two nitrogen atom of 1,2,3-triazole units on (S, β-D)-1 and the oxygen atom of BINOL offered the binding site to metal ion.
To further con rm the high selectivity of uorescent sensor (S, β-D)-1 for discrimination Cu 2+ , the competition experiments were carried out as shown in Fig. 2. The uorescent emission intensities were measured at 376 nm by the treatment of the mixture of 5.0 equiv other different metal ions mixed with same equiv. Cu 2+ to the sensor (20 µM in CH 3 CN), respectively. It was observed that the uorescence intensity of (S, β-D)-1-Cu 2+ have scarce interference with other completive metal ions. Those meant the chiral sensor (S, β-D)-1 could be applied as a speci c Cu 2+ sensor with the tested background competitive ions.

The complexation mechanism of (S, β-D)-1 and Cu 2+
The dose-dependent uorescence response of (S, β-D)-1 to Cu 2+ and the uorescence change induced by Cu 2+ /EDTA were also measured at room temperature, as depicted in Fig. 3. According to the titration, the emission intensity at 376 nm decline gradually during the concentration of copper ion enlarged from 0 to 4.0 equiv, and it reach a low point upon the concentration of 4 equiv. It stated a good linear relationship between the maximal uorescence intensity and the concentration of Cu 2+ . To demonstrate the coordination process between (S, β-D)-1 and Cu 2+ was reversible or not, the chelating agent EDTA was added. The original uorescence intensity did not lead back with the subsequent addition of an excess of EDTA, revealing that it was irreversible by the coordination of (S, β-D)-1 with Cu 2+ .
In order to con rm the binding a nity among modi ed triazole-linked BINOL-Glucose sensor towards Cu 2+ was calculated to be 3.14 × 10 − 7 mol L − 1 based on "LOD = 3σ / s" by the concentration-dependent uorescence titration experiment (Fig. 4C).
Another evidence to determine the complex formed by 1:1 stoichiometry ratio according to ESI-MS spectra data (Fig. 5). A signi cant molecular ion peak of free (S, β-D)-1 was obtained at m/z = 1191. To further investigate the complex mechanism between the sensor and Cu 2+ , 1 HNMR titration experiments were performed in CD 3 CN as illustrated in Fig. 6. to nd out the further detailed binding information between Cu 2+ with (S, β-D)-1. Upon different equivalents of Cu 2+ (from 0 to 1 equiv.) was added, an obvious downshift of chemical shifts was observed until the amount of Cu 2+ was over 1 equiv.
The proton H d at 7.06 ppm assigned to phenolic hydroxyl disappeared totally when Cu 2+ was added to the solution of the sensor, indicating that the oxygen atom in phenolic hydroxyl was connected with the coordination between Cu 2+ and (S, β-D)-1. But proton H a of the 1,2,3-triazole rings exhibited a remarkable downshift Δδ = 0.11 ppm from 8.07 ppm to 8.18 ppm, indicating copper ion were bound to the nitrogen atoms of the 1,2,3-triazole group. The nuclear magnetic peaks H c, -OCH 2 -linking 1,2,3-triazole groups were represented weak up eld shifts from 4.80 ppm to 4.76 ppm. The results demonstrated that Cu 2+ was selectively coordinated with -OCH 2 -and 1,2,3-triazole rings. Tetrahedron complex may be formed by copper ions as the center. The consequences provided by mass, uorescence titration and NMR spectroscopic analyses were all illustrated the 1 + 1 binding model was formed between the BINOLtraizole-glucose compound and copper ion.
The IR spectrum was also used to identify the binding site as illustrated in Fig. 7. Phenolic hydroxyl exhibited a strong single absorption band at 1227 cm − 1 , according to the weak hydrogen-bonding interaction with the adjacent OCH 2 group. The absorption bands of phenolic hydroxyl in the 1:1 complex of Cu 2+ was weaken sharply, indicating that oxygen of phenolic hydroxyl was coordinated with Cu 2+ .

Conclusions
A novel Cu 2+ -selective uorescence sensor was designed and synthesized by click reaction of glucose azide and BINOL chromophore with high sensitivity and selectivity with scarce interference of other completive metal ions. Cu 2+ exhibited obvious quenching uorescence emission intensity under excited at 287 nm with the color changed from pale yellow to green. The new BINOL-Glucose derivative show a 1:1 stoichiometry with high binding constants and a low detection limit. The Cu 2+ -induced uorescence quenching may be attributed to the intramolecular proton transfer through six-member ring transition state from phenolic hydroxyl groups to the adjacent OCH 2 group in excited-state and a PET effect of the copper 1 + 1 complex.

General Reagents and apparatus
All the analytical grade solvents were distilled before used. Materials were supplied by reagent suppliers or provided by our laboratory's synthesis through the known routes and no further puri cation before used. If no otherwise speci ed, the reagents applied in the chiral synthesis were optical purity. The corresponding metallic nitrates were used to prepare the various metal ions ( was applied to measure the uorescence quantum yield. ESI-MS spectral data were recorded with a Bruker amazon SL Ion Trap Mass spectrometer. AWRS-1B melting point apparatus was used to measure melting points. A Rudolph AUTOPOL IV automatic polarimeter was used to measure the optical rotation.

Contributions
Xiaoxia Sun and Yu Hu designed the methodology for the research experiment and wrote the paper, analyzed most of the data and wrote the paper; Huizhen Wang and Yang Liu performed the research and carried out additional analyses.
Con icts of Interest. The authors decare no con ict of interest.