New ferrocene–triazole derivatives for multisignaling detection of Cu2+ in aqueous medium and their antibacterial activity

Journal Pre-proof New ferrocene–triazole derivatives for multisignaling detection of Cu2+ in aqueous medium and their antibacterial activity

Jianwei Xu, Yongqiang Yang, Huricha Baigude, Haiying Zhao PII:

S1386-1425(19)31270-3

DOI:

https://doi.org/10.1016/j.saa.2019.117880

Reference:

SAA 117880

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

15 September 2019

Revised date:

13 November 2019

Accepted date:

29 November 2019

Please cite this article as: J. Xu, Y. Yang, H. Baigude, et al., New ferrocene–triazole derivatives for multisignaling detection of Cu2+ in aqueous medium and their antibacterial activity, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2018), https://doi.org/10.1016/j.saa.2019.117880

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© 2018 Published by Elsevier.

Journal Pre-proof New ferrocene−triazole derivatives for multisignaling detection of Cu2+ in aqueous medium and their antibacterial activity Jianwei Xua, YongqiangYanga, Huricha Baigudea, Haiying Zhaoa, b, * a College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot 010021, PR China b Inner Mongolia Key Laboratory of Fine Organic Synthesis, Hohhot 010021, PR China *Corresponding author. Fax: +86 471 4995123. E-mail address: [email protected]

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ABSTRACT Ferrocene–based naphthalene or quinoline receptors 1-4 linked by triazole were designed and synthesized. Their recognition properties of metal cations have been investigated systematically in aqueous environment. Upon addition 1 equiv of Cu2+ ion, receptors 1 (C23H19FeN3O) and 2 (C22H18FeN4O) showed fluorescent turn-off, enhanced absorption and colour variations. At the same time, receptors 1 also caused the perturbation of redox potential after addition 1 equiv of Cu2+ ion. Therefore, receptors 1 and 2 behaved as naked-eye chemosensors and fluorescent probes for Cu2+ without interference by other ions and with low detection limit. In addition, receptor 1 could also be considered electrochemical sensor for Cu2+ having excellent sensitivity and selectivity. However, increasing the molecules flexibility resulted in the lower selectivity of ion recognition in the case of receptors 3 (C24H21FeN3O) and 4 (C23H20FeN4O). Furthermore, this series of compounds were nontoxicity and receptor 1 exhibited certain antibacterial activity.

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Keywords: Ferrocene; Triazole; Multisignaling sensor; Cu2+ ion; Biological activity

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1. Introduction Construction and development of multichannel chemical sensors for detecting toxic heavy metal ions is a significant research area because heavy metal ions are applied to many fields and have a serious impact on the human health and environment [1-4]. Among the various metal cations, Cu2+ ion is regarded as a very harmful environmental pollutant [5]. On the other side, Cu2+ is also a kind of indispensable nutrient for aquatic media and animals which participates in the functioning of various enzymes and blood formation [6-8]. However, excessive copper intake can lead to some diseases, for instance, renal liver damage, epileptic seizures and Parkinson's disease [9,10]. Hence, the research of chemosensors for rapid and sensitive monitoring of trace Cu2+ in the environment has aroused great attention during the past few years [11,12]. Fluorescence emission and UV−visible spectroscopy studies are usually used to detect toxic metal ions. For sake of improve the performance of chemical sensors, some researchers attempt to insert a redox center near the ion binding site in the sensor [13]. In this sense, ferrocene stands out among many redox active entities because of its strong electron donation and reversible one-electron redox properties [14]. Various chemosensors with ferrocene moieties for cations detection have been designed and applied, which induces a significant positive movement of the redox potential of ferrocene (Fc/Fc+) when guest cations are introduced to their solution [15]. Ferrocene−triazole derivatives with a photoactive chromophore or fluorophore are one of the systems that have proved to be versatile chemosensors specially for transition-metal cations, like Pb2+, Ni2+, and Hg2+, etc [16-23], while they were hardly used for recognition of Cu2+ ion [24,25]. Therefore, it is a considerable challenge to construction triazolylferrocene-based multisignaling receptors for the monition of Cu2+ in supramolecular chemistry. On the other hand, though promising results have been achieved in the field of chemosensors for anions and cations in recent years, few people pay attention to the toxicity of sensors themselves. Ferrocene is

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non-toxicity, neutral nature, and chemical stability under physiological conditions, but in some chemosensors, the photoactive chromophores linked to ferrocene, such as pyrene [26,27], benzothiazole [28], carbazole [29], are toxic. If the sensor itself is toxic, using it to recognize ions can easily cause secondary pollution, which is not the result we expected. In fact, some ferrocene derivatives have enhanced biological activity as compare with other organic molecules [30] due to their biocompatibility, lipophilicity as well as redox properties [31-33], which have already been shown to be successful novel antitumour, antiparasitic, antibacterial, and antimalarial alternatives [34-38]. Therefore, it is more valuable to design a non-toxic multi-signal sensor with biological active. Herein, we report new ferrocene–based naphthalene or quinoline receptors linked by 1,2,3-triazole (Scheme 1). The fluorescence and electrochemical properties of receptors 1 and 2 showed significant changes in aqueous environments after complexing with metal ions, and they can be applied for the visible chemosensors of Cu2+. Furthermore, the cytotoxicity and antibacterial activity of these receptors were evaluated.

Scheme 1. Synthesis of receptors 1-4.

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2. Experimental section 2.1. Instrumentation and materials Ferrocenyl azide 1 [39] and azidomethylferrocene 2 [40] were synthesized according to the literatures. HRMS spectra were obtained using a Bruker QTOF mass spectrometer. Bruker-ALPHA spectrometer (KBr pellets) and Advance 500 Bruker spectrometer were used to measure FTIR and NMR spectra, respectively. Edinburgh FLS920 spectrophotometer and Shimadzu UV2600 spectrophotometer were used to measure fluorescence and UV-visible (UV-vis), respectively. A Metrohm AUTOLAB PGSTAT302 analyzer was used for electrochemical studies. A Bruker APEX II CCD diffractometer (λ = 0.71073 Å) was used for X-ray diffraction measurement of single crystal 2 at room temperature. Crystal data and structure refinement parameters are placed in Table S1. Selected bond distances and angles are listed in Table S2. 2.2. Absorption and emission studies Stock solutions of receptors 1-4 (1 × 10−5 M, 10 μM) and metal ions (salts of perchlorate, 2 × 10-2 M) were prepared in H2O/CH3CN (V/V, 1:4) and distilled water, respectively. In titration experiments, the cation stock solution was gradually dripped into 2 mL solution of receptor (5 μM) with a micro-liter syringe. In selective experiments, 1 μL (10 μM) stock solution of cation was added into a 2 mL solution of receptor (10 μM) for each solution to be tested. 2.3. Electrochemical studies Three-electrode method was used in cyclic voltammetry (CV) and differential pulse voltammetry (DPV) spectra response experiments of receptors to metal ions, including a working electrode of glassy carbon, a platinum counter electrode, and an reference electrode of Ag/AgCl couple (immersion in KCl saturated

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solution). The response experiments of receptor to metal ion were performed at room temperature in CH3CN/H2O (4:1, V/V, 1 × 10-4 M) solutions at a scan rate of 0.1 V s-1. The dissolved oxygen in the solution was removed by bubbling nitrogen before the experiment. The supporting electrolyte was n-Bu4N[PF6] (0.1 M). 2.4. Antibacterial test Escherichia coli (E. coli, ATCC 8099, Gram-negative bacteria) was selected as representative bacteria for the assessment of antibacterial activity of synthetic compounds. Before the experiment, E. coli was incubated in a broth medium on Luria−Bertani (LB) substrate at 37 °C for 24 h, and the glassware and samples need to be sterilized. Into a 1.5 mL centrifuge tube was placed 100 μL of E. coli suspension (1×104 CFU mL−1) and 900 μL sample suspension (1 mg/mL in distilled water). After incubating by shaking (200 rpm) in a rotary shaker for 2 h, the antibacterial action was terminated by introducing Na2S2O3 aqueous solution (900 μL of 0.3 wt%). 1 mL of dilute suspension was then distributed evenly on LB substrate and incubated at 37 ◦C. The viable colonies on the plate were recorded 12 hours later. Use the following formula to calculate bacterial reduction [41]: Bacterial reduction (%) = ((N − M) / N) × 100% Where M and N are the number of viable colonies of the treated and blank samples, respectively. 2.5. Cell culture and cytotoxicity measurement The cytotoxicity of the synthetic compounds was evaluated using PC12 cells as test cells. The medium consisted of DMEM/F12 containing 10% FBS, 1% streptomycin and 1% penicillin. On 96-well plate, 100 μL cell suspension with density of 1.0 × 104 was planted in each well and cultured at 37 ◦C for 24 h in 5% CO2 atmosphere, then dimethyl sulfoxide (DMSO) solutions of synthetic compound (5 μL) with a concentration of 1, 10, 20 μM were put into per well. All conditions were performed in triplicate. DMSO alone was also included as a solvent toxicity control. 24 h later, MTT method was used to detect cell viability by recording the absorption spectra at 450 nm. Cell viability was calculated by Eq. [42] Cellviability (%) = (B(testcells) /B(controlcells)) × 100% 2.6. Synthesis of receptors 1 - 4 Triazolylferrocene derivative (1.0 mmol), 1-(prop-2-yn-1-yloxy)naphthalene [43] or 8-(prop-2-yn-1-yloxy)quinoline [44] (1.0 mmol) and tetrahydrofuran (THF, 20 mL) were placed into a flask and stirred to dissolve, then the aqueous solution (8 mL) of sodium ascorbate (4.0 mmol, 0.78 g) as well as aqueous solution (6 mL) of CuSO4•5H2O (0.32 g, 2.0 mmol) were slowly added at ambient temperature, respectively. After stirring overnight, the substrate was treated with NH4OH (10 mL), then extracted using dichloromethane two times. The combined organic phases were washed with brine and water, dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by column chromatography over silica gel eluted with CH2Cl2/EtOAc (4:1, V:V). Receptor 1, m.p. 179-183 ºC, yield 85%, Rf = 0.21 (EtOAc / CH2Cl2 1:4, V:V); 1H NMR (CDCl3, 500 MHz) (δ ppm): 8.28 (s, 1H, napthol), 7.82 (d, 2H, J = 7.0 Hz, napthol), 7.49 (d, 2H, J = 6.0 Hz, napthol), 7.47 (s, 1H, triazole), 7.40 (t, 1H, J = 6.6 Hz, napthol), 6.99 (d, 1H, J = 6.5 Hz, napthol), 5.45 (s, 2H, CH2), 4.98 (s, 2H, Cp), 4.41 (s, 2H, Cp), 4.34 (s, 5H, Cp); 13C NMR (CDCl3, 125 MHz) (δ ppm): 153.97, 144.31, 134.61, 127.58, 126.51, 125.85, 125.74, 125.35, 122.34, 121.99, 121.00, 105.68, 93.69, 70.20, 66.74, 62.22; FTIR (KBr, ν, cm-1) 3090, 2922, 1628, 1459, 1096, 1054, 765, 496; ESI-TOF HRMS, m/z: [M]+ for C23H19FeN3O: Calcd 409.0872, found 409.0867. Receptor 2, m.p. 164-167 °C, yield 83%, Rf = 0.26 (EtOAc / CH2Cl2, 1:4, V:V); 1H NMR (CDCl3, 500 MHz) (δ ppm): 9.03 (s, 1H, quinoline), 8.23 (m, 2H, quinoline), 7.52 (s, 2H, quinoline, triazole), 7.45 (d, 2H, J = 8.1 Hz, quinoline), 7.38 (d, 1H, J = 6.2 Hz, quinoline), 5.64 (s, 2H, OCH2), 4.84 (s, 2H, Cp), 4.24 (s, 2H, Cp), 4.17 (s, 5H, Cp); 13C NMR (CDCl3, 125 MHz) (δ ppm): 149.48, 144.05, 136.55, 133.61, 129.57, 126.99,

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121.75, 120.72, 120.36, 110.51, 99.99, 93.70, 70.34, 70.21, 62.38; FTIR (KBr, ν, cm-1) 3074, 2922, 1566, 1459, 1102, 1054, 807, 501; ESI-TOF HRMS, m/z: [M+Na]+ for C22H18FeN4NaO: Calcd 433.0722, found 433.0710. Receptor 3, m.p. 49-51°C, yield 87%, Rf = 0.15 (EtOAc / CH2Cl2,1:2, V:V); 1H NMR (CDCl3, 500 MHz) (δ ppm): 8.21 (d, 1H, J = 8.2 Hz, napthol), 7.78 (d, 1H, J = 8.1 Hz, napthol), 7.58 (s, 1H, triazole), 7.49-7.43 (m, 3H, napthol), 7.35 (t, 3H, J = 7.9 Hz, napthol), 6.95 (d, 1H, J = 7.6 Hz, napthol), 5.36 (s, 2H, OCH2), 5.31 (s, 2H, CH2), 4.27 (s, 2H, Cp), 4.22 (s, 2H, Cp), 4.17 (s, 5H, Cp); 13C NMR (CDCl3, 125 MHz) (δ ppm): 154.27, 144.46, 134.82, 127.79, 126.75, 126.13, 125.93, 125.55, 122.30, 121.12, 105.75, 81.14, 69.41, 66.24, 62.78, 50.46; FTIR (KBr, ν, cm-1) 3085, 2920, 1557, 1458, 1097, 1047, 768, 486; ESI-TOF HRMS, m/z: [M]+ for C24H21FeN3O: Calcd 423.1029, found 423.1017. Receptor 4, m.p. 46-49°C, yield 81%, Rf = 0.18 (EtOAc / CH2Cl2, 1:4, V:V); 1H NMR (CDCl3, 500 MHz) (δ ppm): 8.92 (d, 1H, J = 2.7 Hz, quinoline), 8.11 (d, 1H, J = 8.1 Hz, quinoline), 7.68 (s, 1H, triazole), 7.38-7.42 (m, 3H, quinoline), 7.32 (d, 1H, J = 7.4 Hz, quinoline), 5.52 (s, 2H, OCH2), 5.26 (s, 2H, CH2), 4.23 (s, 2H, Cp) 4.18 (s, 2H, Cp), 4.13 (s, 5H, Cp); 13C NMR (CDCl3, 125 MHz) (δ ppm): 154.12, 149.60, 144.17, 140.52, 136.38, 29.78, 127.09, 123.07, 121.93, 120.48, 110.34, 80.91, 69.37, 69.25, 63.22, 50.44; FTIR (KBr, ν, cm-1) 3089, 2922, 1578, 1456, 1099, 1045, 768, 510; ESI-TOF HRMS, m/z: [M+H]+ for C23H21FeN4O: found 425.1055, Calcd 425.1059. 3. Results and discussion 3.1. Synthesis Ferrocene derivatives 1–4 (Scheme 1) were synthesized by the "click reaction" of azido-ferrocene derivatives and ethynyl appended naphthalene or quinoline in H2O/THF at ambient temperature. The yields of compounds 1-4 were 80-85%. Their structures were characterized by the common analytical techniques (FTIR, NMR, HRMS). The HRMS spectra displayed [M]+ or [M + H]+ or [M + Na]+ signals for compounds 1-4, which are consistent with the calculated value. Furthermore, X-ray diffraction analysis also established the composition of compound 2 (CCDC 1911579). Fig. 1 shows the molecular structure of 2. As shown in Table S2, the band length of N(1)–N(2) (1.345(2)) was longer than that of N(2)–N(3) (1.311(2)) indicating double-bond feature of the later. The substituted Cp and quinoline ring deviated 21.838˚ and 79.286˚ from 1,2,3-triazole plane, respectively.

Fig. 1. Crystal structure of receptor 2. 3.2. UV–vis spectra studies Receptors 1-4 displayed good UV–vis absorption properties which could be used for assessment their recognizing behaviors to metal ion. The peaks at the range of 200 - 250 nm for the receptors 1-4 are due to electronic transitions of π-π* in quinoline or naphthalene rings (E1 band) and Fe(d)-π* of ferrocene, and the peaks at 250 - 350 nm attributed to π-π* transitions (E2 band) is likely to superimpose with the B band of aromatic rings [45]. To assess the recognizing properties of these receptors to metal ion, we first investigated the absorption response behaviors of compounds 1 and 2 in CH3CN/H2O (4:1, V/V, 10 μM) to various ions such as Pb2+, Mn2+, Mg2+, Ca2+, Cu2+, Fe2+, Cd2+, Zn2+, Ni2+, Hg2+, Co2+, K+, Li+, Ag+ and Na+ ions (Fig. 2). The absorbance spectra of Cu2+ free solution was also measured as a control sample. The structural difference

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between compounds 1 and 2 is that compound 1 contains a naphthalene chromophore, whereas compound 2 contains a quinoline chromophore. Interestingly, compounds 1 and 2 exhibited similar selectivity in metal ions recognition though their structures were different. That is, the absorption spectra of the solutions 1 or 2 showed significant changes in the intensity in the presence of Cu2+ ion (1 equiv) and those only displayed slight changes when other monovalent and divalent metal ions were added separately (Fig. 2a and 2b). Then receptors 1 and 2 were titrated with Cu2+ and detected by absorption spectra. As shown in Fig. 3, the absorption bands centred at 209 nm (116800 M−1 cm−1) for 1 and at 202 nm (86600 M−1 cm−1) for 2 increased in the intensity accompanied by slight bathochromic shifts to 211 nm (169400 M−1 cm−1) and 205 nm (171400 M−1 cm−1) respectively when Cu2+ ions were dripped gradually into the solution of receptors 1 or 2 up to 1 equiv. In addition, the bathochromic shift of the absorption band at about 278 nm (20600 M−1 cm−1) for 1 was also observed. However, the copper salt free with the same concentration as the ligand only displayed very weak absorption at the range of 200 - 250 nm and no absorption at other bands. Thus it can be seen that the changes in the absorption spectra of receptors is due to the interaction between Cu2+ ion and the receptors, not just only the result of a linear combination of their spectra. Furthermore, a new notable increase band and a weak absorption band centred at 306 nm and 464 nm respectively appeared with gradual addition of Cu2+ ions to the solution 2 up to 20 equiv. (Fig. 3b) which might be attribute to the band of ligand-metal charge transfer (LMCT) mediated by quinoline [28]. According to this was the color variation of receptors 1 and 2 from yellow to green after adding 2 equiv of Cu2+ ion, as shown in Fig. 4 and Fig. S1. Because the solution of Cu2+ ion was almost colorless at the concentration of 200 μM, so the color change was caused by the interaction of ligand and ion, which could be applied for the “naked-eye” recognition of Cu2+. From the UV−vis titration profile, the binding constant values of 1 and 2 with Cu2+ can be determined using the well-known Benesi−Hilderbrand (BH) equation [46]. As shown in Supplementary Fig. S2, the binding constant (K) determined from the increasing absorption intensity for 1-Cu2+ at 211 nm and 2-Cu2+ at 238 nm were 6.8 × 105 M−1 and 5.97 × 104 M−1, respectively [13]. In addition, we deduced that 1 and 2 with Cu2+ formed 1:1 and 2:1 stoichiometry respectively from Job’s plots shown in the insert figure of Fig. 3a and 3b. The lowest detectable limit of 1-Cu2+ and 2-Cu2+ (Supplementary Fig. S3) were also calculated and reached 2.5 × 10−7 M and 1.71 × 10−6 M, respectively.

Fig. 2. UV–vis spectra changes of chemosensors 1 (a) and 2 (b) in the presence of various metal ions (1 equiv, 10 μM). Next, we examine the effect of rigid and flexibility of molecules on the ability of ion recognition. Receptors 3 and 4, inserted a methylene between ferrocene and triazole, were synthesized and used as new chemosensors in the recognition experiment. As noted from Fig. S4, No obvious changes appeared in the absorption spectra of 3 and 4 when 1 equiv of other competitive ions including Ag+, K+, Na+, Li+, Cd2+, Mn2+, Ni2+, Hg2+, Co2+, Ca2+, Zn2+, Mg2+ was added separately. Though the absorption intensity of receptors 3 and 4

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increased notably with introduction of 1 equiv of Cu2+, both Pb2+ and Fe2+ also induced obvious changes of absorption bands. So receptors 3 and 4 have lower selectivity for the metal ions recognition compared with receptors 1 and 2. The possible reason is that flexible molecules can adjust their structure easily by rotation, so they can complex more metal ions.

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Fig. 3. Changes of UV–vis spectra for 1 (a) and 2 (b) in CH3CN/H2O (4:1, V/V, 5 μM) upon titration with Cu2+. Insert shows the Job's continuous variation plots.

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Fig. 4. Visual color variations of solution 1 in CH3CN/H2O (4:1, V/V, 100 μM) on the addition of various metal ions (2 equiv).

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3.3. Fluorescence spectroscopic studies Fluorescence spectrum was also applied for the investigation of binding ability of Cu2+ with the receptors. Because these compounds show the strongest excitation at 366 nm in the excitation spectra, 366 nm is chosen as the excitation wavelength in the fluorescence spectroscopic experiments. As presented in Fig. 5a and Fig. S5, receptors 1 and 2 (10 μM) show an emission band at 403 nm (quantam yield Φ = 0.033) and 407 nm (Φ = 0.036) nm respectively in CH3CN/H2O (4:1, V/V) when excited at λex = 366 nm. Upon the separate addition 1 equiv of Ag+, Na+, Li+, K+, Ca2+, Mn2+, Mg2+, Fe2+, Ni2+, Zn2+, Pb2+, Co2+, Hg2+ and Cd2+ ions, both 1 and 2 exhibited a negligible fluorescence response. However, introduction the same amount of Cu2+ ion induced a significant fluorescence decrease (1: Φ = 0.014, 2: Φ = 0.011) indicating the interaction between Cu2+ ion and the receptors 1 and 2. Fluorescence quenching of receptors may be due to the electron transfer between O or N-atoms of receptors and vacant orbital of Cu2+ in the excited state [1]. Higher selectivity to Cu2+ ion in the presence of other competitive species was an important index for evaluating the performance of chemical sensors. Therefore, the competition experiments to recognize Cu2+ ion for sensors were also carried out (Fig. 5b and Fig. S6). Upon addition of the mixture containing Cu2+ ion (1 equiv) and other interfering cation (1 equiv) to the solutions of sensors 1 or 2, the fluorescence decrease of the receptor was similar to that caused by adding Cu2+ only, regardless of whether the coexisting interference ions were equal (10 μM) or excessive (10 equiv, see Fig. S6), which indicated that the specific selectivity of sensors 1 and 2 to Cu2+ was not interfered by other competitive cations. In the case of receptors 3 and 4, a significant fluorescence decrease appeared when 1 equiv. of Cu2+ ions were added to the solution, but slight fluorescence decrease was also observed when 1 equiv. of

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Fig. 5. (a) Changes of fluorescence spectra for chemosensors 1 in CH3CN/H2O (4:1, V/V, 10 μM) in the presence of metal ions (1 equiv). λex = 366 nm. (b) Response of fluorescence spectra for receptors 1 to 1 equiv of Cu2+ and coexistent metal ions in CH3CN/H2O (4:1, V/V, 10 μM), λex = 366 nm. Fluorescence titration experiments can further confirm the sensing properties of 1 and 2 to Cu2+. As shown in Fig. 6a, when Cu2+ ions were dripped gradually into the solution of sensor 1, the fluorescence intensity weakened slowly and reached a equilibrium state when 2.0 equiv. of Cu2+ ion was used, which indicated that there was a certain degree of interaction between recepors and Cu2+. For sensor 2, similar fluorescence decrease was observed at 380-480 nm when Cu2+ (0–1 equiv.) were dripped gradually into the solution. To our surprise, an increase of red-shift emission band centred at 494 nm (Φ = 0.039) accompanied by an isoemissive point at λ = 435 nm appeared on further addition of Cu2+ from 1 to 2 equiv. to the solution 2 (Fig. 6b). This phenomenon is consistent with the solution luminescence of the sensor 2 from dim to bright green under ultraviolet lamp in the presence of Cu2+. Thus, we reasoned that the titration reaction of sensor 2 might consist of two steps. That is to say, it is possible that the ligand complexes with copper ions at first and then self-assembles to form quinoline dimer induced by higher concentration of Cu2+, causing the Excimer/Exciplex effect [47,48].

Fig. 6. Fluorescence titrations spectra of sensors 1 (a) and 2 (b) with Cu2+ in CH3CN/H2O (4:1, V/V, 10 μM), λex = 366 nm. 3.4. Electrochemical studies Ferrocene based electrochemical sensors have been widely investigated to detect metal cations. The selective recognition of metal cations for receptors 1 and 2 has been studied by CV and DPV in CH3CN/H2O (4:1, V/V, 100 μM). The formal redox potentials of Fc/Fc+ in receptors 1 and 2 appeared at E1/2 = 0.775 V (ΔE (Epa - Epc) = 78 mV) and E1/2 = 0.778 V (ΔE = 81 mV) respectively, which exhibited a reversible oxidation

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process. Moreover, the influence of scanning rate on redox potential of representative receptors 1 was slight in a range from 20 to 200 mVs-1 (Fig. S8), and a diffusion-controlled process was supposed from the linear relationship between the peak currents and square root of scan rates [49]. On separate addition of Ag+, K+, Na+, Li+, Ca2+, Pb2+, Co2+, Zn2+, Mn2+, Cd2+, Ni2+, Fe2+, Mg2+ and Hg2+ ions, CV and DPV of receptor 1 did not change significantly. (Fig. 7a and 7b). However, upon addition Cu2+ ions (1 equiv) to receptor 1, great changes have taken place in both redox potential and current of DPV and CV. The titration experiments of DPV and CV (Fig. S9) could confirm the interaction between receptors and Cu2+ ion. When Cu2+ (0–3.0 equiv) was incrementally dripped into the solution of 1, the peak current gradually decreased and the redox potentials shifted obviously. Similar phenomena displayed in DPV of 1, so the receptor 1 was an excellent electrochemical sensor for recognition of Cu2+ ions with specific selectivity. However, when above ions were separately dripped into the solution of 2 (Fig. S10), Co2+, Ni2+, Hg2+, Fe2+ ions besides Cu2+ also made the redox potential of CV change largely. The possible reason is that the introduction of nitrogen atom in quinoline ring increases the bonding site, which can stabilize more metal ion complexes. Moreover, electrochemical experiments are sensitive to this effect. So the receptor 2 was not a good electrochemical sensor for ions recognition.

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Fig. 7. CV (a) and DPV (b) curves of receptor 1 mixed with various metal ions (1 equiv) in CH3CN / H2O (4:1, V/V, 100 μM). 3.5. NMR titrations binding studies In order to speculate the binding mechanism between the receptors and Cu2+ ion and to further confirm the results of spectroscopic and electrochemical experiments, 1H NMR titration of Cu2+ was performed in CD3CN for the receptor 1 (Fig. S11) and in DMSO-d6 for the receptor 2 (Fig. 8). In the 1H NMR spectra of free receptor 1, two methene protons Ha resonated at δ 5.44 ppm as a singlet. In addition, the naphthalene protons Hb and Hc resonated at δ 7.17 ppm and 8.27 ppm respectively, and the triazole ring proton He resonated at δ 8.3 ppm as a singlet. Next, Cu2+ ions were dripped gradually into the solution of compound 1 (up to 1.0 equiv), as expected, significant upfield shifts of these signals were observed: ΔHa 2.80 ppm, ΔHb 2.65 ppm, ΔHc 2.61 ppm. To our surprised, the triazole ring proton He disappeared. Similarly, in the case of compound 2, on gradual addition of Cu2+ ion (from 0 to 0.5 equiv), obvious downfield shifts assigned for methene protons (ΔHa' 0.46 ppm), quinoline (ΔHb' 0.89 ppm, ΔHc' 0.50 ppm) and triazol proton (ΔHe' 0.69 ppm) were also observed. The signal changes were attributed to the interaction between receptors and Cu2+ ions [16]. However, some signals weakened gradually even disappeared due to the paramagnetism of Cu2+ ion or the poor solubility of formed complex as more Cu2+ ions were added. The possible binding mode of ligand and ion can be concluded from these signal changes and is shown in Fig. 9. The O and N atoms in receptor 1 might bind with Cu2+. In the case of receptor 2, N atom in quinoline ring might also bind with Cu2+ besides O atom and N atom in triazole ring.

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Fig. 8. 1H NMR spectra of 2 titrated with Cu2+ ions in DMSO-d6.

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Fig. 9. Proposed binding mode for 1 and 2 with Cu2+.

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3.6. Cytotoxicity assay If an excellent chemosensor is nontoxicity or low-toxicity, it could be used in an environmentally friendly way. To evaluate the cytotoxicity of these 1,2,3-triazole ferrocene receptors, we treated PC12 cells with 100 μL of receptors 1 and 2 solutions in medium containing three concentrations (1 μM, 10 μM, 20 μM, Fig. 10). Surprisingly, both compounds 1 and 2 had no cytotoxicity on PC12 cells within the studied concentration range. On the contrary, the survival rate increased slightly after treatment with the receptors, with the cell viabilities of 111-114% for receptor 1 and 100-103% for receptor 2 at a concentration of 1 μM - 20 μM, compared with the nontreated (NT) group. The above results indicated the receptors 1 and 2 expressed excellent biocompatibility [50].

Fig. 10. Cytotoxicity assays of PC12 cells incubated with various concentrations of receptors 1 and 2.

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3.7. Antibacterial evaluation Since these sensors are non-toxic, they will have wider application value if they have certain biological activity. Therefore the antibacterial activities of the receptors were detected preliminarily via the plate counting method using E. coli as representative bacteria. The experimental results are shown in Fig. 11. The photographs of LB agar cultural plates taken after antibacterial test displayed the surviving E. coli treated and untreated with the water suspension of samples (1 mg/mL). The survival bacterial colonies grew on LB agar plate and aggregated into the white pellets. On the cultural plates in the presence of receptor 2, the bacterial colonies were dense and their number exceeded 6.8% compared with the control plates, which indicates the receptor 2 containing a quinoline ring has no antibacterial activities to E. coli. In contrast, it was encouraging to see that the number of surviving colonies declined obviously (bacterial reduction of 66%) when they were treated with the suspension of receptor 1, suggesting that receptor 1 has a certain antimicrobial activity toward E. coli [51].

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Fig. 11. Images of E. coli colonies on culture plates upon 2 h exposure to control, receptors 1 and 2. Conclusions In summary, we have prepared a series of ferrocene-triazole receptors appended naphthalene (1 and 3) or quinoline ring (2 and 4). Receptors 1 and 2 behaved as naked-eye chemosensors and fluorescent probes for Cu2+ even coexistence with other metal ions. Moreover, receptor 1 could also be considered electrochemical sensor for Cu2+ with very high sensitivity and selectivity. In the case of receptors 3 and 4, however, the selectivity of ion recognition was lowered with increasing the flexibility of molecules. Furthermore, this series of compounds were nontoxicity and have certain biological activity. Receptor 1 shows certain antimicrobial activities toward E. coli. Thus, the kinds of receptors can be selected as multi-responsive chemosensors in aqueous environment for potential practical application in chemical and biological fields. Acknowledgements

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Journal Pre-proof Declaration of interests √ The authors declare that they have no known competing financial interests or personal relationships that □ could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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CRediT author statement Jianwei Xu: Conceptualization, Methodology, Writing- Reviewing and Editing Haiying Zhao: Data curation, Writing- Original draft preparation. Huricha Baigude: Visualization, Investigation, Supervision. YongqiangYang: Software, Validation.

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Highlights • Ferrocene–based naphthalene or quinoline receptors 1-4 linked by 1,2,3-trizaole were designed and synthesized. • This kind of compounds behaved as very selective and sensitive colorimetric, electrochemical and fluorescent chemosensors for Cu2+ ion in an aqueous environment. • These receptors had nontoxicity and receptor 1 displayed certain antimicrobial activity toward E. coli.

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