Cyanide and biothiols recognition properties of a coumarin chalcone compound as red fluorescent probe

Accepted Manuscript Cyanide and biothiols recognition properties of a coumarin chalcone compound as red fluorescent probe

Yatong Sun, Yanyan Shan, Ning Sun, Zhipeng Li, Xiangwen Wu, Ruifang Guan, Duxia Cao, Songfang Zhao, Xun Zhao PII: DOI: Reference:

S1386-1425(18)30731-5 doi:10.1016/j.saa.2018.07.071 SAA 16340

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

26 April 2018 7 July 2018 24 July 2018

Please cite this article as: Yatong Sun, Yanyan Shan, Ning Sun, Zhipeng Li, Xiangwen Wu, Ruifang Guan, Duxia Cao, Songfang Zhao, Xun Zhao , Cyanide and biothiols recognition properties of a coumarin chalcone compound as red fluorescent probe. Saa (2018), doi:10.1016/j.saa.2018.07.071

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ACCEPTED MANUSCRIPT Cyanide and biothiols recognition properties of a coumarin chalcone compound as red fluorescent probe Yatong Suna, Yanyan Shana, Ning Suna, Zhipeng Lia, Xiangwen Wub, Ruifang Guana, Duxia Caoa,*, Songfang Zhaoa,*, Xun Zhaoa School of Materials Science and Engineering, University of Jinan, Jinan 250022,

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a

College of Chemistry, Chemical Engineering and Materials Science, Collaborative

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b

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Shandong, China

Innovation Center of Functionalized Probes for Chemical Imaging, Key Laboratory of

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Molecular and Nano Probes, Ministry of Education, Shandong Normal University,

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Jinan 250014, Shandong, China *Corresponding author.

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E-mail addresses: [email protected] (D. Cao), [email protected].

Abstract: A novel coumarin chalcone derivative 1 was designed, synthesized and

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characterized by nuclear magnetic resonance spectra and high resolution mass

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spectrum. The photophysical and recognition properties of the compound as red fluorescent probe for cyanide and biothiols including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) have been discussed systematically. Red fluorescence probe 1 was able to achieve rapid and selective identification for cyanide anion and biothiols in aqueous solutions with red fluorescence quench. In addition, the recognition mechanism of 1 was demonstrated by in situ 1H NMR. The compound has two potential nucleophilic sensing sites including carbon-carbon double bond and

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ACCEPTED MANUSCRIPT 4-position of coumarin. The results indicate that cyanide anions can be bonded to these two sites to afford 2:1 bonding product. But biothiols only are bonded to carbon-carbon double bond by Michael addition reaction. The bonding of both cyanide and biothiols to the probe disrupts intramolecular charge transfer and leads to

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fluorescence quench.

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Keywords: Coumarin chalcone; Cyanide anion; Biothiol; Michael addition

1. Introduction

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As is known to all, cyanide is extremely toxic for the organism that a small

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amount can result in death [1-3]. Cyanide can form a stable complex with oxidase in cytochrome, which inhibits the function of the enzyme and blocks cell oxygen and

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then leads to cell choking. However, the production of cyanide is really large because

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of its extensive use in industrial field such as papermaking, textile, synthetic resins, gold extraction reagents and so on [4-7]. At the same time, in the fire accident,

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cyanide also can be released and cyanide poisoning arises from smoke inhalation [8].

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Therefore, the research of rapid and sensitive fluorescent probes for cyanide attracted a lot of interest to the scientists [9-14]. Over the past years, coumarin derivatives as fluorescent probes for cyanide have attracted increasing interests due to their advantageous photophysical properties and high selectivity toward cyanide anions (CN-) [15-18]. Amino acids are the basic substances that make up the protein. Biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), are a class of important

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ACCEPTED MANUSCRIPT amino acids, which contain sulfhydryl group (-SH) in living system. They play vital roles in a lot of biological processes, including protein synthesis, metabolism and so on [19-22]. Cysteine is a non-essential amino acid, which can be transformed from methionine in the organism. An abnormal level of cysteine is associated with slowed

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growth, liver damage, oxidative damage and so on [23-27]. Homocysteine is an

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important intermediate product in methionine and cysteine metabolism. High levels of

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homocysteine in the blood will cause cardiovascular disease, stroke, cognitive dysfunction, and even Alzheimer's disease, schizophrenia and so on [28,29].

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Glutathione, which presents in almost every cell of the body, is a tripeptide containing

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a γ-amide bond and a sulfhydryl group. The decrease of glutathione is a potential activation signal for apoptosis and can lead to some health problems [20-32].

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Therefore, the development of monitoring these three biothiols has attracted great

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attention. Herein, a coumarin chalcone red fluorescent probe with two potential nucleophilic addition sensing sites is reported, which can detect both cyanide and

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biothiols and distinguish them with different spectral phenomenon in aqueous solution.

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The input of bromine atom is based on its electron-accepting ability and can increase Michael reaction activity. 2. Experimental Section 2.1. Chemicals and instruments The

title

compound

was

synthesized

by

condensation

reaction

with

7-(diethylamino)coumarinaldehyde and 5-(bromine)4-(hydroxyl)acetophenone as starting materials (Scheme 1). 7-(diethylamino)coumarinaldehyde was synthesized

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ACCEPTED MANUSCRIPT according to literature procedure [33]. 5-(bromine)4-(hydroxyl)acetophenone, cysteine, glutathione and other 17 amino acids including phenylalanine (Phe), alanine (Ala), glycine (Gly), glutamic acid (Glu), methionine (Met), arginine (Arg), tyrosine (Tyr), leucine (Leu), proline (Pro), tryptophan (Trp), serine (Ser), threonine (Thr),

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aspartic acid (Asp), valine (Val), isoleucine (Ile), histidine (His) and glutamine (Gln)

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(the structures of the amino acids were shown in Table S1) were purchased from

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Aladdin Reagents. Homocysteine was purchased from TCI Development Co. Ltd. Tetra(n-butyl)ammonium cyanide was purchased from Shanghai Reagents. Nuclear

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magnetic resonance spectra were measured on a MercuryPlus-400 spectrometer. High

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resolution mass spectrum was carried out on an Agilent Q-TOF6510 spectrometer.

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2.2. Synthesis and characterizations

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Scheme 1. Synthetic routes to the title compound 1.

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1.07 g (5.0 mmol) of 5-(bromine)-4-(hydroxyl)acetophenone and 1.23 g (5.0

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mmol) of 7-(diethylamino)coumarinaldehyde as starting materials with 20 mL anhydrous ethanol as solvent were added in 50 mL of round flask. 0.4 mL (0.36 g, 5.0 mmol) of pyrrolidine was added as catalyst. After the mixture was stirred for 12 h at room temperature, red precipitates was formed. The precipitates was filtered, washed three times with ethanol and purified via silica gel column chromatography (dichloromethane/petroleumether, 3:1, v/v). 1.92 g of compound 1 as red solid was obtained with a yield 87%. M.p. 215-216 °C. 1H NMR (400 MHz, DMSO-d6), δ

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ACCEPTED MANUSCRIPT (ppm): 1.15 (t, J = 7.2 Hz, 6H), 3.50 (t, J = 7.2 Hz, 4H), 6.62 (d, J = 2.4 Hz, 1H), 6.82 (d, J = 9.2, 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 9.2 Hz, 1H), 7.67 (dd, J = 8.8, 2.4 Hz, 1H) 7.75 (d, J = 15.2 Hz, 1H), 8.01 (d, J = 15.2 Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H), 8.56 (s, 1H), 12.28 (s, 1H). 13C NMR (100 MHz, CDCl3), δ: 12.65, 45.29,

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97.12, 109.16, 109.91, 110.62, 114.55, 120.46, 120.65, 121.72, 130.48, 132.34,

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138.86, 141.93, 147.44, 152.49, 157.02, 160.25, 162.62, 193.61. MS for (M+H)+,

2.3. Photophysical and recognition properties

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Calcd exact mass: 442.0654, found 442.0628.

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UV-vis absorption and fluorescence spectra of the compound in various solvents

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with C = 10 μM were recorded on a Shimadzu UV-2550 spectrophotometer and HORIBA Scientific FM4 fluorescence spectrophotometer, respectively. Fluorescence

an

Edinburgh

FLS920

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on

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quantum yields (Φ) of the compound in solution were measured by absolute method fluorescence

spectrometer.

The

solution

of

tetra(n-butyl)ammonium cyanide (TBACN) and biothiols (Cys, Hcy or GSH) in

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DMSO:PBS (4:1, v/v, pH = 7.4) was used as CN- and biothiols source, respectively.

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3. Results and discussion 3.1. Photophysical properties of the compound

Fig. 1. UV-vis absorption and fluorescence spectra (λex = 486 nm) of compound 1 in various solvents with C = 10 μM.

In order to further understand the photophysical properties of compound 1, the

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ACCEPTED MANUSCRIPT solvent effect was firstly discussed. As shown in Fig. 1, UV-vis absorption spectra of the compound exhibits typical single-peak in common solutions except for double-peak in hexane. With the increase of the polarity of solvent, the maximum absorption peak of compound 1 is red-shifted slightly and the absorbance decreases

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gradually. The fluorescence spectra and quantum yield of the compound show

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dramatic change with the solvents. The compound shows weak fluorescence emission

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with low fluorescence quantum yields (< 0.01) in all the investigated solvents except DMSO. It is surprising that DMSO solution of compound 1 exhibits strong

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fluorescence emission in red light region at 600 nm with high fluorescence quantum

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yield (0.22), which may be attributed to its strong polarity and intermolecular hydrogen bonding reaction with hydroxyl group in the compound.

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3.2. Spectral response to cyanide anions

Fig. 2. UV-vis absorption (a) and fluorescence spectra (λex = 486 nm, b) of 1 in

concentration of TBACN.

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DMSO:PBS (4:1, v/v, pH = 7.4) with C =10 μM upon the addition of different

Based on the strong fluorescence of the compound in DMSO, DMSO:PBS (4:1, v/v, pH = 7.4, 10 μM) mixture solvent was used for the spectral titration experiment. UV-vis absorption and fluorescence spectra of 1 after the addition of different concentration of TBACN are shown in Fig. 2. As can be seen in Fig. 2(a), with the addition of CN-, the characteristic intense charge-transfer absorption band of

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ACCEPTED MANUSCRIPT compound 1 at 486 nm gradually decreases with slight red shift (about 17 nm). When 50 equiv. CN- is added, compound 1 is bonded with CN- completely and the color changes from orange to light pink. The red fluorescence turn-off response of 1 is shown in Fig. 2(b). With the excitation at 486 nm, the maximum fluorescence

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intensity at 590 nm decreased about 6 times compared to the original fluorescence

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after the saturation. From the insert illustration we can see that when the reaction

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system is saturated, the original red fluorescence almost completely disappears. Spectral change indicates that the bonding between the probe and CN- induces the

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break of charge transfer and the decrease of original absorption and fluorescence

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band.

The reaction rate of the compound for cyanide was measured and calculated. The

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reaction reaches saturation with 50 equiv. of CN- after 12 min and rate constant k is

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calculated to be 5.55 × 10-3 s-1 (Fig. S1). pH dependence of the probe with or without CN- was investigated. From Fig. S2, we can see that absorption and fluorescence

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spectra are almost unchanged in pH range 2.0-9.0, which indicates that the probe is

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stable in this pH range. After the addition of CN-, the absorption and fluorescence peaks change are similar with original absorption and fluorescence peak decreasing in pH range 3.0-9.0. The pH study indicates that the probe can work stably in DMSO:PBS (4:1, v/v, pH = 7.4) solution.

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ACCEPTED MANUSCRIPT Fig. 3. Fluorescence spectra (λex = 486 nm, a) and fluorescence intensity at 590 nm (b) of 1 (10 μM) responding to various anions (500 μM) and CN- (500 μM) in DMSO:PBS (4:1, v/v, pH = 7.4).

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To explore the selectivity of red fluorescent probe 1, the influence of other anions

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including F-, Cl-, Br-, I-, CO32-, HSO4- and SCN- on the response of 1 to CN- was also

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examined. As displayed in Fig. S3 and Fig. 3, compound 1 only shows obvious response to CN- by absorption and fluorescence spectra. With respect to other anions,

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the absorption and fluorescence spectra exhibited negligible changes. Furthermore,

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the competitive experiments were conducted in the presence of CN- over the other anions (Fig. 3). The absorption and fluorescence spectra of 1 with CN- was not

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influenced by the addition of competing anions. Then the compound 1 exhibits good

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selectivity and interference immunity.

The detection limits [34] of UV-vis absorption and fluorescence changes for CN-

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in DMSO:PBS (4:1, v/v, pH = 7.4) were 1.00 μM and 0.32 nM, respectively (the

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method for detection limit was provided in Supporting Information). World Health Organization (WHO) requires that the concentration of cyanide in drinking water must be less than 1.9 μM [35]. The above data is lower than the WHO guideline and indicated that red fluorescent probe 1 possess good sensitivity to CN-. 3.3. Spectral response to biothiols It was found that red fluorescent probe 1 can recognize not only CN- but also biothiols in DMSO:PBS solution. Therefore, the identification for biothiols by 1 was

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ACCEPTED MANUSCRIPT also studied systematically. By means of titration tests, the recognition responses of compound 1 for Cys, Hcy and GSH are similar. Thus, we take Cys identification as an example for a detailed discussion. With the increase of Cys, as shown in Fig. 4(a), the absorption peak of 1 at

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486 nm decreased gradually and a new absorption peak at 400 nm was formed, which

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is different to CN-. It can be seen from the insert illustration that compound 1 changes

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from orange to light yellow under natural light. With 486 nm as the excitation wavelength, the red fluorescence at 590 nm gradually decreases with the titration of

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Cys (Fig. 4(b)). It is believed that bonding of biothiols to the probe also results in

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break of intramolecular charge transfer and then the decrease of absorption and fluorescence. The reaction reaches saturation with 40 equiv. of Cys after 12 min (Fig.

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S6). Rate constant is calculated to be 4.1 × 10-3 s-1. Rate constants for Hcy and GSH

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are 3.6 × 10-3 s-1 (Hcy) and 3.13 × 10-3 s-1 (GSH), respectively. Compared with Hcy and GSH, the probe exhibits faster response for Cys. As shown in Figs. S7-S9, to the

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addition of biothiols, the stable absorption and fluorescence response pH range are

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4.0-8.0. Then the probe can also stably response biothiols in DMSO:PBS (4:1, v/v, pH = 7.4) solution.

Fig. 4. UV-vis absorption (a) and fluorescence spectra (λex = 486 nm, b) of 1 in DMSO:PBS (4:1, v/v, pH = 7.4) with C = 10 μM upon the addition of different concentration of Cys.

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ACCEPTED MANUSCRIPT The detection limits of UV-vis absorption changes for Cys, Hcy and GSH in DMSO:PBS (4:1, v/v, pH = 7.4) were 1.02 μM, 2.51 μM and 3.0 μM, respectively. The detection limits of fluorescence changes were 0.46 nM, 0.86 nM and 0.73 nM,

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respectively. Therefore, compound 1 also has good sensitivity to biothiols.

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Fig. 5. Fluorescence spectra (λex = 486 nm, a) and fluorescence intensity at 590

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nm (b) of 1 (10 μM) responding to various anions (400 μM) and Cys (400 μM) in DMSO:PBS (4:1, v/v, pH = 7.4) : 1, blank; 2, Phe; 3, Ala; 4, Gly; 5, Glu; 6,

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Met; 7, Arg; 8, Tyr; 9, Leu; 10, Pro; 11, Trp; 12, Ser; 13, Thr; 14, Asp; 15,

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Val; 16, Ile; 17, His; 18, Gln.

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The selectivity of red fluorescent probe 1 for biothiols were also explored in

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DMSO:PBS (4:1, v/v, pH = 7.4) solution. As can be seen in Fig. 5 and Fig. S10, except Cys, Hcy and GSH, compound 1 showed almost no response to the other 17

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amino acids in absorption and fluorescence spectra. Moreover, the presence of other

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amino acids did not have a significant effect on the identification of Cys, Hcy and GSH in DMSO:PBS (4:1, v/v, pH = 7.4), respectively (Fig. 5 and Figs. S11-S13). The above results indicated that 1 possesses excellent selectivity and interference immunity for biothiols. 3.4. Recognition mechanism of CN- and biothiols Because of the difference of fluorescent probe 1 for CN- and biothiols in the absorption spectra changes, the reaction mechanism of 1 for these analytes may be

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ACCEPTED MANUSCRIPT different. In order to investigate thoroughly the mechanism, in situ 1H NMR was tested in DMSO-d6. From Fig. S14 we can see that with the addition of CN-, peaks related to vinylic protons 8.01 ppm (d, J = 15.2 Hz, 1H, Hb) and 7.75 ppm (d, J = 15.2 Hz, 1H, Hc) disappear and two sets of new peaks at 6.07-6.09 ppm (Hb) and

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3.75-3.80 ppm (Hc) appeared. Meanwhile, the proton signal of 4-coumarin group at

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8.55 ppm (Ha) disappeared and a new proton signal at 5.26 ppm appeared. In situ 1H

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NMR results indicate that CN- is bonded to carbon-carbon double bond in conjugated bridge by Michael addition and 4-position of coumarin by nucleophilic addition,

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which disrupts intramolecular charge transfer and lead to the fluorescence decrease.

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From the above, the reaction mechanism of red fluorescent probe 1 for CN- is shown in Fig. 6. To further verify the reaction mechanism, job’s plot curve was also detected.

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As can be seen in Fig. 7, the intersection of two fitted lines lies between 0.6 and 0.7,

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which further indicates the complexation between 1 and CN- is 1:2. 2-Mercaptoethanol is a model compound of biothiols [36], in situ 1H NMR of 1

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was tested in DMSO-d6 using 2-mercaptoethanol and Cys. As shown in Fig. S15, only

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vinylic protons peaks at 8.01 ppm (Hb) and 7.75 ppm (Hc) disappear and two sets of new peak at 4.45 ppm (Hb) and 3.76-3.78 ppm (Hc) appear. Ha only has slight shift from 8.55 ppm to 7.90 ppm. 1H NMR titration indicates that biothiol is only bonded to carbon-carbon double bond in conjugated bridge of fluorescent probe 1 by Michael addition reaction [36]. 1H NMR of the probe with Cys was also measured and its 1H NMR change is similar to 2-Mercaptoethanol (Fig. S16). That is to say, the complexation between 1 and biothiols is 1:1, which can also be proved by the job’s

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ACCEPTED MANUSCRIPT plot curve (Fig. 7 and S17). Therefore, the reaction mechanism of red fluorescent probe 1 for biothiols is also shown in Fig. 6. The new absorption peak at 400 nm may be from coumarin group. The higher sensitivity of the compound for Cys than Hcy and GSH may be due to its smaller molecular size and lower steric effect, which is

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believed that it is more favorable to nucleophilic addition reaction.

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biothiols.

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Fig. 6. The reaction mechanism of fluorescent probe 1 for CN- and

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Fig. 7. Job’s plot curve of compound 1 to CN- and Cys.

4. Conclusion

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In conclusion, a novel coumarin chalcone derivative 1 was synthesized and characterized by nuclear magnetic resonance spectra and mass spectra. The photophysical and recognition properties of compound 1 as red fluorescent probe

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have been systematically discussed. Fluorescent probe 1 could detect both CN- and

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biothiols in aqueous solutions and possess excellent sensitivity, selectivity and interference immunity. In addition, the recognition mechanism of fluorescent probe 1 for these two analytes were also discussed. By in situ 1H NMR, we consider that not only Michael addition between 1 and CN- occur, but also CN- is added to the 4-position of coumarin. Compared to CN-, the interaction between red fluorescent probe 1 and biothiols is only Michael addition reaction. The fluorescent probe can recognize CN- and biothiols with different spectral change, and then can distinguish - 12 -

ACCEPTED MANUSCRIPT them. The bonding of CN-/biothiols to carbon-carbon bond of probe 1 by Michael addition interrupts the intramolecular charge transfer and then results in the decrease of absorption peak and fluorescence quench. Acknowledgements

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This work was supported by the National Natural Science Foundation of China

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(21672130, 21601065), and the Natural Science Foundation of Shandong Province

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(ZR2017MEM006), the China Postdoctoral Science Foundation Grant (2018M631603) and Colleges and Universities Science and Technology Foundation of Shandong

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Province (J16LA08).

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ACCEPTED MANUSCRIPT Scheme 1. Synthetic routes to the title compound 1. Fig. 1. UV-vis absorption and fluorescence spectra (λex = 486 nm) of compound 1 in various solvents with C = 10 μM. Fig. 2. UV-vis absorption (a) and fluorescence spectra (λex = 486 nm, b) of 1 in DMSO:PBS (4:1, v/v, pH=7.4) with C = 10 μM upon the addition of different concentration of TBACN.

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Fig. 3. Fluorescence spectra (λex = 486 nm, a) and fluorescence intensity at 590 nm (b) of 1 (10 μM) responding to various anions and CN- (500 μM) in DMSO:PBS (4:1, v/v, pH = 7.4).

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Fig. 4. UV-vis absorption (a) and fluorescence spectra (λex = 486 nm, b) of 1 in DMSO:PBS (4:1, v/v, pH=7.4) with C = 10 μM upon the addition of different concentration of Cys.

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Fig. 5. Fluorescence spectra (λex = 486 nm, a) and fluorescence intensity at 590 nm (b) of 1 (10 μM) responding to various anions and Cys (400 μM) in DMSO:PBS (4:1, v/v, pH=7.4) : 1, blank; 2, Phe; 3, Ala; 4, Gly; 5, Glu; 6, Met; 7, Arg; 8, Tyr; 9, Leu; 10, Pro; 11, Trp; 12, Ser; 13, Thr; 14, Asp; 15, Val; 16, Ile; 17, His; 18, Gln.

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Fig. 6. The reaction mechanism of fluorescent probe 1 for CN- and biothiols.

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Fig. 7. Job’s plot curve of compound 1 to CN- and Cys.

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ACCEPTED MANUSCRIPT

Highlights • A coumarin chalcone derivative as red fluorescent probe for CN- and biothiols was synthesized. •

The compound can detect CN- and biothiols in aqueous solution with high selectivity.

•

CN- is bonded to C=C and 4-position of coumarin to afford 2:1 bonding

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The compound recognize biothiols by Michael addition reaction

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•

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product.

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Figure 1

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