Colorimetric and NIR fluorescence probe with multiple binding sites for

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Colorimetric and NIR fluorescence probe with multiple binding sites for distinguishing detection of Cys/Hcy and GSH in vivo Kangming Xiong, Fangjun Huo, Jian-Bin Chao, Yongbin Zhang, and Caixia Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04485 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on November 28, 2018

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Analytical Chemistry

Colorimetric and NIR fluorescence probe with multiple binding sites for distinguishing detection of Cys/Hcy and GSH in vivo Kangming Xiong,‡ Fangjun Huo,† Jianbin Chao,† Yongbin Zhang,† Caixia Yin*, ‡ ‡

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Shanxi University, Taiyuan 030006, China.; †Research Institute of Applied Chemistry (RIAC), Shanxi University, Taiyuan, 030006, China. *Corresponding author. * E-mail: [email protected]. Tel/Fax: +86-351-7011022. ABSTRACT: Although some progress has been made in distinguishing detection of biothiols, NIR biothiol fluorescent probes for simultaneous distinguishing detection of Cys, Hcy and GSH in vivo have not been reported. The design of these probes involves the introduction of NIR fluorophores and multiple binding sites, so the integrated design of probes remains a challenge. Although Cys, Hcy and GSH have the common functional group: a sulfydryl group and an amino group, due to their differences in spatial structure, they may react with multiple binding sites probes to produce different reaction products in different bonding mechanisms, resulting in the different colors and fluorescent signals changes of the system. Therefore, multiple binding sites fluorescent probes can realize their discrimination detection. For NIR fluorescent probe, it is easier to realize in vivo imaging to promote the research of biothiols in clinical diagnosis. In our work, not only multiple binding sites were constructed in the compound, but also NIR fluorophores were introduced. This enables the probe to not only efficiently distinguish detection of Cys/Hcy and GSH but also achieve fluorescence imaging in vivo. We believe this result is a milestone in the discrimination detection of biothiols.

Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play important roles in many physiological processes and are closely related to many diseases.1-3 Because fluorescent sensing possesses operational simplicity and high sensitivity, a lot of fluorescent probes have been developed in recent years to detect these biothiols.4-18 Among these biothiol fluorescent probes, only a few of them can simultaneously distinguish Cys/Hcy/GSH.19-25 As we known, Cys, Hcy and GSH each possesses a sulfhydryl functional group and an amino group which bring about great challenges in discriminating them from each other in biological samples using chemical reaction method. Nevertheless, owing to their differences in spatial structure, they may react with multiple binding sites fluorescent probes to produce different reaction products in different bonding mechanisms, resulting in the different colors and fluorescent signals changes of the system. Therefore, the multiple binding sites fluorescent probes are most likely to realize the simultaneous distinguishing detection of Cys, Hcy and GSH. Moreover, compared with conventional fluorescent probes, NIR fluorescent probes have unique advantages in tracking molecular processes in vitro and in vivo.26 In large part it is because high tissue auto-fluorescence taking place from indigenous biomolecules in the living systems does not interfere with NIR emission and NIR photons can penetrate relatively deeply into tissues and they cause less damage to biological samples.27 So far, some NIR fluorescent probes have been reported.28-30 In 2014, Guo et al. developed a multiple binding sites fluorescent probe that could selectively detect Cys and GSH via

different emission channels.19 And the probe was utilized to simultaneously imaging of Cys and GSH in COS-7 cells using different emission channels. Although their work showed an excellent perspective for simultaneous detection of Cys/Hcy/GSH, The system showed a short-wavelength emission. In 2017, Yin et al. reported a Minireview on various strategies for the design of fluorescent probes for simultaneous detection of Cys, Hcy and GSH. 31 In 2018, Yin et al. designed a multiple binding sites fluorescent probe that could simultaneous detection of Cys, Hcy and GSH.32 And the probe was applied to the fluorescence images of endogenous Cys, Hcy, GSH and exogenous Cys, Hcy and GSH in BEL-7402 cells. Although their probe realized simultaneous detection of Cys/Hcy/GSH, The system still did not achieve NIR detection. In addition, their probe was not utilized to in vivo imaging. In our study, we presented a near infrared fluorescent probe with three potential binding sites for simultaneous sensing Cys/Hcy and GSH (Scheme 1). Different bonding mechanisms between probes and biothiols (substitution−rearrangement−cyclization for Cys/Hcy, and substitution−cyclizatioin for GSH) lead to the corresponding adducts, enabling Cys/Hcy and GSH to be selectively detected. Moreover, the probe was successfully utilized for in vitro cellular imaging and in vivo imaging in mice of exogenous Cys, Hcy and GSH.

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Scheme 1. Simultaneous Sensing of Cys, Hcy and GSH Based on Three Potential Reaction Sites of Probe

EXPERIMENTAL SECTION Materials and Methods. All chemicals were bought from Aladdin (Shanghai, China) and used without further purification. Deionized water was used to prepare all aqueous solutions. All reactions were monitored by thin-layer chromatography (TLC). All spectroscopic measurements were performed in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). The UV–vis absorption spectra were collected on Agilent 8453 spectrophotometer. Fluorescence spectra were recorded on Hitachi F-7000 spectrophotometer. The ability of probe to detect Cys/Hcy/GSH within living cells was also evaluated by laser confocal fluorescence imaging using Airyscan confocal laser scanning microscope. 1H NMR, 13C NMR spectra were recorded on a Bruker AVANCE-600 MHz and 150 MHz NMR spectrometer, respectively. ESI-MS was measured with AB Triple TOF 5600 plus System. An IVIS spectrum imaging system (PerkinElmer, Massachusetts, USA) was used for fluorescent imaging in animal experiments. General Procedure for Fluorescence and UV-Visible Measurements. The stock solution of probe (2 mM) was

prepared in CH3OH. Stock solutions (2×10-1 M) of sodium salts (I-, SO42-, CO32-, F-, NO2-, Br-, PO43-, S2-, HSO3-), and amino acids (L-Leu, L-Iso, L-Ala, L-Val, L-Pro, L-Hyp, L-Thr, L-Tyr, L-Asp, L-Arg, Hcy, Cys and GSH) were prepared in deionized water. These stock solutions were further diluted to required concentration for measurement. Test solutions were prepared as follows: proper probe solution and proper analyte’s solution were added into a test tube, and the solution was diluted to 2 mL using PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). Spectra data were recorded in an indicated time at room temperature. Cell Culture. The HepG-2 cells were provided by Institute of

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Biotechnology of Shanxi University. Cells were grown in CMEM medium supplemented with 10 % FBS (Fetal Bovine Serum) and 1% antibiotics at 37 ºC in humidified environment of 5% CO2. Cytotoxicity Assays. Cell cytotoxicity was evaluated by CCK-8 assay. HepG-2 Cells were grown in CMEM medium supplemented with 10 % FBS (Fetal Bovine Serum) and 1% antibiotics at 37 ºC in humidified environment of 5% CO2. Cells were cultivated in a 96-well plate until 50-70% confluence, and then incubated with different concentrations of probe (0-50 μM) for 10 h, followed by CCK-8 assays (n= 5). Untreated assay with CMEM (n = 5) was also conducted under the same conditions. In Vitro Cellular Imaging. HepG-2 Cells were plated on 6well plate at 5×106 cells per well and allowed to adhere for 12 hours. The cells were washed with phosphate buffered saline (PBS) and then incubated with probe (10 μM) in CMEM medium for 30 min at 37 °C and washed three times with PBS. To detect exogenous thiols, cells were pretreated with NEM (0.5 mM) for 30 min, subsequently incubated with Cys/Hcy/GSH (220 μM, 30 min) and probe (10 μM, 30 min) respectively, then washed with phosphatebuffered saline (PBS) for three times. Cells were imaged by Airyscan confocal laser scanning microscope. λex = 405 nm, λem = 465-525 nm for the blue channel; and λex = 514 nm, λem = 550-620 nm for the green channel, λem = 700-750 nm for the red channel. In Vivo Imaging. Mice were anesthetized using isoflurane and maintained in an anesthetized state throughout the imaging procedures. The solutions of Cys, Hcy and GSH (220 μM) were injected into three places of peritoneal cavity of mice, followed by intraperitoneal injection of probe (10 μM), respectively. Mice were imaged with IVIS imaging system using 475 nm excitation light source and an emission filter of 560 ± 10 nm with images collected after 0 min, 30 min, 45 min, 60 min, 75 min and 90 min, respectively. Calculation of the Detection Limit (LOD). (1) LOD = 3σ/S

(2) σ: standard deviation of the blank solution. is the mean of the blank measures. is the values of blank measures. n is the number of tested blank measure (n = 15). S: the slope of the linear calibration plot between the absorption and the concentration of Cys, Hcy and GSH, respectively.

RESULTS AND DISCUSSION Design and Synthesis of Probe. On the basis of the reported 7-(Diethylamino)coumarin derivatives fluorescent probes with multiple binding sites for distinguishing biothiols, we have designed a new 7-(Diethylamino)coumarin derivative fluorescent probe for distinguishing biothiols with larger maximum absorption wavelength (Table S1). The detailed synthetic method and characterization data can be seen in Supporting Information. Proposed Responding Mechanism of Probe for Distinguishing Cys/Hcy and GSH. The proposed responding mechanisms are showed in Scheme 2 and explained as following. This probe has three potential reaction sites: the reactive chloro atom in site 1, michael acceptor α, β-unsaturated bond in site 2, the unsaturated bond of indole moiety in site 3. The chloro atom


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Analytical Chemistry in site 1 is reactive and facilitates the thiol-halogen SNAr nucleophilic aromatic substitution between probe and thiols to produce 1a/2a/3a. For 1a and 2a, the following rearrangement will lead to 1b/2b.9 However, if the reaction only stays at rearrangement stage, it seems to be difficult to distinguish Cys and Hcy because of 1b and 2b having similar optical properties. As a Michael acceptor, the α, β-unsaturated bond enables an intramolecular cyclization between the sulfhydryl group and the vicinal site 2 in 1b and 2b, which will lead to 1c and 2c. This made it is possible to discriminate between Cys and Hcy. Because the reaction to form a 7-membered ring (for 1c) should be kinetically favored compared with that of the formation of 8membered ring (for 2c)6, which correspond to the spectral response difference. For 3a, because of the unstable 10membered macrocyclic transition state, it is difficult for 3a to undergo the aforementioned intramolecular rearrangement to form the corresponding amino-coumarin-hemicyanine.9 Alternatively, it is possible that the free amino group in 3a will attack sites 3 or 2 to form the 14- or 12-membered ring products 3b or 3c. According to the currently reported literature,19,32 the probe reacts with GSH to produce only compound 3b. In summary, if the mechanisms we propose are reasonable, it will be possible to achieve the discrimination of Cys, Hcy, and GSH. In order to further study the responding mechanism, we performed the corresponding 1H NMR and HRMS characterization of the probe+Cys, probe+Hcy, and probe+GSH (Figure S2 and S3). From Figure S2, we can find that after adding Cys or Hcy into probe, the protons Ha of probe+Cys and probe+Hcy display the obvious upfield shifts when compared with Ha of probe. In fact, this is consistent with our proposed cyclization reactions (Scheme 2A and 2B), which leads to the increased electron density around Ha in 1c or 2c and causes the upfield shifts. And these facts also confirm that 2b can transform to 2c, even though the transformation process is slow. For adduct probe+GSH, the proton Ha of probe+GSH displays no chemical shift when compared with Ha of probe. And the result also confirms that the free amino group in 3a does not attack sites 2 to form product 3c. Furthermore, the compounds of 1c, 2c, and 3b were observed in the corresponding HRMS experiments (Figure S3). In a word, the above results agree well with our proposed reaction mechanism. Scheme 2. Proposed Responding Mechanisms of Probe for Distinguishing Detection Cys/Hcy and GSH

UV-Vis Spectra for Probe Sensing Cys/Hcy/GSH. Initially, we used probe to detect Cys/Hcy/GSH by utilization of concentration-dependent and time-dependent UV-vis spectra in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). As shown in Figure 1, the UV-Vis spectra of free probe show a main absorption at 578 nm. Upon addition of Cys, the initial absorption peak at 578 nm decreases dramatically while a new peak at 417 nm appears with a 161 nm blue shift (Figure 1a), which indicates that the conjugation between the coumarin and indole is broken due to the Michael addition of Cys to site 2 in probe. Correspondingly, the reaction time between probe and Cys is within 60 min in time-dependent UV-vis spectra (Figure 1b). For Hcy, the concentration-dependent UV-vis spectrum is similar to Cys in the beginning, along with the decrease of the initial absorption peak at 578 nm and a simultaneous increase of a new broad absorption peak at 417 nm (Figure 1c). However, the absorption at 417 nm increases very slowly due to kinetically unstable 8-membered ring transition state (Scheme 2B). As the case of Cys, the poor absorption at 417 nm should be assigned to 2c. Correspondingly, the reaction time between probe and Hcy is within 90 min in time-dependent UV-vis spectra (Figure 1d). Then we investigated the UV-vis spectra performance of GSH. As shown in Figure 1e, initial absorption peak at 578 nm decreases after the addition of GSH, and a new absorption peak is found at 409 nm with a 169 nm blue shift, which indicates that the nucleophilic addition of the amine group in GSH to site 3 in probe (Scheme 2C). Correspondingly, the absorption at 409 nm should be assigned to 3b. The reaction time between probe and GSH is within 80 min in time-dependent UV-vis spectra (Figure 1f). It can be seen from the experimental above results that the probe has high selectivity for Cys and GSH.



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(a) (e)

(b)

(c)

(d)

(f)

Figure 1. (a)-(b) concentration-dependent and time-dependent absorption spectra of probe (10.0 μM) upon addition of Cys (170 μM) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). (c)-(d) concentration-dependent and time-dependent absorption spectra of probe (10.0 μM) upon addition of Hcy (520 μM) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). (e)-(f) concentration-dependent and time-dependent absorption spectra of probe (10.0 μM) upon addition of GSH (240 μM) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). Inset: visual UV-Vis color change photos for Cys, Hcy and GSH. Fluorescence Spectra for Probe Sensing Cys/Hcy/GSH. Next, we studied the fluorescence spectra of probe upon addition of Cys, Hcy, and GSH, respectively, via concentrationdependent fluorescence spectra in the same conditions (Figure 2). First, we selected 417 nm as excitation wavelength to detect Cys/Hcy/GSH (Figure 2a-c). With the excitation, the emission peak at 489 nm was remarkable enhancement upon addition of Cys (36 μM) or Hcy (52 μM). By comparison, GSH (38 μM) caused the emission peak at 489 nm a little enhancement. Second, we selected 514 nm as excitation wavelength to detect Cys/Hcy/GSH (Figure 2d-f). With the excitation, the emission peak at 724 nm was gradually decreased and a new emission peak appeared at 564 nm upon addition of Cys (0-220 μM) or Hcy (0520 μM) or GSH (0-240 μM). By comparison, Cys (220 μM) and Hcy (520 μM) elicited more obvious emission enhancement than GSH (240 μM) when monitoring at 564 nm. Third, we selected 555 nm as excitation wavelength to detect Cys/Hcy/GSH (Figure 2g-i). For excitation at 555 nm, the emission peak at 724 nm was gradually decreased and a new emission peak appeared at 600 nm upon addition of Cys (0-220 μM) or Hcy (0-520 μM) or GSH (0-240 μM). By comparison, Cys (220 μM) and Hcy (520 μM) elicited more obvious emission enhancement than GSH (240 μM) when monitoring at 600 nm. From the above


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Analytical Chemistry experimental results, the multiple binding sites probe can simultaneously distinguish detection of Cys/Hcy and GSH.

Figure 2. (a)-(c), concentration-dependent fluorescence spectra of probe (2 μM) in the presence of Cys (0-36 μM), Hcy (0-52 μM) and GSH (0-38 μM) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v), λex= 417 nm, slit (nm): 10/10; (d)-(f), concentration-dependent fluorescence spectra of probe (10 μM) in the presence of Cys (0-220 μM), Hcy (0-520 μM) and GSH (0-240 μM) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v), λex= 514 nm, slit (nm): 20/20; (g)-(i), concentrationdependent fluorescence spectra of probe (10 μM) in the presence of Cys (0-220 μM), Hcy (0-520 μM) and GSH (0-240 μM) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v), λex= 555 nm, slit (nm): 20/20.

Figure 3. Absorption spectra of probe (10 µM) upon addition of 5.2 mM various amino acids (L-Leu, L-Iso, L-Ala, L-Val, L-Pro, L-Hyp, L-Thr, L-Tyr, L-Asp, L-Arg), Cys (0.52 mM), Hcy (0.52 mM) and GSH (0.52 mM) as well as 5.2 mM representative ions (I-, SO42-, CO32-, F-, NO2-, Br-, PO43-, S2-, HSO3-) in PBS (pH 7.4, 10 mM)/CH3OH=1600/400 (v/v). Cytotoxicity Experiments. The CCK-8 assays for different concentrations of probe (0, 1, 2.5, 5, 10, 20, 30, and 50 μM) were also carried out, and the results showed that the concentration of probe below 30 μM had low cytotoxicity to living HepG-2cells within 10 h (Figure S5). In Vitro Cellular Imaging. We evaluated the capability of probe to selectively sense endogenous Cys, Hcy and GSH in living HepG-2 cells. First, the cells were pretreated with probe (10 μM) for 30 min, then three kinds of fluorescence were observed from red emission channel, green channel and blue channel in living cells respectively (Figure 4a, e, i,), indicating that probe was responsive to intracellular Cys, Hcy and GSH. Second, when cells were pretreated with NEM (0.5 mM, 30min), then incubated with Cys/GSH/Hcy (220 μM, 30 min) and probe (10 μM, 30 min) respectively, marked increase in green emission (Figure 4f-g) and blue emission (Figure 4j-k) were observed for Cys and Hcy. And slight increase in green emission (Figure 4h) and blue emission (Figure 4l) were observed for GSH. These results are consistent with their respective fluorescence spectra in Figure 2. In addition, marked increase in blue emission (Figure 4r-s) were also observed for Cys and Hcy. These results agree well with the specificity of probe for Cys/Hcy and GSH and also demonstrate the potential of probe to detect intracellular Cys/Hcy and GSH simultaneously from different emission channels.

Detection Limits for Cys, Hcy and GSH. As shown in Figure S4, it is found that the absorbtion responses of probe (10 µM) at 578 nm are linearly proportional to the amount of Cys and Hcy from 0 to 100 µM (Figure S4b-c). The detection limits for Cys and Hcy were determined as 2.965 μM and 6.140 μM based on S/N = 3, respectively. The absorbtion response of probe (10 µM) at 578 nm is linearly proportional to the amount of GSH from 0 to 180 µM (Figure S4d). The detection limit for GSH was calculated to be as 6.847 μM based on S/N = 3. In consideration of these results, probe could be applied to sense intracellular Cys/Hcy/GSH. Selective Response of Probe to Cys/Hcy/GSH. Further, we studied the selectivity of probe toward other biologically related amino acids and representative anions through UV-Vis spectra in the same conditions (Figure 3). Experimental results demonstrate that probe can simultaneously and selectively sense Cys/Hcy, and GSH over other various analytes.

Figure 4. Confocal fluorescence images of Cys, Hcy and GSH in HepG-2 cells. λex=405 nm, λem=465-525 nm for the blue channel; and λex=514 nm, λem=550-620 nm for the green channel, λem=700750 nm for the red channel.

In Vivo Imaging. In order to further expand the application scope of the probe, we applied it for in vivo imaging of exogenous Cys, Hcy and GSH in mice. The solutions of Cys, Hcy and GSH (220 μM) were injected into three places of



peritoneal cavity of mice (“a” position for Cys, “b” position for Hcy and “c” position for GSH), followed by intraperitoneal injection of probe (10 μM), respectively. As shown in Figure 5A, no fluorescence was observed in the control group (injection of Cys/Hcy/GSH but no probe). Subsequently, a bright fluorescence signal was detected from “a” position of mice and a slightly bright fluorescence signal was detected from “c” position of mice after 30 minutes of the probe injection (Figure 5B). During the following 45, 60, 75 and 90 min, the fluorescence intensities of “a” and “c” positions were almost invariable, but the fluorescence intensities of “b” position were increased over time (Figure 5C-F). These results demonstrated that probe could be used to simultaneously sense Cys, Hcy, and GSH in mice.

Figure 5. In vivo imaging of exogenous Cys, Hcy and GSH in the peritoneal cavity of mice using probe. (A): the control group (injection of Cys/Hcy/GSH but no probe). (B-F): timedependent images of mice via injection of Cys/Hcy/GSH and probe. Mice were imaged with IVIS imaging system using 475 nm excitation light source and an emission filter of 560 ± 10 nm.

CONCLUSION In summary, we have designed a NIR fluorescence probe with three binding sites for simultaneous sensing Cys, Hcy and GSH. The probe displays high selectivity and high sensitivity for Cys/Hcy and GSH. Moreover, the probe is able to simultaneously monitoring Cys/Hcy and GSH in HepG-2 cells and in mice. All these results are based on multiple binding sites and near infrared probe. We hope that the multiple binding sites NIR fluorescent probe can inspire the exploration to simultaneously sense Cys, Hcy and GSH in vivo to promote the research of them in biomedicine and diagnostics.

ASSOCIATED CONTENT Supporting Information Experimental details and additional spectroscopic data as noted in text. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Caixia Yin *E-mail: [email protected]

Author Contributions These authors contributed equally to this work.

ACKNOWLEDGMENTS

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We thank the National Natural Science Foundation of China (No. 21672131, 21775096, 21705102), Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061) and Scientific Instrument Center of Shanxi University, Institute of Pharmacology and Toxicology Academy of Military Medical Sciences PLA, Peop. Rep. China. We also thank Dr Y. S. Yao for his assistance in in vivo imaging.

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