Catechol amide derivatized polyhydroxylated fullerene as potential chelating agents of radionuclides: Synthesis, reactive oxygen species scavenging, and cytotoxic studies

Journal Pre-proof Catechol amide derivatized polyhydroxylated fullerene as potential chelating agents of radionuclides: Synthesis, reactive oxygen species scavenging, and cytotoxic studies

Tian Zheng, Xinru Wan, Qingchun Zhang, Bo Jin, Ru-Fang Peng PII:

S0162-0134(19)30497-0

DOI:

https://doi.org/10.1016/j.jinorgbio.2019.110921

Reference:

JIB 110921

To appear in:

Journal of Inorganic Biochemistry

Received date:

26 July 2019

Revised date:

8 October 2019

Accepted date:

11 November 2019

Please cite this article as: T. Zheng, X. Wan, Q. Zhang, et al., Catechol amide derivatized polyhydroxylated fullerene as potential chelating agents of radionuclides: Synthesis, reactive oxygen species scavenging, and cytotoxic studies, Journal of Inorganic Biochemistry (2018), https://doi.org/10.1016/j.jinorgbio.2019.110921

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© 2018 Published by Elsevier.

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Catechol amide derivatized polyhydroxylated fullerene as potential chelating agents of radionuclides: Synthesis, reactive oxygen species scavenging, and cytotoxic studies Tian Zheng a, b, Xinru Wan a, b, Qingchun Zhang a, Bo Jin*, a, b Ru-Fang Peng*, a, b a

State Key Laboratory of Environmental-friendly Energy Materials, Southwest University of Science and Technology, Sichuan Mianyang 621010, China.

b

Department of Chemistry, School of Materials Science and Engineering, Southwest

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University of Science and Technology, Mianyang 621010, China.

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E-mail: [email protected]; [email protected]; Tel/Fax:86-816-6089399.

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Abstract

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Radionuclide internal contamination can induce chemical and radioactive intoxication

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and produce harmful free radicals in vivo. At present, administration of chelating agents is the most effective treatment against nuclide contamination. However,

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traditional studies on chelating agents have ignored the damage caused by free

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radicals to the body. The present study aimed to develop a type of a bifunctional sequestering agent that can chelates nuclides and scavenges free radicals simultaneously. Therefore, a novel catechol amide-derivatized polyhydroxylated fullerene was designed and prepared. The poor water solubility of fullerene was ameliorated by chemically modifying hydrophilic catechol amide and multiple hydroxyl groups, and obtaining high water-soluble fullerene derivatives. The affinities of chelators were investigated via sulfochlorophenol competitive complexing method and antioxidant capacities were examined by electron paramagnetic resonance. The results revealed the good complexation of the designed and synthesized chelating agent with uranyl ions; and its efficiency in scavenging hydroxyl radicals. This 1

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chelating agent showed extremely low toxicity and notable protective effect against oxidative stress on A549 cells. Besides, in U(VI)-exposed A549 cells, immediate treatment with catechol amide-derivatized polyhydroxylated fullerene significantly decreased the lactate dehydrogenase (LDH) release by inhibiting the cellular U(VI) intake, promoting the intracellular U(VI) release and inhibiting the production of

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intracellular reactive oxygen species (ROS). These results suggest that this fullerene derivative may be a valuable in vivo antioxidant and radionuclide decorporation agent.

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Keywords: tetradentate catechol derivative; polyhydroxyfullerene; nuclide chelation;

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free radical scavenging; cytotoxicity

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1. Introduction

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With the development of science and technology, actinides have become widely

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used in nuclear, civilian, and military applications[1,2]. These radioactive elements pose an increased health risk to the body[3]. As a result of nuclear leakage and

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proliferation caused by unexpected events, radionuclide can be ingested through the respiratory tract, wound and epidermis, which in turn produce harmful free radicals that can induce apoptosis[4]. Application of chelating agents is the most eff ective method for removing radioactive elements, chelating agents form complexes with these radioactive elements to accelerate their excretion through urine and feces[5,6]. Numerous

chelating

agents,

including

catecholamides[7-11],

hydroxylpyridones[12-16], and azamacrocyclic derivatives[17,18], have been synthesized and investigated for their complexing capacities with radioactive elements. Catecholamide ligands have attracted widespread attention because of their high

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affinity and potential clinical applications as decorporation agent for heavy metal ions[19-21]. Leydier et al. reported the preparation of several sulfocatecholamide (CAMS) ligand based calixarene and evaluated their uranyl removal capabilities by using log Kcond up to 16.3 at pH=7.4[7]. Leydier et al. further synthesized a series of modified ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) derivatives with substituted amines and observed that EDTA-CAMS

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displays high complexation efficiency with a uranyl cation at neutral pH[22]. Queiros

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et al. demonstrated the synthesis of the new tripodal hexadentate chelator (catTHC)

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with CP256 as skeleton and catechol as unit; the stability of the complex was

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analyzed under different pH conditions, catTHC/Fe3+ (log β110=36.7) exhibiting [23] . 110=34.4)

Therefore, catecholamine can

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higher stability than ligand CP256 (log β

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be a good candidate as a radionuclide chelating agent. However, almost all studies regarding chelating agents excluded free radical scavenging ability and disregarded

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the inherent risks of free radicals. Thus, actinides must be eliminated from the body by applying non-toxic water-soluble chelating agents with antioxidation capabilities to scavenge free radicals.

Fullerenes and their derivatives have been thoroughly explored for various biomedical applications[24,25]. Given its unique electron-deficient structure, fullerene has been regarded as a “free radical sponge” and has been demonstrated to be a valuable candidate as a free radical scavenger[26,27]. However, the solubility of fullerene derivatives may limit their applications in biological systems. Fullerenes are generally modified in order to solve the problem of aqueous insolubility[28]. Studies

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have shown that increasing the polar groups on fullerene molecule, reduces its hydrophobic nature, without compromising its biological activity[29]. Poly hydroxylated derivatives of fullerene, generally known as fullerenols (C60(OH)x), are highly hydrophilic in nature and can perform intracellular penetration[30]. The free radical scavenging capability of fullerenols has been extensively studied. For example, Chiang et al. discovered that fullerenol exhibits a good scavenging effect on

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superoxide free radicals[31]. Sun et al. have indicated that fullerenols can not only

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scavenge superoxide free radicals, but also exhibit good scavenging effect on

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hydroxyl radicals[32]. Wang et al. described the preparation of polyhydroxylated

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gadofullerenol, which can scavenge active oxygen free radicals better than fullerenol,

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and determined its unique biological activities, such as anti-pulmonary fibrosis, action

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and cell protection[33, 34]. However, the application of modified fullerene derivatives to promote radionuclide emissions has been barely studied.

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Therefore, a rational compound composed of fullerene and catechol was designed in this study. The catechol group with good chelating property was introduced into the fullerene carbon cage, and numerous hydroxyl groups were further introduced to render the compound highly water soluble. This novel sequestering agent possesses nuclide complexation and good free radical scavenging capability. To the best of our knowledge, such fullerene derivatives remain unreported. 2. Materials and methods 2.1. Materials C60 was purchased from Puyang Yongxin Fullerene Technology Co.,Ltd. The

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organic reagents were commercially available from Aladdin and used without purification. Anhydrous solvents were purchased from Chengdu Kelong Chemical Reagents

Co.,

Ltd,

China.

CH2Cl2

was

distilled

before

use.

5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Sigma-Aldrich. Uranyl nitrate was brought from Hubei ChuShengWei Chemistry Co., Ltd, China.

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Cell Counting Kit-8 (CCK-8) was obtained from MedChemExpress Co., Ltd, China. Flash chromatography was performed on 300-400 mesh silica gel from Qingdao

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Hailang. Other reagents and solvents were all commercially available and used as

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received.

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2.2. Preparation of catechol amide derivatized polyhydroxylated fullerene

Scheme 1 Synthesis of catechol amide-derivatized polyhydroxylated fullerene Scheme 1 presents the synthetic route of catechol amide-derivatized polyhydroxylated fullerene. Tetradentate ligand 4 was prepared in accordance with

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Journal Pre-proof previously published methods[35,36]. Bingel reaction was used to synthesize single-addition catechol amide-derivatized fullerene. In brief, 72.0 mg C60 (0.1 mmol) was fully dissolved in 50 mL of toluene solution, and 10 mL of anhydrous dichloromethane containing 0.1 mmol 4, 49.7 mg CBr4 (0.15 mmol), and 23 L DBU (0.15 mmol) was then added. The mixture was magnetically stirred for 30 min in ice

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bath. The yellowish brown solid 5 was obtained after vacuum rotary evaporation to remove the solvent and purified by silica gel chromatography (chloroform/ethanol =

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30:1). Approximately 51 mg catechol amide-derivatized fullerene 5 (0.04 mmol) was

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then dissolved in 40 mL chloroform. Approximately 0.4 mL dichloromethane

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containing 0.1 M BBr3 was then added drop-wise to the previous solution with

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continuous stirring at -15 oC. The reaction mixture was stirred for 12 h at room

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temperature until discoloration of the chocolate-brown chloroform solution, followed by brown-colored precipitation. The precipitate 6 was collected and washed 3-4 times

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in sequence with chloroform, deionized water, and ethanol. The precipitate was then dried and stored for further use. Phase-transfer-catalyzed reaction was used to synthesize water-soluble catechol amide-derivatized polyhydroxylated fullerene[37]. In 10 mL aqueous solution of 50 mg polyethylene glycol and 20 mg sodium hydroxide, the catechol amide-derivatized fullerenes 6 solution of toluene/dimethylformamide (25:2 v/v) was added drop-wise with continuous stirring at room temperature. The reaction mixture was stirred continuously until discoloration of brown solution, followed by formation of a dark brown precipitate. The solvent was removed by vacuum rotary evaporation. Approximately, 50 mL pure water was then added, and

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the mixture was stirred for another 5 min, filtered and the insoluble substance was removed. The brown filtrate was concentrated, methanol was added, and catechol amide-derivatized polyhydroxylated fullerene 7 was precipitated from the water solution. The reprecipitation procedure was repeated thrice. 2.3. Characterization of catechol amide derivatized polyhydroxylated fullerene

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Nuclear magnetic resonance (NMR) spectra were recorded with Bruker AVANCE 600 spectrometers in CDCl3 or CD3OD. Fourier-transform infrared (FT-IR)

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spectra were recorded on a Nicolet 380 FTIR spectrophotometer (Thermo Fisher

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Nicolet, USA) in KBr with a 4 cm-1 resolution from 400 cm-1 to 4000 cm-1. An

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ultraviolet-visible (UV-Vis) spectrophotometer (Thermo Scientifc Evolution 201,

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USA) with a double-beam light source from 190 nm to 1100 nm was used. Mass

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spectrometry (MS) was conducted using Biflex Ⅲ MALDI-TOF. X-ray photoelectron spectroscopy (XPS) (Thermo VG 250, USA) was also performed.

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2.4. Complexation studies

A screening method was performed using the method developed by Taran et al.[38] based on competitive uranium binding with sulfochlorophenol (SCP) as ingrain agent. Affinity constants towards uranyl ion were determined through competitive displacement between the ligand and complex UO2SCP. The ligand complexation constant (Kconcd) was evaluated and compared with the values of complexes, Kconcd SCP/UO2. The effect of competitive ligand addition was visualized by naked-eye observation or quantified by UV-Vis spectroscopy by staining at 690 nm using a 96-well absorbance reader.

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Journal Pre-proof SCP (100 μM), SCP/UO22+ (100 μM), and 7 solution (400 μM) were prepared by dissolving the reagents in buffer solution at pH 5.5, 7.4, and 9.0, respectively. The solution containing 200 μL SCP/UO22+ (100 μM ) and 50 μL 7 solution (400 μM) was added to the pores of a clean 96-well plate. All experiments were performed in duplicate. Control experiment: SCP alone (200 μL of 100 μM SCP, 50 μL of buffer), SCP/UO22+ (200 μL of 100 μM SCP/UO22+ and 50 μL of buffer solution), 7 alone

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(200 μL of buffer and 50 μL of 400 μM 7), and SCP/7 solution (200 μL of 100 μM

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SCP, and 50 μL of 7) were set as control groups to calculate the percentage of

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SCP/UO2 displacement or to prevent that the competitive ligand from interfering with

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the UV-Vis properties of SCP. All solutions were mixed evenly and stored at room

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temperature for 36 h. Absorbance measurements were performed on an absorbance

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plate reader by staining at 690 nm. SCP displacement rate can be calculated using Eq.s (1), and log Kcond can be calculated as the F. Taran[38].

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Displacemen t (%) 

A 690 A SCP  A blank

(1)

2.5. Scavenging of hydroxyl radical The scavenging capacity of hydroxyl radicals by catechol amide-derivatized polyhydroxylated fullerene 7 was detected by electron paramagnetic resonance (EPR). H2O2 was applied to produce massive hydroxyl radicals under UV irradiation, and DMPO was employed as the radical capturer. In brief, a solution containing 100 μL DMPO (100 mM) and 50 μL of H2O2 (1 M) was separately mixed with 50 μL catechol amide-derivatized polyhydroxylated fullerene 7 at various concentrations (0, 25, 50,

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Journal Pre-proof and 100 M). The solutions were first irradiated under 500 W UV light for 4 min, and the X band EPR spectrum was then recorded in the dark. 2.6. Cell viability assessment For cytotoxicity detection, pulmonary adenocarcinoma (A549) cells were cultured in 96-well plates at 37 °C for 3 h with various concentrations of catechol

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amide-derivatized polyhydroxylated fullerene 7 (50, 100, 150, and 200 μM). The cells were then treated with 1 mM H2O2 for 1 h. The medium was then superseded with

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fresh medium and incubated in the dark at 37 °C for 24 h to assess cell viability. Cell

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viability was evaluated by the activity of mitochondrial dehydrogenase using a

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CCK-8 (MCE, Shanghai). The absorption value (optical density) at 450 nm was

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recorded using a 96-well plate reader (iMark microplate reader, Bio-take, Taiwan).

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Each treatment was repeated thrice. Cell viability was calculated as follows: OD sample  OD blank

OD control  OD blank

100 %

（2）

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Cell Vability (%) 

2.7. Fluorescence co-staining and imaging A549 cells were cultured with the samples at various concentrations (50, 100, 150, and 200 μM) for 3 h. The cells were co-stained with calcein acetoxymethyl ester (calcein AM)/propidium iodide (PI) in the dark for 15 min and then washed with phosphate-buffered saline. Fluorescence imaging was performed with a fluorescence microscope. A 490 nm laser was used to excite calcein AM and PI, and fluorescence signals were recorded from 500 nm to 530 nm for calcein AM and from 600 nm to 630 nm for PI. 2.8. Measurements of lactate dehydrogenase (LDH). 9

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To evaluate U(VI)-induced cell injury, the release of LDH into the medium was detected with the production of pyruvic acid-2,4-dinitrophenylhydrazones, utilizing lactate as a substrate. Briefly, A549 cells grown on a 35 mm dish were cultured with 400 μM UO2(NO3)2·6H2O and various concentrations catechol amide-derivatized polyhydroxylated fullerene 7 (50, 100, 150 and 200 μM ) for 24 h. The untreated cells

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and U(VI)-exposed alone cells were set up as the controls. At the end of the incubation period, the liquid supernatant were collected. According to the LDH

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activity assay kit (Guangzhou Weibo Technology Co., Ltd., Guangzhou, Guangdong

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Province, China), the activity of LDH was detected at λ = 450 nm on a 96-well plate

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reader (iMark microplate reader, Bio-take, Taiwan).

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2.8. Statistical analysis

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Data obtained from various studies were subjected to statistical analysis for inference. One-way analysis of variance was employed for all comparisons, as is most

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prescribed for such data sets[39]. Unless otherwise mentioned, the level of significance was p<0.05. All studies were repeated thrice, unless mentioned and the data are presented in the form of mean ± standard error. 3. Results and discussion 3.1. Characterizations The synthesis and characterization of compounds 2-4 were presented in Supporting Information (SI). Figure 1a presents the UV-Vis spectra of compounds 5 and 6; the UV-Vis spectra displayed a characteristic peak at 430 nm, which corresponds to the diagnostic absorption for the 1,2-adduct of C60[40, 41]. As shown in

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Figure 1b, given the introduction of numerous hydroxyl groups, the UV-Vis spectra of hydroxylated fullerene 7 differed remarkably from those of compounds 5 and 6. Hydroxylated fullerene 7 mainly exhibited a marked absorption band with a maximum at 190 nm, corresponding to the characteristic absorption peak of fullerenol[22]. Fig. 1

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Figure 2 (a, b, and c) portray the FT-IR spectra of catechol amide-derivatized

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fullerene 5, deprotection product 6, and polyhydroxylated catechol amide-derivatized

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fullerene 7, respectively. As shown in Figure 2, catechol amide remained on fullerene

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after hydroxylation. The characteristic adsorption peaks at 3435-3434 cm-1 were

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attributed to the -OH and -NH stretching vibrations of fullerenol and catechol amide. The characteristic absorption peak at 2927-2918 cm-1 of saturated C-H was observed,

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and the absorption peak at 1720-1635 cm-1 indicates stretching vibration of C=O and

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C=C. In addition, the small peaks at 1535-1375 cm-1 were ascribed to the skeleton vibration of benzene ring.

Fig. 2

Figure 3 portrays the 1H NMR spectra of fullerene derivative 5, which showed that catechol amide was successfully grafted on fullerene cage. As shown in Figure 3, the aromatic region of catechol amide was observed in the region of of 7.6054 to 6.9801. Peaks at  of 8.4924 and 8.3751 were ascribed to -CO-NH- of catechol amide. Peaks at  of 3.9246 to 3.6966 showed the -OCH3 and -CH2-CH2- groups of catechol amide. High-resolution mass spectrum (HRMS) was used to further verify the structure of compound 5 (see SI, Figure S5). The calculated value of compound 5 was 11

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m/z=1234.2056, and the observed value was m/z=1234.2066. Given the poor solubility of the compound 6, further characterization cannot be performed. Given the uncertainty in the number of hydroxyl groups, we have attempted to characterize compound 7 by NMR and MS without obtaining the expected results. Fig. 3

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To further confirm the structure of catechol amide-derivatized polyhydroxylated

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fullerene 7 and calculate the number of hydroxyl groups, we conducted XPS on the

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modified derivative 7. The XPS survey scan spectra (Figure 4a) of the peak of

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catechol amide-derivatized polyhydroxylated fullerene 7 indicated binding energies at

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531.9, 399.3, and 285.3 eV, which were ascribed to O (1s), N (1s), and C (1s), respectively. As shown in Figure 4b, the peaks at 289.4 eV were ascribed to the C of

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catechol and alkyl groups, whereas those, at 285.3 eV were ascribed to sp2.28 C of

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fullerene[42]. The peaks at 531.9 eV were ascribed to the O of hydroxyl groups (Figure 4c). The N (1s) peaks at 399.3 eV showed that the fullerenes were modified with catechol amide, which contained the benzamide groups in Figure 4d. The average substituted number of hydroxyl groups on fullerene can also be calculated by atom percent (at %) of fullerene derivatives 7 from XPS. The average numbers of hydroxyl groups was 26. Fig. 4

3.2. Complexation studies Fig. 5

Figure 5 shows that, absorption intensity of SCP increased with the increase of 12

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addition amounts of 7. The addition of a stronger ligand 7 than SCP displaced the equilibrium towards the formation of UO2-7 (Figure 5), decreased the absorbance value of complex SCP/UO2, and changed the color of medium. The ligand complexation constant (Kconcd) was evaluated and compared with the values of complexes Kconcd SCP/UO2. The catechol amide-derivatized polyhydroxylated

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fullerene 7 was then screened in a parallel manner on microtiter plates for their uranium binding properties under various pH conditions, that is, acidic, neutral, and

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basic conditions at pH 5.5, 7.4, and 9.0, respectively, by using SCP as a reference

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chelate (see SI, Tables S2-3 ). The results are summarized in Table 1. From the

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results, Kconcd enhancement was observed in basic conditions in accordance with

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previous findings[43], displaying remarkable displacement and a larger complexation

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efficiency compared with EDTA and DTPA[22]; compoud 7 exhibited sufficient

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affinities to uranium decorporation.

Table 1

3.3. Hydroxyl radical scavenging capability Fig. 6

In this study, we employed EPR techniques to systematically provide direct evidence that catechol amide-derivatized polyhydroxylated fullerene 7 can effectively scavenge free radicals. Hydroxyl radicals were selected as representative substances for discussion. As shown in Figure 6a, catechol amide-derivatized polyhydroxylated fullerene 7 exhibited excellently capability to eliminate hydroxyl radicals by reducing the intensity of DMPO-OH adduct. The EPR signal of DMPO-OH adduct intensity

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was reduced by the addition of various concentrations of fullerene derivative 7 (Figure 6a). Reductions in signal intensities were approximately 28.69%, 55.44%, and 73.26% at concentrations of 25, 50 and 100 M, respectively (Figure 6b). The above results showed that catechol amide-derivatized polyhydroxylated fullerene 7 scavenged most of the generated hydroxyl radicals (ca.55%), implying a slightly poorer condition than that observed for C60(OH)22 (ca.66%)[20], but superior to that of

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C70-OH NPs (ca.48%) at the same concentration (50M)[21]. The introduction of

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catechol amide groups increased the steric resistance on the fullerene sphere, and this

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finding might be associated with the compound’s reduced capacity to scavenge free

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radicals.

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3.4. Protective effect against cellular oxidative stress Fig 7

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To assess the potential cytotoxicity of compound 7 to cells, we quantitatively

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determined the relative viabilities of A549 cells after incubation with compound 7 for 24 h under various concentrations (50-200 μM). A549 cells in the control group was incubated without H2O2 or fullerene derivative 7. Compared with control group, treatment of A549 cells with 1 mM H2O2 for 2 h resulted in remarkable cellular toxicity, which was measured as the loss of mitochondrial dehydrogenase activity. Concentration dependent protection against cytotoxicity was observed following treatment with fullerene derivative 7. A549 cells presented higher viabilities than the blank group at 0 μM after H2O2 treatment, suggesting the low sample cytotoxicity of the studied compound (Figure 7). Compound 7 exhibited notable protective effects

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against H2O2-induced cytotoxicity. We also conducted fluorescence microscopy for visual confirmation of cytotoxicity. After catechol amide-derivatized polyhydroxylated fullerene 7 treatment, A549 cells were stained with calcein AM and PI to distinguish live (green) and dead (red) cells. From the fluorescence images (Figure 8), no interference was observed

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with the addition of fullerene derivative 7. Most of the cells were green, whereas

exposure of compound 7.

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Fig. 8

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several red cells were also observed, indicating that most cells survived in the

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The above results indicate that catechol amide-derivatized polyhydroxylated

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fullerene 7 exhibited low cytotoxicity and an evident protection against cellular oxidative stress at a low concentration. Derivatization of the fullerene cage with

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carboxyl and hydroxyl groups dramatically decrease in the cytotoxicity and reactive

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oxygen species generation of ROS[44,45], agreeing with the results of our research. 3.5 Measurements of lactate dehydrogenase (LDH). Fig. 9

To confirm the chelating agents 7 can effectively protect against cell damage caused by U(VI), the release of LDH into the medium was detected. A549 cells in the control group was incubated without UO2(NO3)2·6H2O or fullerene derivative 7. A549 cells in the blank group (0 M) was treated with 400 M U(VI) without fullerene derivative 7. Compared with control group, incubation of A549 cells with 400 M U(VI) for 48 h increased cell injury and augmented LDH release by 3-folds (Fig. 9). Concentration dependent protection against cytotoxicity was observed 15

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following treatment with fullerene derivative 7. When cells were co-treated with U(VI) and various concentration of chelating agents 7, LDH release was significantly decreased with increasing catechol amide-derivatized polyhydroxylated fullerene 7 concentration. The above results show that chelating agent 7 could protect cells against oxidative stress from UO2 exposure.

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4. Conclusion

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In this work, we demonstrated the straightforward preparation of novel catechol amide-derivatized fullerene as potential radionuclide chelator. The plurality of

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hydroxyl groups were excellently introduced to solve the water-solubility problem of

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fullerene. The affinities of the ligands for radionuclides were investigated using SCP

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competitive method at physiological pH, and the results showed the higher efficacy of

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ligands for uranyl ion than traditional chelating agents (EDTA and DTPA). The EPR spin-trap technique provided direct in vitro evidence that fullerene derivative possess

agents

do

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excellent radical scavenging capability, which is a property that traditional chelating not

amide-derivatized

possess.

fullerenes

Compared showed

with

C70-OH

superior

nanoparticles,

radical-scavenging

catechol capability.

Consequently, the authors proposed that fullerene-based chelating agent 7 with the bi-function of accelerating the U(VI) removal and scavenging the radical or antioxidant effects can enhance the protective effects against cells and tissue damage induced by internal exposure of radionuclides. This may be a effective treatment modality for radionuclides internal contamination. The prepared fullerene derivative can be applied to facilitate radionuclide decorporation in vivo, and is expected to

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feature potential as a radionuclide chelator. 5. Experimental Section 5.1. General All compounds used were reagent grade or better, solvents were used as received unless otherwise specified. The following regents were used as received, C60 (99.5+%, Crop.),

1,8-diazabicyclo[5.4.0]undec-7-ene,

malonyl

dichloride,

CBr4,

of

MER

2,3-dimethoxybenzoic acid were purchased by Aladdin. The water was ultrapure

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water (resistivity≤18.25 MΩ), other reagents were analytical grade or better.

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Whenever necessary, solvent were dried according to standard methods. All the

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silical-gel column.

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chemical recations were TLC controlled, and purify by the chromatography on a

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5.2. Synthesis and characterized

2

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Synthesis of N-(N’-tert-butyloxycarbonylethane diamino)-2,3-bis(methoxyl)benzamide

A mixture of 2,3-bis(methoxyl)benzoic acid 2 (2.18 g, 6.5 mmol), N-hydroxybenzotriazole (HOBt, 0.14 g, 1.0 mmol) and dicyclohexylcarbodiimide (DCC, 1.62 g, 7.8 mmol) was dissolved in 80 mL CH2Cl2 and stirred for 30 min. Then 1.04 g (2-aminobutyl) carbamic acid tert-butyl ester 1 (6.5 mmol) was added dropwise and the reaction proceeded at room temperature overnight. The obtained white solution was filtered to remove the dicyclohexylurea, and the filtrate was concentrated in vacuo. The residue was chromatographed on a silica gel column (ethyl acetate: petroleum ether, 2:3) to give 2 as yellow solid (2.52 g, 80%). Rf = 0.4. FTIR (KBr) v (cm-1): 3367 (N-H) 2939 (-CH2-), 1633 (C=O), 1574 (C-N-H), 1538, 1471, 1386, 1313, 1265, 1227, 1128, 1087, 992, 811, 754, 620. 1H NMR (600 MHz, CDCl3): δ 8.20 (s, 1H, CO-NH), 7.68 (m, 1H, Ar-H), 7.14 (m, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 17

Journal Pre-proof 3.90 (m, 6H, O-CH3, 3.57-3.60 (m, 2H, CH2), 3.38 (m, 2H, CH2), 1.42 (s, 9H, CH3). Synthesis of N-(3-aminopropyl)-2,3-bis(methoxyl)benzamide 3 A mixture of TFA (trifluoroacetic acid)/CH2Cl2 (20 vol%, 15 mL) was dropped into

a

solution

of

N-(N-tert-butyloxycarbonylethane

diamino)-2,3-bis(benzyloxy)benzamide 2 (0.95 g, 2.0 mmol) in CH2Cl2 (15 mL) at 0 o

C, and the obtained solution was stirred at room temperature for 3 h. Then, the

solution was concentrated in vacuo, and the residue was dissolved in 0.5 mL of hot ethanol with 30 mL ether added and the white crystal was precipitated. Then the was

filtered

to

give TFA

salt

as

white

solid.

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diamino)-2,3-bis(benzyloxy)benzamide,

N-(N-tert-butyloxycarbonylethane

of

solution

N-(aminoethyl)-2,3-bis(benzyloxy)-benzamide, TFA salt (1.96 g, 4 mmol) in 20 mL

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1.25 M NaOH aqueous solution was stirred 20 min. CH2Cl2 extraction (20 mL × 3),

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the organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to dryness to give N-(aminoethyl)-2,3-bis(methoxyl) benzamide 3 as clear oil (1.37 g,

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90%). 1H NMR (600 MHz, CDCl3): δ 8.29 (s, 1H, CO-NH), 7.68 (s, 1H, Ar-H), 7.16-7.11 (m, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 3.91 (s, 3H, O-CH3), 3.89 (s, 3H,

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O-CH3), 3.53 (t, J = 6.0 Hz, 2H, CH2), 2.94 (d, J = 6.1 Hz, 2H, CH2), 1.57 (s, 2H); 13C

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NMR (150 MHz, CDCl3): δ 165.50 (C=O), 152.56 (Ar-C), 147.44 (Ar-C), 126.87 (Ar-C), 124.31 (Ar-C), 122.59 (Ar-C), 115.25 (Ar-C), 61.28, 56.04, 42.50, 41.5. Synthesis of N,N’-bis(N’’-(aminoethyl)-2,3-bis(benzyloxy)benzamido)malonamide 4 A mixture of malonyl dichloride (0.28 g, 2 mmol) and Et3N (0.2 g, 2 mmol) was dissolved in 20 mL CH2Cl2 and then dropped to a solution of 3 in 20 mL CH2Cl2 under ice bath and vigorous stirring conditions. The stirring was maintained at room temperature for 5 h. Purification on silica gel (ethanol:CH2Cl2, 1:15) afforded 4 as clear oil (1.3 g, 80%). Rf = 0.7. FTIR (KBr) v (cm-1): 3434 (N-H), 2938 (-CH2-), 1639 (C=O), 1579 (C-N-H), 1534, 1473 (benzene ring), 1265, 1075, 748, 592; 1H NMR (600 MHz, CDCl3): δ 8.29 (s, 2H, CO-NH), 7.88 (s, 2H, Ar-H), 7.58 (d, J = 8.0 Hz, 2H, Ar-H), 7.10 (d, J = 8.0 Hz, 2H, Ar-H), 7.01 (d, J = 8.1 Hz, 2H, Ar-H), 3.87 (m, 12H, O-CH3), 3.58 (t, J = 5.8 Hz, 4H, CH2), 3.47 (d, J = 5.9 Hz, 4H, CH2), 3.22 (s, 2H, CH2).

13

C NMR (150 MHz, CDCl3): δ 168.04 (C=O), 166.65 (C=O), 152.57, 18

Journal Pre-proof 147.52, 126.46, 124.33, 122.43, 115.44 (Ar-C), 61.32, 56.02, 42.79, 39.70, 39.32. Synthesis of N,N’-bis(N’’-(aminoethyl)-2,3-bis(benzyloxy)benzamido)malonamide C60 derivative 5 N,N’-bis(N’’-(aminoethyl)-2,3-bis(benzyloxy)benzamido)malonamide (52 mg, 0.1 mmol) 6a was dissolved into CH2Cl2 (10 mL) and C60 (72 mg, 0.1 mmol) dissolved into toluene (50 mL), then CBr4 (49.7 mg, 0.15 mmol), DBU(23 L, 0.15 mmol) was added. The resulting mixture was stirred magnetically for 30 minutes in ice bath. The yellowish brown solid 5 (12.6 mg, 11%) was obtained after purification

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by silica gel chromatography (chloroform/ethanol = 30:1). FTIR (KBr) v (cm-1)：3434

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(N-H), 2925, 1719 (C=O), 1656 (C-N-H), 1576, 1523, 1458, 1384 (benzene ring), 1263, 1076, 1048, 752, 526. 1H NMR (600 MHz, CDCl3) δ 8.49 (s, 2H), 8.38 (s, 2H),

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7.60 (d, J = 8.0 Hz, 2H), 7.07 (t, J = 8.0 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 3.92 (s,

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6H), 3.85 (s, 6H), 3.75 – 3.69 (m, 8H). UV-Vis (CHCl3) λ/nm(log ε): 275, 330, 428. (MALDI-TOF) m/z calcd for C85H30N4O8 [M]+ 1234.2045, found 1234.2066.

derivative 6

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Synthesis of N,N’-bis(N’’-(aminoethyl)-2,3-bis(hydroxyl)benzamido)malonamide C60

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N,N’-bis(N’’-(aminoethyl)-2,3-bis(benzyloxy)benzamido)malonamide

C60

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derivative (51 mg, 0.04 mmol) 5 was dissolved into CHCl3 (40 mL) then the 0.4 mL CH2Cl2 containing 0.1 M BBr3 were added drop-wise with continuous stirring at 0 oC. The reaction mixture was magnetically stirred for 12 h until the discoloration of chocolate-brown colored chloroform solution, followed by brown colored precipitation. The precipitate 6 was collected and washed 3-4 times with chloroform, deionized water and ethanol, respectively. Later the precipitate was dried and stored for further use. FTIR (KBr) v (cm-1): 3435 (N-H), 2927, 1638 (C-N-H), 1535, 1460, 1370 (benzene ring), 1251, 1087, 744, 526. UV-Vis (CHCl3) λ/nm (log ε): 255, 326, 430. Synthesis

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Polyhydroxy-N,N’-bis(N’’-(aminoethyl)-2,3-bis(hydroxyl)benzamido)malonamide C60 derivative 7 Phase-transfer catalyzed reaction was used for synthesizing water soluble 19

Journal Pre-proof catechol amide derivatized polyhydroxylated fullerenes. In 10 mL aqueous solution of 50 mg PEG 4000 and 20 mg sodium hydroxide, catechol amide-derivatized fullerenes 6 solution of toluene/DMF (25:2 v/v) was added drop-wise with continuous stirring at room temperature. The reaction mixture was kept for stirring until the discoloration of brown solution, followed by dark brown precipitation. The solvent was removed by vacuum rotary evaporation, 50 mL pure water was added, stirred for another 5 minutes, filtered, the insoluble substance was removed, brown filtrate was concentrated,

and

methanol

was

added

and

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Polyhydroxy-N,N’-bis(N’’-(aminoethyl)-2,3-bis(hydroxyl)benzamido)malonamide C60

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derivative 7 was precipitated from the water solution. The reprecipitation procedure was repeated thrice. The final product 7 was obtained as the offwhite powder (32 mg,

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1375 (benzene ring), 1250, 1049, 599.

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65%). FTIR (KBr) v (cm-1): 3433 (N-H), 2918, 1720 (C=O), 1635 (C-N-H), 1455,

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Conflicts of interest

Acknowledgements

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The authors declare that they have no conflict of interest

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We are grateful for financial support from the Natural Science Foundation of China (51572230), Key Projects of the Pre-research Fund of the General Armament Department (project no. 6140720020101), National Defense Technology Foundation Project (project no. JSJL2016404B002) and the Institute of Chemical Materials, China Academy of Engineering Physics (project no. 18zh0080).

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[44] C.M. Sayes, A.M. Gobin, K.D. Ausman, J.J. Mendez, L. West, V.L. Colvin, Biomaterials. 26 (36) (2005) 7587-95. [45] C.M. Sayes, J.D. Fortner, W. Guo, D. Lyon, A.M. Boyd, Nano Lett, 4 (10) (2004)

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Fig. 1. UV-Vis spectrum of (a) compounds 5 and 6; (b) compound 7

Fig. 2. FT-IR spectra of (a) compounds 5, (b) 6, and (c) 7

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Fig. 3. 1H NMR spectrum of compound 5

Fig. 4. X-ray photoelectron spectra of catechol amide-derivatized polyhydroxylated fullerene 7: (a) Survey scan spectra of O, N, and C from 1000 eV to 0 eV, (b) curve-fitted high-resolution scans of C (fullerene, catechol, and alkyl), (c) curve-fitted high-resolution scans of O (1s), and (d) curve-fitted high-resolution scans of N (1s) 26

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Fig. 5. UV-Vis spectra of SCP-UO2 and SCP following the addition of increased amounts of

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7

Fig. 6. (a) EPR spectra of hydroxyl radicals captured by DMPO after treatment with various concentrations of 7 (b) Percentage of scavenging or inhibition of hydroxyl radicals

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Fig 7. Cell viability of A549 cells incubated with catechol amide-derivatized polyhydroxylated

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fullerene 7 at various concentrations (50-200 M) for 24h

Fig. 8. Fluorescence images of A549 cells co-stained with calcein AM and PI after incubation with catechol amide-derivatized polyhydroxylated fullerene 7 at various concentrations (50-200 M). Untreated cells were used as control.

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Fig. 9. Effects of catechol amide-derivatized polyhydroxylated fullerene 7 at various

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concentrations (50-200 M) on the LDH release of A549 cells exposed to U(VI). Untreated cells

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were used as control.

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Journal Pre-proof Table 1. Kconcd of 7-UO22+ at several pH values (displacement value range)

Log Kcond U-L/(pH) 5.5

7.4

9.0

15.5-16.2 (25.63%)

18.3-18.8 (34.03%)

EDTA

< 8 (0-20%)

< 13 (0-20%)

< 16 (0-20%)

DTPA[22]

< 8 (0-20%)

< 13 (0-20%)

< 16 (0-20%)

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< 10.5 (18.96%) [22]

7

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A novel high water-soluble catechol amide-derivatized polyhydroxylated fullerene was designed and prepared. The performance test suggested that this fullerene derivative may be a valuable in vivo antioxidant and radionuclide decorporation agent.

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Tian Zheng a, Xinru Wan b, Qingchun Zhang a, b, Bo Jin*, a, b, Ru-Fang Peng*, a, b

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Title Catechol amide derivatized polyhydroxylated fullerene as potential chelating agents of radionuclides: Synthesis, reactive oxygen species scavenging, and cytotoxic studies

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Graphical abstracts

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Highlights

1) A novel fullerene-based potential radionuclide chelator was firstly prepared. 2) The higher efficacy of ligands for uranyl ion than traditional chelating agents was observed. 3) The chelator showes superior radical-scavenging capability.

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4) The chelator exhibits extremely low cytotoxicity and protective effect against oxidative stress.

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5) The chelator is expected to feature potential as a radionuclide chelator applying to human

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body .

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