Copper-Driven Deselenization: A Strategy for Selective Conversion of

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Biological and Medical Applications of Materials and Interfaces

Copper-driven Deselenization: A Strategy for Selective Conversion of Copper Ion to Nanozyme and Its Implication for Copper-Related Disorders Ashish Chalana, Ramesh Karri, Ranajit Das, Binayak Kumar, Rakesh Kumar Rai, Himani Saxena, Ashish Gupta, Mainak Banerjee, Kunal Kumar Jha, and Gouriprasanna Roy ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16786 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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ACS Applied Materials & Interfaces

Copper-Driven Deselenization: A Strategy for Selective Conversion of Copper Ion to Nanozyme and Its Implication for Copper-Related Disorders Ashish Chalana§, Ramesh Karri§, Ranajit Das§, Binayak Kumarǂ, Rakesh Kumar Rai§, Himani Saxenaǂ, Ashish Gupta†, Mainak Banerjee§, Kunal Kumar Jha§, and Gouriprasanna Roy*§ §Department

of Chemistry, ǂDepartment of Life Sciences, School of Natural Sciences, Shiv Nadar University, NH91, Dadri, Gautam Buddha Nagar, UP 201314, India KEYWORDS: nanozyme • copper selenide • imidazole-based selones • oxidative stress • copper toxicity ABSTRACT: Synthetic organic molecules which can selectively convert intracellular copper (Cu) ions to nanozymes with an ability to protect cells from oxidative stress are highly significant in developing therapeutic agents against Cu-related disorder like Wilson’s disease. Here we report 1,3-bis(2-hydroxyethyl)-1H-benzoimidazole-2-selenone (1) which shows an remarkable ability to remove Cu ion from glutathione, a major cytosolic Cu-binding ligand, and thereafter converts it into copper selenide (CuSe) nanozyme that exhibits remarkable glutathione peroxidase (GPx)-like activity, at cellular level of H2O2 concentration, with an excellent cytoprotective effect against oxidative stress in hepatocyte. Cudriven deselenization of 1, under physiologically relevant conditions, occurred in two steps. The activation of CSe bond by metal ion is the crucial first step followed by cleavage of the metal-activated CSe bond, initiated by the OH group of N(CH2)2OH substituent through neighboring group participation (deselenization step), resulted the controlled synthesis of various types of Cu2-xSe nanocrystals (nanodiscs, nanocubes and nanosheets) and tetragonal Cu3Se2 NCs, depending upon the oxidation state of the Cu ion used to activate the CSe bond. Deselenization of 1 is highly metal selective. Except Cu, other essential metal ions including Mn2+, Fe2+, Co2+, Ni2+ or Zn2+ failed to produce metal selenide under identical reaction conditions. Moreover, no significant change in the expression level of Cu-metabolism-related genes, including metallothioneines MT1A, is observed in liver cells co-treated with Cu and 1, as opposed to the large increase in the concentrations of these genes is observed in cells treated with Cu alone, suggesting the participation of 1 in Cu homoeostasis in hepatocyte.

1. INTRODUCTION The copper (Cu), an essential trace element to humans, is involved in numerous biological processes in our body. However, the excess Cu is equally detrimental as it produces hydroxyl radical (OH) from H2O2 via Fenton-type reactions, and thereby causes oxidative damage to proteins, lipids, and nucleic acids. The intracellular Cu concentration is, thus, strictly maintained by Cu storage proteins metallothioneines (MTs), Cu exporting proteins ATP7A and ATP7B, and intracellular Cu trafficking chaperones ATOX1 and CCS, and endogenous thiol, glutathione (GSH).1,2 The majority of cytosolic Cu is bound to GSH, the most abundant intracellular Cu binding ligand of low molecular mass in living cells and is known to be a major contributor to Cu exchangeable pool in the cytosol.3-6 Mutation of ATP7B gene results non-functional of ATP7B protein causing Cu over-load in tissues including liver, brain of patients with Wilson’s disease (WD) and excess Cu has been implicated in the progression of neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases.7-9 Medical therapy in WD involves lifelong treatment with Cu chelators (penicillamine, trientine) that bind Cu directly in

blood and tissues and facilitate its excretion.10 However, chelation therapy is not always efficient for symptomatic neurological patients and has harmful side effects and,11,12 thus, efforts were made to discover tissue specific chelators.13,14 An antagonism between Cu and Se in the biological system is reported in the literature.15-17 Inorganic selenium, sodium selenite (Na2SeO3), is known to provide significant protection against Cu-induced oxidative damage of DNA, protein, lipid and reduce Cu load in tissues including liver and kidney of animals fed high levels of Cu.18-20 However, the exact mechanism by which Na2SeO3 exerts its protective effect against Cu-induced toxicity is not clear. Several reports suggested the possible formation of copper selenide (CuSe hereafter) in tissues by Na2SeO3, which might reduce the bioavailability of Cu in the cellular system or help in excretion of excess Cu from body through bile.21-22 Unfortunately, Na2SeO3 does not reach to organs in its intact form and it is taken up by all organs after being metabolized to selenide in plasma,23-24 which again has strong tendency to form metal selenide with other essential metal ions.25 Thus, inorganic selenium, in general, is considered to be more toxic26,27 than

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organoselenium compounds which, recently, have attracted considerable attention due to their important role in many biochemical processes such as antioxidant, anticancer activity, and more recently their ability to reduce metal related toxicity.28-31 Moreover, organoselenium compound can easily be tuned for organ or tissue-specific delivery. GSH being a major Cu-binding ligand plays crucial role in Cu homoeostasis by transferring Cu to many proteins including ATOX1, CCS and MT in cell.4 Thus, the present study is aimed at the development of a new organoselenium compound which can selectively converts Cu ion into a product (rather than simple chelation), that is relatively redox inactive, biocompatible and have an antioxidant enzyme-like activity in controlling the intracellular H2O2 concentrations. This may have an enormous significance in combating excess Cu related disorders like Wilson’s, Alzheimer’s, and Parkinson’s diseases and, to the best of our knowledge, no such type of molecule has been reported in the literature so far. Herein we report a new selenium precursor 1 (Figure 1), having N-(CH2)2OH substituent at the 5-membered heterocyclic ring, which show remarkable ability to selectively convert Cu ion (Cu2+ or Cu+) to CuSe nanocrystal (NC hereafter), under physiologically relevant conditions, that exhibited excellent cytoprotective effect in hepatocyte against oxidative stress by degrading intracellular H2O2 concentration, similar to an antioxidant enzyme glutathione peroxidase (GPx). The activation of CSe bond followed by deselenization of 1 is highly selective to Cu ion (Cu2+ or Cu+). Other essential metal ions including Mn2+, Fe2+, Co2+, Ni2+ or Zn2+ failed to produce corresponding metal selenide under identical reaction conditions. Additionally, we show that the kinetics of deselenization of 1 and the morphology of the final product CuSe NCs can easily be controlled by simply changing the oxidation state of Cu ion. To the best of our knowledge, for the first time, here we report Cu-driven CSe bond cleavage, initiated by OH group of N-(CH2)2OH substituent, leading to the controlled production of cubic Cu2-xSe nanodiscs, nanocubes and nanosheets or tetragonal Cu3Se2 NCs under various reaction conditions.

confirmed the formation of Cu2-xSedc (Figure 2c) with binding energy values of Cu2p3/2, Cu2p1/2 and Se3d are 932.32, 951.96 and 54.70 eV, respectively.33 X-ray diffraction peaks at 26.8, 31.0, 44.6, 52.9, and 71.6 can be assigned as (111), (200), (220), (311), (331) planes of cubic Cu2-xSedc (JCPDS # 06-680), Figure 2e.34

Figure 1. Structures of 1-4 (a) and 5-7 (e). UV-Vis spectra of deselenization of 1 induced by CuOAc at 37 C (b) and the corresponding t50 values obtained in the reactions of 1/CuOAc and 1/Cu(OAc)2 (c). (d) TEM images and the scheme for the synthesis of Cu2-xSedc, Cu2-xSecu and Cu2-xSesh NCs, obtained from the reactions of 1/Cu(OAc)2, in the presence or absence of oleylamine (OA) under various reaction conditions. Table 1. Initial rates of deselenization of 1 and 2. SR NO.

Reactions

Temp.

t50

1a 2a 3b 4b 5b 6b 7b 8b

1/Cu(OAc)2 1/CuOAc 1/Cu(OAc)2 1/Cu(OAc)2 1/Cu(OAc)2 1/CuOAc 2/(CuOAc)2 2/CuOAc

37 °C 37 °C -5 °C 10 °C 37 °C 37 °C 37 °C 37 °C

12.5 h 8.9 h 35 h 6h 25 min 19 min 58 min 26 min

2. RESULTS AND DISCUSSION 2.1 Synthesis of CuSe NCs via Degradation of Cu(selone) Complexes. In order to develop a molecule which can selectively convert Cu ions (Cu+ or Cu2+) to biologically somewhat inert redox inactive CuSe species, at low concentration under physiologically relevant conditions, we have synthesized various types of imidazole and benzimidazole-based selones 1-7 with different N-substituents at the 5 member heterocyclic ring. When a light blue solution of Cu(OAc)2 (100 M) was treated with 1, in 1:1 molar ratio at 37 C, the color of the reaction mixture immediately turned to yellowish green, indicating the formation of Cu(selone) complex (max 318 nm, Figure 1b). However, to our surprise, we observed the gradual decrease of concentration of Cu(selone) complex and subsequently formation of black precipitates into the above reaction solution over time, which were isolated after completion of the reaction and characterized thoroughly. TEM, SEM, EDX and powder X-ray diffraction (XRD) patterns confirmed the formation of Cu2-xSe nanodiscs (Cu2-xSedc hereafter) with an average size of ca. 58 nm (Figures 1, 2 and S1).32 X-ray photoelectron spectroscopy (XPS) data further

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a[1]

Initial Rate (Mh-1) 3.9 ± 0.4 x 10-4 8.6 ± 0.5 x 10-4 2.5 ± 0.8 x 10-4 1.1 ± 0.14 x 10-3 7.0 ± 0.21 x 10-3 10.5 ± 0.1 x 10-3 3.7 ± 0.42 x 10-3 5.3 ± 0.12 x 10-3

= [Cu+/2+] = 100 M; b[1] = [2] = [Cu+/2+] = 0.01 M.

Characterization of supernatants of the above reaction mixture of 1/Cu(OAc)2 revealed the formation of the corresponding ketones 8 and 10 [m/z ([M+H]+): 223.1009 (8), 265.1165 (10)], and diselenide 12 (Figure 2b and Figure S2). The CSe bond cleavage of 1 was found to be critically dependent upon the nature of the metal ion (hard or soft) used to activate this bond. Cu+ being a soft Lewis acid than Cu2+, the deselenization reaction of 1 occurred 2.2 times faster in the presence of Cu+ ion than Cu2+ ion (Table 1, entries 1 and 2; Figure 1c). Interestingly, we found that the morphology of the NCs obtained in the reaction of 1 and Cu ion is critically


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dependent on the oxidation state of the Cu ion used. X-ray diffraction analysis of NCs obtained in the reaction of 1 and Cu+ acetate, CuOAc, confirmed the formation of umangite Cu3Se2 phase through fast-nucleation, instead of stable Cu2-xSe phase, Figure 2f. X-ray diffraction peaks at 25.0°, 27.3°, 28.4°, 31.1°, 36.6°, 42.4°, 45.2°, 46.5°, 48.2°, 53.1°, 61.6°, 73.9 can be assigned as (101), (200), (111), (210), (211), (002), (221), (112), (301), (212), (401) planes of tetragonal Cu3Se2 phase (JCPDS # 190-402).35

(- 5 C). Rate of CSe bond cleavage of 1 (or 2) induced by Cu+ or Cu2+ increased with increasing the reaction temperature. In contrast, the treatment of Cu(OAc)2 or CuOAc with other derivatives of 1 (5-7) that are lacking N-(CH2)2OH substituent failed to produce CuSe NCs under identical reaction conditions, at room temperature (21 C), indicating that the N-(CH2)2OH substituent of 1 or 2 plays a very crucial role in the deselenization process.

Figure 3. Structures of 16-18 (a) and ORTEP images of 15 (b) and 19 (c). Figure 2. (a) Mechanism for the formation of Cu2-xSe, cyclic intermediate-I, and 8-11. (b) Structures of 8-15. XPS (c) and energy-dispersive X-ray absorption spectroscopy pattern (d) of Cu2-xSedc obtained in the reaction of 1/Cu(OAc)2 at 21 °C. XRD patterns of Cu2-xSe (e) and Cu3Se2 (f) phase NCs.

Likewise, the treatment of Cu(OAc)2 with another derivative of 1 (2) having N-(CH2)2OH substituent, in 1:1 molar ratio, afforded 9 and 11 [m/z ([M+H]+): 193.0974 (9), 235.1068 (11)], 13 and Cu2-xSe NCs under identical reaction conditions (Figure S1). However, the initial rate of deselenization of 2 induced by Cu2+ was found to be almost 1.9 times slower than the initial rate of deselenization of 1, under identical reaction conditions (Table 1, entries 5 and 7). Additionally, like 1, the initial rate of deselenization of 2 induced by Cu+ acetate was 1.4 times faster compared to the initial rate obtained by Cu2+ acetate and, again, we have observed the formation of Cu3Se2 NCs when Cu+ acetate was used to activate C=Se bond of 2. Initial rates of CSe bond cleavage of 1 and 2 induced by Cu(OAc)2 or CuOAc at various concentrations and temperatures are shown Table 1 and the deselenization graphs are shown in Figures 1c and S3. Significantly, we noticed that the CSe bond cleavage and the gradual formation of CuSe NCs in the reaction of 1 and Cu(OAc)2 or CuOAc occurred even at low temperature

2.2 Mechanism of Copper-driven Deselenization and the Formation of CuSe NCs. In order to elucidate the mechanism and the role of acetate ion (weak base), we employed other Cu salts like CuCl2 or CuSO4 in our study. The treatment of 5, devoid of any N-(CH2)2OH substituent, with CuCl2 afforded an oxidized product, diselenide 18, which was accompanied by the concomitant reduction of Cu2+ to Cu+ followed by the formation of complex 17 (Figures 3 and S5) and no CuSe formation was observed, in this case, at 21 C.36,37 To our surprise, CuSe formation was also not observed in the reaction of 1 or 2 with CuCl2 or CuSO4 (10 mM), in 1:1 molar ratio, even after 4 h of stirring at 21 C. Instead, we observed the formation of the corresponding Cu(selone) complexes (14 and 15) and the corresponding diselenides (12 or 13). Slow evaporation of the methanolic solution of 2/CuCl2 afforded nice single crystals of 15. However, in the presence of 1.2 equiv of weak base (such as NaOAc, NEt3, oleylamine (OA) or histidine), 15 degraded and yielded CuSe NCs (Figure S4 and Table S1). However, the addition of excess Cu-binding ligand into the reaction solution slowed down the deselenization process. For instance, the treatment of 1 (10 mM) with 1 equiv of Cu(OAc)2 in 5 mL of methanol-OA (1:1) afforded cubic-shaped Cu2-xSe (Cu2-xSecu



hereafter) at 21 C with an average size ca. 18 nm (Figure 1d). However, the same reaction when performed only in OA (5 mL), no Cu2-xSe formation was observed, indicating that the presence of large excess of Cu-binding ligand preventing the activation of CSe bond. Nevertheless, at high temperature (175 C) the above reaction ([1]:[Cu(OAc)2] = 1:1) produced cubic-shaped Cu2-xSe (Cu2-xSecu hereafter) with average size ca. 20 nm, Figure S4. With 0.5 equiv of 1 ([1]:[Cu(OAc)2] = 0.5:1) at 175 C for 10 min afforded nice monodispersed Cu2-xSe NCs (avg. size ca. 33 nm), Figure 1d. Upon increasing reaction time from 10 to 40 min hexagonal-shaped Cu2-xSe nanosheets (Cu2xSesh hereafter) were isolated (avg. size ca. 246 nm). In contrast, Cu3Se2 phase was obtained in the reaction of 1 and CuOAc, in 1:1 molar ratio, at high temperature (175 C), Figure S4, confirming the reproducibility of umangite Cu3Se2 phase even at high temperature.

2.3 Crystallographic Studies, NMR Spectroscopy and Quantum Chemical Calculation. X-ray structure of 15 revealed that –OH group of N-(CH2)2OH substituent did not form any direct bond with the Cu center and remained free in the solid-state (Figure 3). 1H NMR studies also confirmed that the resonance corresponding to the –OH group of 2 (or 1) did not disappear after gradual addition of Cu(OAc)2 (0.25 – 1 equiv), Figure S6. These observations strongly suggest that the –OH group of 1 or 2 remained free in solution even after complexation with Cu(OAc)2. Crystal structure of 19, obtained from the reaction of the sulfur analogue of 1 (16) and CuCl2, showed that the one oxygen atom (O1) of N-(CH2)2OH substituent coordinated to the Cu center whereas the other –OH group remained free in the solid-state. From DFT calculations of selones and their Cu(selone) complexes, it is evident that free selone 1 or 2 exists as zwitterion, in which the Se atom carries a negative charge and the heterocycle carries delocalized positive charge, with CSe bond order is close to in between single and double bond, ranging from 1.34–1.38 (towards more single bond character), Figure S8, Table S2.38 Upon coordination to the Cu center through the p-type of Se lone pairs (HOMO and HOMO-1 of selone can form CuSe dative covalent bond), the CSe bond length of 15 increased slightly (0.007–0.021 Å) compared to that in free 2 (d(C2Se) in 2 = 1.844 Å;39 d(C2Se) in 15 = 1.865 Å and 1.851 Å). Accordingly, the CSe bond order of 15 further moved closer to the single bond (BO: 1.18 and 1.19). Thus, as a result of this electron donation from Se to Cu center, the overall positive charge of the benzimidazole ring, including C2 atom, of Cu-bound selone increased substantially (more zwitterionic form after coordination to metal center) in comparison to the free selone, leading to the facile cleavage of the Cu-activated CSe bond initiated by neighboring group participation of -OH group of N(CH2)2OH substituent, in the presence of weak base, resulted CuSe NCs and unstable intermediate–I, (Figure 2a),39 which converted into stable products 8–11 upon reacting with base. In contrast, the imidazole-based selone 3 or 4 (10 mM),40 in spite of having N-(CH2)2OH substituent, failed to produce CuSe when treated with 1 equiv of Cu(OAc)2 at 21 C (Figure S4h), indicating that the stabilization of conjugated benzimidazolebased cyclic intermediate–I is an additional driving force to cleave the Cu-activated CSe bond of 1 (or 2).

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2.4 Copper Selective Deselenization. Next we investigated whether other essential metal ions including Mn2+, Fe2+, Co2+, Ni2+ or Zn2+, have similar ability, like Cu (mostly present in +1 state in cellular system), to activate the CSe bond and facilitate the deselenization reaction under identical reaction conditions. To our surprise, treatment of 1 (100 M) with MCl2 (M = Mn2+, Fe2+, Co2+, Ni2+ or Zn2+), in 1:1 molar ratio, in the presence of 1.2 equiv of weak base, NaOAc, did not produce any metal selenide (MSe) even after 48 h at 37 C (Table 2, Figure 4), indicating that these essential metal ions, unlike Cu, failed to activate the CSe bond of 1 under identical reaction conditions. The softness values (), simply the inverse of the hardness (), of Mn2+, Fe2+, Co2+, Ni2+ or Zn2+ ions are found to be relatively lower compared to the softness value of Cu+ ion and, thus, the interaction between the relatively hard metal ions and the soft Se center of 1 is weaker in nature compared to the interaction between the Cu+ ion (soft) and the Se center (soft), leading to the almost no change in the zwitterionic form of the metalbound selone compared to free selone (vide supra), causing almost impossible to cleave poorly activated CSe bond by neucleophilic oxygen atom of N-(CH2)2OH substituent, in the presence of weak base. These observations clearly suggest that the CSe bond cleavage of 1 (i.e. deselenization reaction) is critically dependent upon the hardness () of the metal ion (and possibly the other factors including the counter anion effect, as mentioned above).

Figure 4. (a) Deselenization of 1 (100 M) induced by copper and other essential metal ions (1 equiv) in the presence of weak base NaOAc (1.2 equiv), in PBS buffer (pH 7.4)/MeOH (1:1) at 37 C. (b) Reaction vials of 1/M+/2+. Table 2. Deselenization of 1 by some essential metal ions. SR NO. 1a 2a 3a 4a 5a 7a

Reactions

Metal

1/CuCl/NaOAc 1/MnCl2/NaOAc 1/FeCl2/NaOAc 1/CoCl2/NaOAc 1/NiCl2/NaOAc 1/ZnCl2/NaOAc

Cu+ Mn2+ Fe2+ Co2+ Ni2+ Zn2+

Hardness ()c 6.28 9.02 7.24 8.22 8.50 10.88

Initial Rate (Mh-1) 9.5 ± 1.1 x 10-4 -b -b -b -b -b

= [Cu+] = [M2+] = 100 M, where M = Mn, Fe, Co, Ni, Cu or Zn, 1.2 equiv of NaOAc; bdid not observe MSe formation at 37 °C; c values taken from Ref 41,42. a[1]

2.5 Removal of Copper(I) ion from Cu(1)2GS complex.

The majority of cytosolic Cu is bound with tripeptide GSH in the form of Cu+-GSH complex (binding constant ~10-12 M),3 which is a major contributor to Cu exchangeable pool in cytosol and, thus, we investigated the ability of 1 to remove Cu+ from Cu+-GSH complex (synthesized by following the literature procedure)43,44 and convert it into biologically inert CuSe under physiologically relevant conditions. The immediate formation


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of [Cu(1)2(GS)] complex was observed after treatment of 1 into the solution of Cu+-GSH complex in PBS buffer (pH 7.4). However, complex [Cu(1)2(GS)] was unstable and gradually degraded over time at 37 C (rate of deselenization was 0.99  10-3 M h-1) and formed round-shaped Cu3Se2 (Cu3Se2(GSH) hereafter), as illustrated in Figures 5a-d, Figure S9.

2.6 Role of 1 in Copper Homoeostasis in Liver Cell. In order to examine whether 1 playing any role in Cu homoeostasis in hepatocyte we have investigated the changes in expression levels of major Cu-responsive genes including Cu storage proteins MT1A and MT2A, Cu exporting ATPases ATP7A and ATP7B, Cu chaperone ATOX1 (responsible for the incorporation of Cu into Cu/Zn superoxide dismutase SOD1), and Cu binding protein SOD1.4,5 Real-time RT-PCR experiments were performed to examine the changes in expression profile of each gene after treating HepG2 cells with 100 M Cu [Cu(OAc)2 was used] and the combination of 1 and Cu (1/Cu hereafter), Figure 5e-l. Significant changes in expression level of Cu-responsive genes were observed in cells treated with Cu (100 M) compared to the untreated (UT) cells.45-49 However, no change at all (ATP7B and ATOX1) or a slight change in expression level of MT1A, MT2A or ATP7A was observed when cells were treated with 1/Cu, in 1:1 molar ratio. With excess amounts of 1 ([1]/[Cu] = 2), no change in expression level of MT1A or ATP7A was observed (Figures 5k,l, S23). Similarly, the increase of SOD1 concentrations in cells treated with 1/Cu is less (1.5-fold with respect to UT cells) in comparison to the increase of SOD1 concentrations in cells treated with Cu only (5.3-fold with respect to UT cells). These results suggest that 1 could successfully reduce the bioavailability of intracellular Cu ion concentrations by forming Cu(selone) complexes which are unstable and possibly will degrade into CuSe species over the time, resulting poor or no response of Cu-responsive genes in liver cells. Deselenization of 1 in the presence of copper salt in a complete cell culture media at 37 C suggest that the Cu(selone) complex is unstable at physiologically relevant conditions (Figures S24 and S26, and Table S6). Next, to investigate the internalization of selone 1 into the liver cells, we have incubated HepG2 cells with 1 (100 M) for 4 h and performed HRMS-QTOF analysis with cell lysates after thorough washing of cells with PBS buffer. Identification of 1 in cell lysates, and not in the final washing buffer, by mass spectrometry [1: m/z for (M+H)+ = 287.0329 and 2: m/z for (M+H)+ = 257.0222], Figure S25, indicates the internalization of 1 into liver cells.

2.7 Radical-Scavenging Activity of CuSe NCs. Does CuSe acts as pro-oxidant, like free Cu+ ion which is known to produce radical from H2O2, or antioxidant at intracellular level of H2O2 concentrations? In general, intracellular and extracellular H2O2 concentrations can vary from 0.001–1 M and 0.1–100 M or more, respectively, depending upon the normal and oxidative stress conditions.50-53 Detailed investigation showed that, unlike free Cu ion, CuSe NC (10 ng/L of Cu2-xSedc, Cu2xSesh or Cu3Se2(GSH)) did not oxidize ascorbic acid or showed any copper-mediated oxidative damage of protein and DNA in the presence of low concentration of H2O2, indicating that Cu22+ or xSe or Cu3Se2 is redox inactive in comparison to free Cu Cu+ ion, Figures 6a,b,c. Instead, we found that Cu2-xSedc NC markedly inhibited the protein carbonyl formation induced by Cu2+/H2O2 and showed excellent radical (OH, DPPH, and ABTS) scavenging property in a concentration dependent manner (Figures 6d-f, S10). The production of OH radical has not been detected when Cu3Se2(GSH) (10 ng/L) was treated with low concentrations of H2O2 ( 100 M). However, at high H2O2 concentration (100 mM) Cu2-xSe or Cu3Se2 exhibited OH

Figure 5. (a) HPLC chromatogram of degradation of Cu(1)2(GS) in PBS buffer (pH 7.4) at 37 C. Reaction vials are shown at the right side. (b) Amount of GSH (free) released after completion of reaction. XRD pattern (c) and TEM image (d) of Cu3Se2 {Cu3Se2(GSH)} obtained from the above reaction. Effect of 1 on the expression of Cu-responsive genes in HepG2 cells treated with 1/Cu2+ in 1:1 molar ratio for 24 h (e-j) and 2:1 molar ratio for 8 h (k, l and Figure S23). [Cu2+] = 100 M.

The increase of GSH (free) concentration was observed in the reaction solution after completion of the above reaction (Figure 5b). FT-IR spectra of Cu3Se2(GSH) NCs of ca. size 149 nm (cald.) showed that the some amount of glutathione molecules were also attached at the surface of Cu3Se2(GSH) NCs, Figure S9. Conversely, imidazole-based selone 3 afforded [Cu(3)2(GS)] complex after treating with Cu+-GSH complex. However, complex [Cu(3)2(GS)] was highly stable in solution and did not produce CuSe NCs even after 24 h at 37 C, under identical reaction conditions.



peroxidase-like activity,54 where active sites are considered to be the Cu ions that participate in redox reaction with H2O2 to produce OH radical via Fenton-type reaction leading to the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) with the Michaelis-Menten constant (Km) values 22 mM and 1.5 mM for TMB and H2O2, respectively (Figures 6c, and S12, S13).55,56 Thus, at low H2O2 concentrations, the role of Se ions (Se2-) of {111}-faceted cubic Cu2-xSe, where Se2- anions are located at the corners (eight) and centers (four) of face-centered cubic lattice (fcc) and cationic Cu ions are located within the Se2- fcc cage,57 or tetragonal Cu3Se2, where Se2- sublattice forms a quasi-planer hexagonal layer of Se2- anions that stacked in an alternative ABAB fashion with 3.201 Å interplanar spacing between two layers,35 were investigated thoroughly.

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mM, and [GSH] = 2 mM (Figure 7c). Interestingly, we observed that the Cu2-xSe nanosheet (Cu2-xSesh) exhibited the highest activity among other nanaocrystals, as mentioned in Table 3. The rate of reduction of H2O2 by bigger size Cu2-xSesh is found to be 2 times higher than the rate obtained for smaller size Cu2xSecu. Similarly, the rate of reduction of H2O2 by bigger size Cu3Se2(GSH) is almost 2 times higher than the rate obtained for smaller size Cu3Se2. This suggest that, although the surface to volume ratio decreases with increasing the size of the catalyst the rate is higher for the bigger size of NCs (Cu2-xSesh), indicating that the GPx-like activity of CuSe NCs is morphology dependent and the morphology dependent GPxlike activity is common in case of other nanomaterials as well.51

Figure 6. Effect of Cu2-xSedc on the oxidation of ascorbic acid (a) and on plasmid DNA (b). (c) Production of OH by Cu3Se2(GSH) (10 ng/µL) at different H2O2 concentrations. (d) Scavenging of OH (induced by Cu2+/ascorbate/H2O2) by Cu2-xSedc and Cu2-xSesh. (e) Protein carbonylation by Cu2+/H2O2 and Cu2-xSedc/H2O2. (f) Effect of Cu2-xSedc on the inhibition of protein carbonylation induced by Cu2+/H2O2.

2.8 Glutathione Peroxidase-like Activity of CuSe NCs. Glutathione reductase (GR) coupled assay was employed to study GPx-like activity of CuSe NC by monitoring the decrease of NADPH concentration spectrophotometrically at 340 nm.58 To our surprise, like GPx, Cu2-xSe or Cu3Se2 showed remarkable ability to reduce H2O2 catalytically in the presence of GSH. The initial rates determined at various assay conditions are shown in Figures 7a, b, and Table 3, which suggest the GPx mimicking activity of Cu2-xSe or Cu3Se2 NC. In the absence of at least one of the components in the reaction mixture, Cu2-xSe did not show GPx-like activity. A proportional dependence of initial rate of reduction of H2O2 with first-order reaction kinetics was observed when the concentration of Cu2-xSedc was varied from 0 to 30 ng/µL with keeping the all other required reagents constant, [H2O2] = 100 M, [GR] = 1.35 units, [NADPH] = 0.4

Figure 7. GPx-like activity of Cu2-xSe. (a) Bar diagram showing the initial rates of GPx assay at various assay conditions. Plots of initial rate against various Cu2-xSedc (b) and H2O2 (c) concentrations. (d) Bar diagram showing no significant inhibition of GR by Cu2xSedc/H2O2. (e) FT-IR spectra showing the formation SeOH and SeO bonds after treatment with H2O2. (f) Representation of antioxidant and pro-oxidant activity of Cu2-xSe NC. (g) XPS analysis of Cu2-xSedc after treatment with H2O2 followed by GSH.

To understand the substrate binding at the surface of the Cu2-xSe, the apparent steady-state kinetic parameters were determined by independently varying the concentration of GSH and H2O2 in the presence of the fixed concentration of GR (1.35


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units) and NADPH (0.4 mM), which followed a typical Michaelis-Menten kinetics (Figures 7c, S15, S16). The supernatant liquid obtained after the incubation of Cu2-xSedc for 15 min, did not show any significant antioxidant activity, indicating that reaction occurs on the surface of Cu2-xSedc (Figure S14b). The kinetic parameters obtained from the corresponding Lineweaver-Burk plots showed that there were significant differences in the substrate binding at the surface of Cu2-xSe. For H2O2, the KM and Vmax values obtained for Cu2-1 xSedc (0.95 mM, 66.6 M min ) and Cu2-xSesh (1.15 mM, 80.3 M min-1), respectively (Tables 3 and S3). GPx-like activity of tetragonal Cu3Se2 NC was found to be 2-times less than that of bigger size Cu3Se2(GSH). Table 3. Initial Rates (ν0) for the reduction of H2O2 in the presence of catalysts. Km Vmax SR Initial Rate Catalysts Avg. Size NO. (µM min-1)a H2O2 1 Control 1.61 ± 0.18 2 Cu3Se2 31 ± 6 6.73 ± 0.40 3 Cu3Se2(GSH) 149 ± 12 13.1±0.54 943.23 64.15 4 Cu2-xSecu 20.5 ± 3.4 9.59 ± 0.72 5 Cu2-xSedc 58 ± 8.0 14.7 ± 0.62 958.67 66.66 6 Cu2-xSesh 246 ± 30 20.50 ± 0.71 1154.83 80.3 aConditions: Cu Se or Cu Se (10 ng/µL), H O (100 M), GSH (2 mM), 2-x 3 2 2 2 GR (1.35 unit), NADPH (0.4 mM) in PBS buffer (pH 7.4, 21 °C); Km in µM; Vmax in µM min-1.

It is worthwhile to note that the Cu2-xSedc/H2O2 combination used in this study (GR coupled assay) did not affect the activity of GR as it has been observed in case of CuSO4/H2O2 combination (Figure 7d).59 On the contrary, we noticed that the another metal selenide HgSe inhibited the activity of GR enzyme and did not show any GPx-like activity under identical reaction conditions.60 To further understand the mechanism behind the GPx- like activity of Cu2-xSedc, we have performed several in-situ XPS and FT-IR studies of Cu2-xSedc in the presence of H2O2 and GSH. Upon treatment of H2O2 (100 M) to Cu2-xSedc for 100 sec (time required for the 50% completion of reaction), XPS analysis revealed that the symmetric and narrow shaped characteristic peak of Se2- for Se3d, comprising of Se3d5/2 and Se3d3/2 (Figure 2c), shifted to higher binding energy above 58 eV, Figure 7g. Almost 70% of the selenide species (Se2-) was converted to an intermediate species with higher oxidation state than Se2-, with a large percentage of ca. 46% being converted to Se6+ (tentatively identified as SeO3), a smaller percentage of ca. 7% being converted to Se4+ (tentatively identified as SeO2), and ca. 17% being converted to other unstable intermediate oxidized products of selenium species (Table S4).61 However, the large broad peak corresponding to the various oxidation products of selenium species was substantially disappeared upon addition of GSH, with ca. 21% reversal of oxidized species to Se2species after addition GSH. On the other hand, we found that the change of Cu+/Cu2+ ratio upon treatment of H2O2 (100 M) to Cu2-xSedc for 100 sec is negligible {Cu+/Cu2+ (untreated) = 0.46 vs Cu+/Cu2+ (treated) = 0.43}. Formation of SeOH and SeO bonds has also been confirmed by FT-IR analysis (Figure 7e); the large down-field shift of OH stretching frequency (2364 cm-1), compared to the free OH is indicative the presence of strong hydrogen bonding.62,63 Peaks at 1238, 1150, 930, 720, 637, 563, and 535 cm-1 were associated with the OH bending, SeO stretching and bending, respectively, for selenium

oxidized species like SeOH, SeO2, and SeO3.64-66 Moreover, similar type of characteristic peaks for the corresponding oxidized species of selenium were observed in FT-IR when selenium nanoparticle was treated with H2O2 under identical reaction conditions (Figure S20). Interestingly, addition of GSH brought back oxidized-Se species to Se2- again and completed the catalytic cycle (Figure 7e for Cu2-xSedc and Figure S20 for selenium nanoparticle). Furthermore, Cu2-xSedc was found to be capable of performing multiple cycles of H2O2 reduction without loss of catalytic activity (Figure S14), similar to the other nanozyme, V2O5 nanomaterial, reported previously in the literature.51

2.9 Protection of Liver Cells from H2O2-induced Oxidative Stress by CuSe NCs. The in vitro antioxidant GPx-like activity of the Cu2-xSe or Cu3Se2 prompted us to investigate the cytoprotective effect of these NCs in hepatocytes. First, we investigated the internalization of CuSe NCs into the liver cells. HepG2 cells were incubated with Cu2-xSedc (25-200 ng/µL) for 6 h and the presence of Cu2-xSedc NCs in the cells were evaluated by flow cytometric analysis (FACS) of altered side scatter intensity (SSC) resulting due to the differential density of the intracellular Cu2-xSedc accumulation.58,67,68

Figure 8. Cu2-xSe rescue cell viability via efficient ROSscavenging. (a-c) Fluorescent images of ROS levels in HepG2 cells after different treatments. (d-f) Bright field images of corresponding to fluorescent images of a-c. (g) Quantitative analysis of the ROS levels after treating cells with Cu2-xSe NCs (Cu2-xSedc, Cu3Se2(GSH), and Cu2-xSesh). Data is represented as the mean fluorescence intensity. Cell viability (MTT assay) of selones 1 and 2 (h), and Cu3Se2(GSH) and Cu2-xSesh (i). DLS results showing hydrodynamic size of Cu2-xSedc (j) and Cu2-xSedc dissolved in histidine (k).



Detailed experimental procedure is mentioned in the Supporting information. Cells treated with Cu2-xSedc showed a comparable shift in the side scatters pattern (SSC) of the laser beam in comparison to the untreated cells (Figure S27). The change in the SSC pattern clearly indicates the cellular uptake of Cu2-xSedc NCs into the cells. Overproduction of ROS within cells leads to a situation called oxidative stress, and therefore, to examine the ability of CuSe nanozymes to combat oxidative stress in the liver cells we mimic the conditions of oxidative stress by treating HepG2 cells with H2O2 (80 M), which is known to induce ROS in the cells.69 DCFH-DA was employed as a fluorescence probe to measure intracellular levels of ROS. The strong fluorescence signal of DCF showed that the ROS level in H2O2-treated HepG2 cells was remarkably enhanced in comparison to the untreated cells (UT), Figure 8a-f. However, the production of intracellular ROS was significantly inhibited in cells co-treated with 10 ng/L of CuSe and 80 M H2O2, as indicated by much weaker fluorescence signal of DCF, confirming the cytoprotective effect of CuSe nanozymes. The bigger size Cu2xSesh showed higher cyto protective activity (ca. 81% of ROS scavenging ability) than Cu3Se2(GSH) (ca. 67%), Figure 8g. The cell viability assay demonstrated that Cu3Se2(GSH) or Cu2-xSesh NCs did not show cytotoxicity at low concentration (10 - 30 ng/L), Figure 8i. As it is evident from experimental results that selone 1 (or 2) first formed an unstable complex with Cu which gradually degraded to CuSe species in the presence of weak base. Thus we have also checked cytotoxicity of selones and the combination of selone plus Cu salt. Selones 1 and 2 did not show cytotoxicity in liver cell at low concentrations (up to 100 M), Figure 8h. Moreover, HepG2 cells were also co-treated with 1 and Cu2+, in 1:1 molar ratio, and this combination did not result any cytotoxicity up to 100 M concentrations (Figure S21), indicating that the intermediate Cu(selone) complex is not cytotoxic to liver cells at low concentrations and thus, selone 1 could be a potential molecule against Cu-mediated oxidative stress.

2.10 Dissolution of CuSe NCs. Finally, in an attempt to solubilize insoluble CuSe nanozyme we treated Cu2-xSedc with natural amino acids like cysteine, GSH or histidine. Dispersed solution of black nanodiscs of Cu2-xSedc (100 ng/L) formed a clear brown-colored solution after stirring with histidine (20 mM) for 24 h at 37 C, as shown in Figure 8j, k. However, Cu2xSedc NCs were not soluble in phosphate buffer (pH 7.4) in the presence of cysteine or GSH (20 mM). The solubility of Cu2xSedc in histidine solution is due to the binding of histidine through N-atom of 5-member ring at the surface of nanodiscs.70 Dynamic lighting scattering (DLS) analysis revealed 3-times reduction of average hydrodynamic size of dissolved Cu2-xSedc compared to the undissolved nanodisc, from 356 nm to 130 nm. This finding may have a significant implication on understanding the binding of CuSe particles with biomolecules and their possible clearance from cellular system. Insoluble CuSe species may bind at the histidine rich center of proteins which may facilitates its excretion from cellular system.

3. Conclusion In conclusion, we reported the Cu-driven C=Se bond cleavage, initiated by the neighbouring group participation of the O-atom of N-(CH2)2OH group of 1, leading to the controlled release of

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Se2- at low temperature, resulted the synthesis of various types of {111}-faceted face-centered cubic Cu2-xSe or tetragonal Cu3Se2 NCs with remarkable GPx-like activity in degrading intracellular H2O2 concentrations and exhibited significant cytoprotective effect to liver cell against oxidative stress. On the contrary, selone 3 (or 4), in spite of having N-(CH2)2OH group, failed to produce Cu2-xSe NCs at 21 C. We found that the CSe bond cleavage of 1 occurred in two steps at 37 C: The activation of CSe bond induced by Cu ion (first step) followed by the facile cleavage of the Cu-activated CSe bond through neighboring group participation of the O-atom of N-(CH2)2OH substituent (second step) in the presence of weak base. Significantly, we found that the deselenization of selone 1 did not occur in the presence of any other essential metal ions including Mn2+, Fe2+, Co2+, Ni2+ or Zn2+, under identical reaction conditions, indicating that the cleavage of CSe bond 1 is highly Cu selective. Moreover, we showed that the kinetics of deselenization reactions and the morphology of the CuSe products can rationally be controlled by varying the oxidation state of the copper salt. Reaction of 1 (or 2) with Cu2+ acetate, Cu(OAc)2, led to the formation of cubic Cu2-xSe phase, whereas the same reaction with Cu+ acetate, CuOAc, afforded tetragonal Cu3Se2 phase both at room temperature and high temperature. We also showed that the selone 1 successfully reduced the bioavailability of excess Cu ion concentrations and played significant role in Cu homoeostasis in hepatocyte by removing Cu from Cu+-GSH complex and thereafter converted it into Cu3Se2 nanozyme and, as a result, the Cu-responsive genes were not up-regulated in liver cell co-treated with the combination of Cu and 1, as compared to the expression level of those genes in cell treated with excess Cu only. We believe that this new concept of selectively converting Cu into CuSe species may have significant importance in developing potential drug in combating Cu related disorders like Wilson’s disease and Indian Childhood Cirrhosis in future.

ASSOCIATED CONTENT Supporting Information Synthesis methods; HR-ESIMS analysis of compounds; and 13C; Kinetic studies and procedures; procedure of cellbased assays; TEM, SEM, EDX analysis and sample preparation. DFT calculations; Optimized geometries and co-ordinates of the optimized structures; Crystal data in .cif format. 1H,

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions AC carried out maximum experiments mentioned in the article including synthesis of selones, kinetic studies, synthesis and characterization of NCs, cell culture studies, ROS measurement in HepG2 cells etc. RK performed DFT calculations and NMR studies. RD performed GSH binding studies, isolation and characterization of intermediates. BK and AC performed RT-PCR. HS, BK, and AG performed DNA cleavage study. KKJ, MB, and AC performed crystal data collection and structure determination. Designing of all experiments, data analyses, and manuscript writing were done by GPR and AC. All authors approved the final version of the manuscript for submission.


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Funding Sources This work is supported by Shiv Nadar University (SNU) and Indo French centre for the promotion of advanced research (IFCPAR/CEFIPRA, Project Number. 5605-1).

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT We thank to Indo-French Centre for the promotion of advanced research (IFCPAR/CEFIPRA, Project Number. 5605-1). AC, RK, RD, and HS thank to SNU for fellowship. We thank to Dr. Dipak Maity and Mr. Atul for helping to record DLS. We thank Mr. Gourav Bhattacharya and Priya Mandal for helping to record the powder-XRD data. We also thank Dr. Sri krishna Jayadev Magani for helping in Flow cytometry (FACS) experiments. Authors also wish to thank for XPS facility at Micro and Nano Characterization Facility (MNCF), CeNSE, IISc, funded by Ministry of Electronics and Information Technology, DST, Govt. of India.

ABBREVIATIONS TEM, transmission electron microscopy; EDX, energy-dispersive X-ray spectroscopy; SEM, scanning electron microscopy.

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