Synthesis and Molecular Recognition Studies on Small-Molecule

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Synthesis and Molecular Recognition Studies on Small-Molecule Inhibitors for Thioredoxin Reductase Di Zhang,† Zhonghe Xu,‡ Jia Yuan,§ Ying-Xi Zhao,† Zeng-Ying Qiao,† Yu-Juan Gao,† Guang-Ao Yu,*,§ Jingyuan Li,*,‡ and Hao Wang*,† †

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), No. 11 Beiyitiao, Zhongguancun, Haidian District, Beijing, 100190, China ‡ CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, 19 B, Yuquan Road, Beijing, China § Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Central China Normal University, Wuhan, 430079, China S Supporting Information *

ABSTRACT: Thioredoxin reductase (TrxR), which is overexpressed in many aggressive cancers, plays a crucial role in redox balance and antioxidant function, including defense of oxidative stress, control of cell proliferation, and regulation of cell apoptosis. Deactivation of TrxR can destroy the homeostasis of the cancer cells, inducing elevation of reactive oxygen species (ROS) levels and the oxidation of enzymatic substrates. Here, we synthesized and identified a new gold(I) small molecule (D9) that possesses two strong electron-donating moieties, i.e., 4methylphenyl alkynyl and thionyldiphenyl phosphine, exhibiting an enhanced p−π conjunction effect. The resulting compound shows the increased soft Lewis acids and the stability of gold(I). And we demonstrated that D9 could efficiently and specifically inhibit the activity of TrxR in vitro and in vivo, and it could effectively avoid the ligand exchange with albumin that was one of the most abundant proteins in blood. We believe that these comprehensive studies on the relationship between the structure and performance will provide inspiring information on the precise synthesis and design of new compounds for targeting TrxR.



micromolar concentration in vitro.29,30 Despite their in vitro efficiency, the chemical stabilities are poor and systematic toxicities are significant for gold(III) compounds in in vivo applications.4 Furthermore, gold(I) compounds are effective inhibitors of TrxR as well. Gold(I) compounds, e.g., Auranofin,19,20 triphenyl phosphine gold chloride,22 gold sodium thiomalate,31 sodium aurothiosulfate,31 and gold Nheterocyclic carbene complexes,26 have been investigated for their inhibition of the TrxR activity, with half maximal enzyme inhibition concentration (EC50) values ranging from 4 to 4000 nM.4 Notably, alkynyl phosphine gold(I) compounds exhibited high activity of TrxR inhibition in vitro.23 Many gold(I) compounds are practically ineffective in vivo, due to their potentiality of exchanging ligand with biomolecules, which usually leads to the reduction or even loss of their activity.4 Herein, we systematically design and synthesize 55 gold(I) compounds with varying molecular features for targeted inhibition of TrxR. The screened D9 compound exhibits high efficiency and specificity for TrxR in vitro. Moreover, we comprehensively evaluate the inhibition effect of D9 compound

INTRODUCTION TrxR is the electron donor for the enzyme of ribonucleotide reductase which plays critical roles in DNA synthesis, replication, and repair.1,2 Overexpressed TrxR in cancer cells3 is potentially related to the imbalanced deoxynucletide pools and may accelerate the development of the malignant phenotype by gene amplification, genetic rearrangements, and even therapy resistance.4 Recent studies demonstrated that overexpressed TrxR is necessary for growth of cancer cells and development of tumors, such as sustained progression of cell cycle, the evasion of growth-inhibitory signals, and the resistance to necrosis or apoptosis.3,5−7 On the contrary, deactivation of TrxR can destroy the redox homeostasis of cells3,7−9 through the elevation of cellular reactive oxygen species (ROS) levels10−12 and further lead to the inhibition of proliferation, and even induction of necrosis or apoptosis of cells.3,13 Accordingly, numerous TrxR-targeting compounds, such as platinum,14,15 arsenic,16,17 and gold18−20-related chemoprevention agents,4,21−26 have been developed as potential TrxR inhibitors for anticancer treatment. Recent studies suggested gold(III) compounds could be TrxR-targeted drug candidates.27,28 Preliminary results showed that apoptosis of cancer cells was induced by gold(III) compounds with © XXXX American Chemical Society

Received: July 15, 2014

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Figure 1. (a) Structures of a series of elaborately designed gold (I) compounds with host and R radical. (b) Screening of the highly efficient host structures from A, B, C, D, and E with phenyl as R radical. (c) Screening of the highly efficient R radical (1−11). (d) Investigating the highly efficient R radical combined with different host structures to screen the highest efficacy TrxR inhibitors. (e) Screening of the potential inhibitor (dark dash ellipse) of TrxR through designed concentrations (10, 20, and 50 nM) from 15 gold compounds. Error bars in (b), (c), (d), and (e) represent the SD of experimental duplicates.

Table 1. EC50 Values of 15 Gold Compounds to TrxR and the Homologous Enzyme of GR EC50 (nM)

EC50 (nM)

Gold compds

TrxR

GR

A9 A1 B9 C2 C10 C4 C8 C9

∼10 ∼10 ∼10 ∼100 ∼100 ∼100 ∼100 ∼100

>1000 >1000 >1000 >1000 ∼ 50 >1000 >1000 >1000

GR/TrxR >100 >100 >100 >10 ∼ 0.5 >10 >10 >10

TrxR

GR

C1 C5 D7 D8 D9 D10 E9

∼100 >50 ∼100 ∼20 1000 >1000 >1000 >1000 >1000 >1000 >1000

GR/TrxR >10 >20 >10 >50 >100 >10 >10

small energy gap between s, p, and d states, which results in proficient s/d or s/p hybridizations.32 The host structures include the following: phenyl, 4-methylphenyl, 1-hydroxycyclohexane, 4-methoxyphenyl, and 4-fluorophenyl, denoted A, B, C, D, and E, respectively (Figure 1a). These host structures with different electron-donating moieties enhanced the stability of positive gold atom. To further improve the stability of gold atom, we designed a series of phosphines with different degrees of electron-donating as R radical (shown in Figure 1a, R 1−11). Therefore, 55 kinds of gold(I) compounds were synthesized through orthogonal combination of host and radical R structures. All these structures were confirmed by 1H, 13C, and 31P NMR and by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF) (see Supporting Information). In order to identify possible

at enzyme, cells, and animal levels. The merits of D9 are (i) high activity of in vitro TrxR inhibition with nanomolar EC50 value (2.8 nM), which is comparable with the clinically practiced gold(I) compound Auranofin (EC50 = 4 nM);4 (ii) good selectivity and high toxicity toward cancer cells and limited side effects toward normal cells; and (iii) high stabilityit can prohibit ligand exchange with albuminand high antitumor efficiency in vivo.



Gold compds

RESULTS AND DISCUSSION

To realize the specific inhibition of TrxR activity both in vitro and in vivo, we elaborately designed a series of gold(I) compounds with ligands of alkynyl and phosphine. The host structure of gold acetylide compound was combined with alkynyl ligand with the linear geometry which is attributed to a B

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Figure 2. (a) Curves of TrxR activity after incubation with different concentrations of D9. The activity was evaluated through the color changes of TNB monitored through UV/vis absorption. (b) Kinetic curve of TrxR inhibition by D9; (inset) linear fitting of the reaction ratio curve. (c) Specific inhibition of TrxR by D9 was verified through comparison with the homologous enzyme of GR. (d) The stability of D9 was investigated through incubation with BSA at different times. MALDI-TOF was used to monitor the MW changes of BSA. The MW (66.3 kDa) of BSA without gold compounds incubation was set as control. References I, II, and III were [2-(4-methoxyphenyl)ethyn-1-yl] (triphenylphosphine) gold(I); [2phenylethyn-1-yl] (triphenylphosphine) gold(I); and triphenylphosphine gold chloride, and their structures are shown in Supporting Information, Figure S5.

Next, we further investigated the inhibition efficiency and specific targeting capabilities of D9 compound in detail. The concentration-dependent (0−20 nM) TrxR inhibition is shown in Figure 2a. D9 almost completely inhibited the activity of enzyme with the concentration as low as 6 nM. The kinetic studies were also carried out with various concentrations (Figure 2b). On the basis of linear dependence of the reaction rate (R2 = 0.9960), the EC50 value of D9 (Figure 2b inset) was 2.8 nM, which was comparable with the EC50 value of the gold compound (auranofin). The EC50 value of auranofin toward TrxR was approximately 4 nM (see Supporting Information, Figure S4), which was consistent with the previous report.4 And the detailed inhibition specificity of D9 was also evaluated through comparing the inhibition of the activities of TrxR and GR. TrxR activity was reduced by approximately 60% with 2 nM D9 treatment and 80% with 4 nM D9 treatments. Furthermore, the catalytic activity of GR was not significantly inhibited even when the concentration of D9 compound increased to more than 1000 nM. The EC50 value of GR was at least 337-fold higher than that of TrxR (Figure 2c, inset). Therefore, the results of these detailed studies confirmed that D9 had an efficient and specific profile for targeted inhibition of TrxR activity. Previous studies suggested that auranofin or many other gold compounds that quickly exchange the ligands with albumin existed abundantly in blood serum, leading to no significant effect on TrxR inhibition.31 Therefore, the stability

TrxR inhibitors from these gold compounds, the inhibition efficacy of gold compounds was first investigated at two concentrations (50 and 100 nM). The results of the compounds with host structure (D) and radical R (9) are shown in Figure 1b, c, d, and Figure S1. And 15 gold compounds were identified (structures shown in the Supporting Information, Figure S2) with relatively high activity. All these gold compounds similarly bear strong electron-donating moieties, which may result in the delocalization of the metal active center. In addition, the remaining 15 gold compounds were further screened with detailed concentrations (10, 20, 50 nM). D9 compound (dashed rectangle in Figure 1e) with 4-methylphenyl alkynyl gold (host D) and phosphine ligand of thienyldiphenylphosphine (R 9) was found to be the most highly efficient TrxR inhibitor with low inhibition concentration (6 5.1 >6 >6 >6 >6 >6 >6

C1 C5 D7 D8 D9 D10 E9

4.75 1.25 0.55 0.55 0.03 0.51 0.62

>6 >6 0.75 0.56 0.10 0.62 0.6

50% HT-29 cells were killed by D9 with the concentration as low as 0.1 μM (Figure 3d and Supporting Information, Figure S8. D9 had the highest efficacy to inhibit the cell proliferation with minimum values of IC50 for MCF-7 cells (0.03 μM) and HT-29 cells (0.10 μM). As indicated by these results, the inhibition of TrxR can further result in the reduction of cell viability. Hence, we further investigated potential capacities of D9 for anticancer cells in general. The activities of different kinds of cancer cells, including mouth epidermal carcinoma cells (KB), colorectal adenocarcinoma (HT-29), breast cancer cells (MDA MB-231 and MCF-7), human lung cancer cells (A549), and human cervical cancer cells (HeLa), were analyzed after the incubation of D9 with different concentrations (0−1.4 μM). D

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Figure 4. (a) Schematic illustration of the treatment protocol of the mice by D9. D9 (200 μL, 5 mg/kg) was intravescularly injected into BALB/c nude mice bearing a MCF-7 tumor. The mice were treated every 2 days (d = 1, 3, 5, 7, 9, and 11). (b) In vivo antitumor efficacy studies of D9 (5 mg/ kg) with PBS solution as controls. Values were expressed as means ± SD (N = 5). (c) Representative photos of the tumor-bearing mice of two groups after days 1 and 15 of treatment. (d) Body weight changes of the mice during the course of treatments. Values were expressed as means ± SD (N = 5).

cancer cells but has a limited side effect on normal cells. Auranofin and other gold compounds can similarly inhibit the activity of TrxR, while it is still very difficult to achieve an efficient antitumor effect in vivo.31 Therefore, it is very important or crucial to investigate the tumor inhibition of D9 in vivo. In vivo tumor suppression of D9 was carried out using a MCF-7 cells xenografted BALB/c nude mice model. MCF-7 cells (5 × 106) in 100 μL of PBS solution were subcutaneously injected into the right flank of BALB/c nude mice. The mice were randomly divided into two groups (N = 5). The designs of the in vivo antitumor protocol are shown in Figure 4a. When the tumors developed to approximately 80 mm3, D9 was intravenously injected into mice (200 μL, 5 mg/kg) at days 1, 3, 5, 7, 9, and 11 (Figure 4a). The same solutions (PBS with 1% DMSO) without D9 were intravenously injected into mice as control. The injections were performed every 2 days, and treatment was maintain to 15 days. During the process of the treatment, the tumor volumes and body weight were measured every day. The time-dependent tumor volumes of mice treated by D9 were compared with the control group (Figure 4b). It is clear that D9 compound can effectively inhibit the growth of tumors. And the efficacy of inhibition was significantly different after 10 days of treatment. After 15 days of treatment, the tumor inhibition ratio was calculated from the equation as follow:

The cell viability was obtained after 72 h of incubation with D9 compound in complete cell culture medium (with 10% FBS). The proliferation of all cancer cells was completely inhibited by D9 (0.60 μM), and the IC50 values of all cancer cells could be as low as 0.55 μM (Figure S9). PI/Hoechst was used to evaluate the necrosis/apoptosis induced by D9. In this assay, we incubated MCF-7 cells with D9 (0.80 μM) for 4 and 8 h separately, and the necrosis/apoptosis of cells was examined by adding the solution of PI/Hoechst and maintaining the incubation for 15 min. Confocal microscopy was then used for the fluorescent imaging of cells. After 4 h of treatment, there was more than 50% necrosis/apoptosis of cells compared to control (without treatments). And all cells had complete necrosis/apoptosis after 8 h of incubation (see Supporting Information, Figure S10). These results suggested that the efficient anticancer activity of D9 compound may be attributed to its impact of inducing cell necrosis/apoptosis. We further investigated the selectivity of its anticancer cells activity by examining the potential cytotoxicity of D9 compound to normal cells as well as tumor cells. Normal cells (human embryonic kidney cell (293T) and immortal hepatic cell (LO2)) and tumor cells were separately incubated with D9 (0.6 μM). The cell viability was measured through CCK-8 assay after 4 h of incubation in complete medium with 10%-FBS. The cytotoxicity is shown in Figure 3f. D9 has much higher toxicity to tumor cells than normal cells. The tumor cells activity decreased to below 40%, and even 80% tumor cells were completely killed by D9, while the cell viability of normal cells was not significantly affected. These results suggested that D9 can be a potential anticancer drug which can selectively kill

Inhibition ratio (%) = (Vc − Vt) × 100%/Vc

where Vc is the average tumor volume of the control group after treatment with intravascular injections of PBS for 15 days, and E

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Figure 5. (a) The schematic illustration of the TrxR electron transfer processes; verified the inhibition site of TrxR by D9 through the substrates inhibition approaches: the activity of N-terminal (b) and C-terminal sites (c) was investigated through the catalysis of Juglone and 9,10phenanthrenequinone, respectively, after 75 min incubation of TrxR and D9. (d) The irreversible inhibition of TrxR by D9 was carried out through verifying the catalytic activity of the system of TrxR-Trx: the substrates of insulin was added into after 75 min incubation of TrxR and D9. The activity of TrxR in (b), (c), and (d) was evaluated through the NADPH reduction at 340 nm absorption.

Vt is the average tumor volume of treatments with D9. From the results, we can see that D9 compound has effective tumor inhibition in in vivo treatments with high tumor inhibition ratio (IR ≈ 91.5%, see Supporting Information, Figure S11). The representative images of tumor-bearing mice before and after administration are shown in Figure 4c. The biodistribution of D9 compound in major organs was studied by inductively coupled plasma−atomic emission spectroscopy (ICP-AES) analysis of ex vivo tissues. The gold contents in different organs, i.e., tumor, head, lung, liver, kidney, spleen, and heart, were estimated. The results revealed that D9 mainly distributed in spleen (106.5 μg/g), kidney (52.4 μg/g), and liver (41.8 μg/ g), and the concentration of D9 in tumor was 25.3 μg/g after 4 h of treatment (see Supporting Information, Figure S12), which was sufficient to inhibit the proliferation and induce the necrosis/apoptosis of tumor cells. In the control group, the gold concentration was negligible and close to the baseline. Meanwhile, no obvious body weight loss was observed in all groups (Figure 4d), suggesting no acute toxicity of D9 after in vivo treatments. In addition, the toxicity of D9 was evaluated through blood chemistry and the activity of transaminase in liver (ALT and AST) of the mice model (see Supporting Information, Table S1). There was no significant damage after in vivo treatment with D9. Therefore, we successfully demonstrated that D9 has the capability to inhibit tumor proliferation both in vitro and in vivo.

Moreover, we tried to investigate the corresponding mechanism about the TrxR inhibition of D9. TrxR is a selenoprotein with two redox active sites, i.e., between Cys-57 and Cys-64 (N-terminal) and between Cys-497 and CSe-498 (C-terminal); the latter is considered as the binding site of the protein substrate, e.g. Trx. The electron transfer pathway of TrxR was depicted in Figure 5a. Briefly, the electrons are first transferred from NADPH to FAD and subsequently transferred to the disulfide of N-terminal active site. Finally, this dithiol of the N-terminal reduces the C-terminal selenenylsulfide of the enzyme. The selenolthiol has high activity to reduce Trx or other substrates. The substrates inhibition approaches were utilized to investigate the inhibition mechanism of D9 compound. 5-Hydroxy-1,4-naphthoquinone (Juglone) serves as the substrate of the N-terminal redox active motif, and 9,10phenanthrenequinone can receive electrons from redox-active selenolthiol of the C-terminal motif.33−35 D9 compound and TrxR were incubated at room temperature for 75 min in the environment of inert gas, and mixture solution preprepared was added into the solution; finally Juglone and 9,10-phenanthrenequinone (50 μM) were respectively added into the solution. The reduction of NADPH (absorption at 340 nm) was immediately detected. The activity of enzyme was estimated according to the reduction of NADPH. The activity of TrxR to catalyze 9,10-phenanthrenequinone was completely inhibited, suggesting the redox-active selenenylsulfide motif was comF

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was added into the solution of enzyme and then incubated in the environment of inert gas for 75 min at room temperature. The mixture solution (175 μL, 100 mM potassium phosphate, pH = 7.0, 10 mM EDTA, 20 mg/mL BSA, and 0.045 mg/mL NADPH) was added into 96-well plates. The rate of NADPH oxidation was immediately monitored after addition of 9,10-phenanthrene quinone (50 μM) and 5-hydroxy-1,4-naphtoquinone (Juglone, 50 μM). The control experiments were done with the same processes without the addition of gold compounds. To investigate the irreversible inhibition of TrxR, the mixture solutions containing potassium phosphate (50 mM, pH 7.0), EDTA (1 mM), insulin (0.5 mg/mL), NADPH (0.2 mM), and Trx (3 pM) were added into the culture solutions of TrxR and gold compounds. The absorption of NADPH at 340 nm was monitored by microplate reader. Antiproliferation Assay by Gold Compounds. MCF-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humified atmosphere containing 5% CO2. And then the cells were seeded in 96-well plates at a density of 6 × 104 cells per well. After 15 h adhesion, different concentrations of gold compounds (50, 100, and 500 μM for initial screening; 0.1, 0.5, and 1 μM for detailed screening of 15 gold compounds; 0.02−1.4 μM for investigating the IC50 values of different cells) were added into the culture mediums with 72 h of incubation. And then the cells were washed 3 times with phosphate buffer solution (PBS). Finally the cell viability of different treatments was monitored with the cell counting kit-8 assay (CCK-8, see Supporting Information). To investigate the cytotoxicity of D9 compound to tumor cells and normal cells, D9 (0.6 μM) was added into the medium of cells with 4 h of incubation, and then the medium was replaced with the fresh medium without D9 compound and cultured for another 24 h, and the cell viability was evaluated through the kit of CCK-8. In Vivo Antitumor of D9. All animal experiments were performed complying with the NIH guidelines for the care and use of laboratory animals and according to the protocol approved by the Institutional Animal Care. The initial body weight of mice was about 17−18 g. ICP-AES Analysis. The gold distribution in different organs of treated mice was evaluated through the concentrations of gold detected by ICP-AES analysis (ELAN 6100, PerkinElmer SCIEX). Organs were dissected and dissolved into aqua regia. The resulting solution was diluted with 2% HNO3 to constant volumes.

pletely inhibited by D9. However, the catalytic activity of TrxR to reduce Juglone was moderately inhibited. The redox-activity of the N-terminal motif was not severely inhibited by D9. In the control system, TrxR effectively catalyzed the compounds of Juglone and 9,10-phenanthrenequinone without the incubation of D9 compound (Figure 5b and c). Meanwhile, Trx reduction is important to maintain the homeostasis of cells. Insulin is one of the TrxR dependent substrates of Trx, and we used insulin to characterize the catalytic activity of the TrxRTrx system. The results are shown in Figure 5d, and the catalytic activity of the TrxR-Trx system was completely inhibited by D9 compound. These results showed that D9 had the capability to efficiently inhibit the redox-active selenenylsulfide motif of TrxR, and the inhibition was irreversible.



CONCLUSIONS In summary, we designed a novel compound D9 by screening 55 gold compounds. D9 was found to have specific and efficient inhibition of TrxR activity in vitro and in vivo. As indicated by this work, D9 compound exhibits great potential in the application of biomedicine.



EXPERIMENTAL SECTION

Elaborately Designed and Synthesized Gold Compounds. The host structures of gold(I) compounds were elaborately designed with 5 different alkynyl ligands, denoted as A−E: phenyl (A), 4methylphenyl (B), 1-hydroxycyclohexane (C), 4-methoxyphenyl (D), and 4-fluorophenyl (E), which represent classical electron donors and acceptors. Meanwhile, we also synthesized 11 ligands of phosphine, denoted as R radical, with different electron donating degrees. Through orthogonal combination of all these host structures and R radicals, we designed 55 kinds of gold compounds and tested their capabilities for the inhibition of the TrxR activity in vitro and in vivo. The synthetic processes of these gold compounds were as follows: The mixture solution of acetone and water (1:1, v/v, 14 mL) was added to the aqueous solution (4 mL) of chlorauric acid (1.94 mmol) and potassium bromide (1.12 g), and aerated with sulfur dioxide in the ice−water bath. Then sodium acetate and the acetone solution of alkyne were added and reacted for 1 h at room temperature. The yellow precipitates were isolated by filtration and washed with acetone and ether. The obtained compounds of alkyne-gold (0.1 mmol), phosphine ligand (0.1 mmol), and dichloromethane (2 mL) were added into the tube and reacted for 2 h at room temperature. The gold compounds were obtained after recrystallization with n-hexane twice. Screening of High Efficient Inhibitors to TrxR. The activity of TrxR was measured by NADPH-dependent reduction of 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) at room temperature according to the assay of ref 23. Briefly, the samples of gold compounds were dissolved into the solution of dimethyl sulfoxide (DMSO) and then diluted with respective buffers. In the assays of TrxR inhibition, 25 μL-TrxR (0.75 U) and two different concentrations (50 and 100 nM) of gold compounds (25 μL) were added into the 96-well plates and cultured for 75 min at room temperature. Finally, the 175 μL mixture solution (potassium phosphate: 100 mM, pH = 7.0; ethlenediamine tetraacetic acid: EDTA, 10 mM; bovine serum albumin: BSA, 20 mg/mL; and NADPH: 0.045 mg/mL) was added into 96-well plates, and incubation was continued for 0.5 min. DTNB (28 mM) was added into the solution, and the absorption of color changes was immediately measured at 412 nm, monitoring the formation of 5-thio-2nitrobenzoic acid (5-TNB). The control experiments were examined through the activity of TrxR without addition of gold compounds. All assays were carried out in triplicate. Demonstration of the Inhibition Site of TrxR. To determine the inhibition site of TrxR, the assays were conducted according to the protocol of reference.34 Briefly, 25 μL- aliquots of the enzyme solution (5 U/mL) were added into 96-well plates; and 25-μL of potassium phosphate buffer (pH 7.0) containing the gold compounds (50 nM)



ASSOCIATED CONTENT

S Supporting Information *

Experimental materials and methods, Figures S1−S12, Table S1, the molecular formula strings, and 1H, 13C, 31P NMR and MS of gold compounds. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.W.). *E-mail: [email protected] (J.L.). *E-mail: [email protected] (G.A.Y.). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program, 2013CB932701, 2013CB933704) and the 100-Talent Program of the Chinese Academy of Science (Y2462911ZX), the National Natural Science Foundation (21374026, 21304023, 21072071, and 51303036), and the Beijing Natural Science Foundation (2132053). G

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