Mimicking the Active Sites of Organophosphorus Hydrolase on the

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Mimicking the Active Sites of Organophosphorus Hydrolase on the Backbone of Graphene Oxide to Destroy Nerve Agent Simulants Xuejuan Ma,†,‡ Lin Zhang,†,‡ Mengfan Xia,†,‡ Shuangqin Li,†,‡ Xiaohong Zhang,†,‡ and Yaodong Zhang*,†,‡ †

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province and ‡Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, PR China S Supporting Information *

ABSTRACT: Recent global military events, such as the conflict in Syria, have emphasized the need to find effective strategies to rapidly destroy organophosphorus-based nerve agents. In this work, we designed active site-engineered graphene oxide (GO) via polymerization (polymer bead-GOs) as organophosphorus hydrolase (OPH) mimetic hotspots for the rapid degradation of nerve agents. This hybrid catalyst has a high total turnover frequency value of 0.65 s−1 and good stability (94.8% activity maintained after 5 cycles). Mechanism investigations suggested that the high catalytic performance could be attributed to the synergistic effect among the clusters of imidazole and the presence of − COOH groups on the GO surface and Zn2+. KEYWORDS: biomimetic catalyst, organophosphorus hydrolase, nerve agents, graphene oxide, synergistic effect

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oxide (GO)16 and graphene quantum dots (GQDs)17 have been reported as peroxidase-like catalysts. Interesting, the −CO groups on GQDs functioned in the catalytically active sites as peroxidase-like catalysts, and the OC−O− groups were substrate-binding sites; however, −C−OH groups can inhibit the peroxidase-like activity.18 GO provides an excellent platform for the size-controlled functional hybrid materials with enhanced catalytic activity because of the large surface and the functional groups on its surface.19 Different mechanisms, such as electrical coupling,20 anchoring sites,21 and electron transfer,22 are involved in catalysis when GO is used as a substrate to construct hybrid materials as catalyst. From a catalytic design perspective, the active site of OPH contains two zinc ions and their first shell ligands, including the bridging hydroxide, the four histidines (His 55, His 57, His 201, and His 230, from Pseudomonas diminuta, PDB code 1HZY), the Asp 301, and the carboxylated Lys 169 (Figure 1c).23−25 The results of molecular docking using Autodock 4.226 indicated that the active site of OPH is located at the molecular center and the paraoxon bound in the pocket of 1HZY (binding energy: −4.49 kcal/mol) and in the Zn2+−liganded structure (Figure 1c and Figure S1). Given the synergetic effects of four histidines, Asp 301, and two Zn2+ on OPH activity, we constructed the active sites of OPH on the backbone of GO using the method of free radical polymer-

erve agents containing phosphonate ester bonds inhibit the key enzyme acetylcholinesterase, which is involved in neuronal signaling; these nerve agents are among the most toxic chemicals known to mankind.1 Recent global military events, such as several incidents of chemical weapons in Syria,2 have emphasized the need to find effective strategies for the rapid destruction of these banned chemicals. In nature, organophosphorus hydrolase (OPH; EC 3.1.8.1, also phosphotriesterase), found in bacteria, is highly active in detoxification of organophosphorus nerve agents, including pesticides and chemical warfare agents such as sarin, soman, tabun, and VX (Figure 1a).3 Therefore, mimicking the active sites of OPH is strategic for creating a rigid artificial enzyme to degrade organophosphate nerve agents. A number of biomimetic catalysts accelerate nerve agent degradation by hydrolysis of simulants of these nerve agents, including metal complexes,4,5 metal oxides,6−9 molecular imprinting polymer,10 porous organic polymers (POPs),11 and metal organic frameworks.12−14 Despite the progress in biomimetic OPH research, the catalytical performance can rarely keep up with natural OPH because the structure of the natural enzyme is elaborately constructed to precisely control the function. Therefore, fine-constructing and fine-tuning the suitable environment around the catalytically active sites that mimic certain features of natural OPH can be a possible solution. Carbon nanomaterials are regarded as nanozymes because they have shown intrinsic enzymatic activity in several important chemical and biochemical reactions.15 Graphene © XXXX American Chemical Society

Received: June 1, 2017 Accepted: June 13, 2017

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DOI: 10.1021/acsami.7b07770 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. SEM images of (a) GO, (b) GO-HEMA, and (c) polymer bead-GOs and (d) IR spectra of GO, His-GO, GO-HEMA, and polymer bead-GOs.

Figure 1. (a) Structural formula of three organophosphates, as follows: sarin, soman, and paraoxon. (b) Hydrolytic degradation pathway of paraoxon by polymer bead-GOs. (c) Molecular docking of paraoxon with organophosphorus hydrolase.

beads around 2 μm are uniformly deposited on GO sheets. As a control, GOs were covalently functionalized with histidines (His-GO, as detailed in Supporting Information). The SEM image of His-GO (Figure S2) presents the wrinkled structure with the large thickness. The formation of polymer bead-GOs composite was further characterized via IR spectra (Figure 2d), Raman spectroscopy (Figure S3), and TGA weight loss curves (Figure S4). Figure 2d shows the IR spectra of GO, His-GO, GO-HEMA, and polymer bead-GOs. The absorbance bands at 3442 cm−1 are attributed to the O−H stretching bands of hydroxy and carboxylic moieties. The peak of 1637 cm−1 attributed to CC stretch vibration. In the His-GO spectrum, in comparison with GO, the presence of a peak at 1380 cm−1 can be attributed to C−N stretch vibrations,27 thereby confirming the formation of His-GO. The absorbance at 1707 cm−1 assigned to CO carboxyl stretching vibration28 strengthens in the GO-HEMA spectrum compared with GO. A wider peak at 1649 cm−1 appeared between 1707 and 1637 cm−1, the peak at 1380 cm−1 also appeared at the spectrum of polymer bead-GOs. The peak at 3442 cm−1 widens and slightly changes to 3544 cm−1 (N−H stretching vibration), thereby indicating that the imidazole group was successfully grafted to GO. The Raman spectroscopy and TGA analysis of (a) GO, (b) His-GO, (c) GO-HEMA, and (d) polymer bead-GOs also confirm the results (detailed in the Supporting Information). The catalytic activity of polymer bead-GOs was evaluated. Various control samples were investigated by using the less toxic simulant paraoxon (diethyl 4-nitrophenyl phosphate) because of the high toxicity of phosphate nerve agents and the concomitant risk, especially in the vapor phase (Figure 1b). Polymer bead-GOs and all control samples were dispersed in a mixture of acetonitrile and 20 mM Tris-HCl buffer (pH 9.0) (detailed in Supporting Information). Then, paraoxon was added with a final concentration of 2.5 mM. The progress of the catalytic reaction was measured by following the formation of p-nitrophenolate via UV/vis spectroscopy (λmax = 400 nm, Figure 3a). Both pure GO and His-GO show no catalytic activity for the hydrolysis of paraoxon, whereas the polymer bead-GOs sample exhibits obvious catalytic activity (Figure 3b).

ization based on the vinyl group functionalized supported materials. The ligands were truncated, i.e., in principle, only the side chains were kept. The histidines were thereby modeled by imidazoles and the aspartate by the carboxyl groups of GO. Vinyl groups were introduced onto the GO surface by esterification carboxyl groups of GO with 2-hydroxyethyl methacrylate (GO-HEMA) (Scheme S1). Then, with crosslinking agent divinylbenzene (DVB), a novel composite of polymer bead-GOs was obtained by copolymerization in the presence of vinyl group functionalized GO, 1-vinylimidazole (1VI) containing the same imidazole group as histidine, and zinc ions (Scheme 1). Scheme 1. Schematic representation of the synthesis of polymer bead-GOs and the degradation to paraoxon

The detailed synthesis and characterization of polymer beadGOs are demonstrated in the Supporting Information. The morphological structures of GO, GO-HEMA and polymer bead-GOs are shown in Figure 2a−c. GO sheets showed smooth surfaces and layered structures. By contrast, GOHEMA sheets showed dense and highly cross-linked owing to HEMA binding. Figure 2c revealed that homogeneous polymer B

DOI: 10.1021/acsami.7b07770 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

of 4(5)-vinylimidazole−Zn2+−methacrylic acid cluster with a divinylbenzene polymer (Table S1).10 The catalytic activity of polymer bead-GOs is higher than that of a metal complex29 and lower than that of MOF catalyst13 or vacancy-engineered nanoceria.6 However, polymer bead-GOs can destroy paraoxon without chemical additives,12,13 such as N-methylmorpholine6 or N-ethylmorpholine.13 Because not all 1-VI molecules contribute to an active site during the polymerization, and all catalysis reactions occur on the exterior surface of the polymer beads, the actual Nactive is much lower than estimated. The real TOF and catalytic activities of the polymer bead-GOs are therefore much higher than the apparent rates described above. To investigate the reutilization of the polymer bead-GOs, we regenerated the used polymer bead-GOs by removing the substrate or by production in acetonitrile and ethanol, after which Zn2+ was uploaded. The hydrolytic reaction of paraoxon catalyzed by the regenerated polymer bead-GOs was measured under the same condition. The capability of catalyzing hydrolysis decreased about 5.2% after 5 circles (Figure S8). Polymer bead-GOs can be regenerated and maintain their functions when used in paraoxon elimination. A plausible mechanism underlying the high catalytic activity of polymer bead-GOs is as follows. First, the clusters of imidazole were produced on the GO surface by the polymerization of 1-vinylimidazole (1-VI). Thus, the binuclear metal center was formed by the coordination with Zn2+ (Scheme 1) and can then be utilized to activate paraoxon for nucleophilic attack by polarization of the P−O bond as the natural OPH hydrolyzes its substrate.23,24 The formation of a binuclear metal center by polymerization was confirmed by experimental results (Figure 3b, Figure S5). The histidine covalently bound with GO exhibited no catalytic activity after incubation with Zn2+ (Figure 3b) because the steric hindrance effect resulted in difficulty to produce the binuclear metal center if the individual imidazole group was covalently bonded with GO. However, the cluster of imidazole groups on the GO surface facilitates the possible formation of the bimetallic center. The solution of 1-vinylimidazole added with Zn2+ can only slightly catalyze the hydrolysis of paraoxon (Figure 3b) because the binuclear metal center is difficult to produce in this case. Second, polymer beads without modification with GO exhibited lower activity (two-thirds) in paraoxon hydrolysis compared with polymer bead-GOs (Figure 3b). Moreover, the activity of polymer bead-GOs decreased by 27.3% when carboxylic groups on the composite were deactivated by 2bromo-1-phenylethanone (BrPE).18 The two points signify the synergistic effect between polymer beads and GO, thereby indicating that the remaining OC−O− groups on GO may function in concert with the binuclear metal center and substrate-binding sites that shuttle protons from the active site to the bulk solvent during substrate turnover.23 Finally, Zn2+ played an important role in catalysis because polymer beadGOs without incubation with Zn2+ showed 33.3% reduced activity in paraoxon hydrolysis compared with polymer beadGOs incubation with Zn2+. The EDX spectra (Figure S5) of polymer bead-GOs incubated with Zn2+ also indicated the retention of polymer bead-GOs with Zn2+. We are currently designing the new ligand instead of 1-VI to directly introduce the binuclear mental center. In conclusion, we have shown that enzyme mimetic active sites can be generated in the GO surface by polymerization. The active site-engineered GO developed by this method catalyzes the degradation of potentially harmful nerve agents,

Figure 3. (a) Absorbance spectra of the product p-nitrophenolate over time in the presence of polymer bead-GOs. (b) Comparison of OPHlike activity of GO, His-GO, Zn2+ + 1-VI, polymer beads, polymer bead-GOs (without incubation with Zn2+), and polymer bead-GOs. (c) Michaelis−Menten equation curve hydrolysis for paraoxon catalyzed by polymer bead-GOs. (d) Lineweaver−Burk plot of kinetics data of hydrolysis for paraoxon catalyzed by polymer bead-GOs.

To investigate the effect of the Zn2+ and imidazole group on the catalytic activity of artificial enzyme, the catalytic activity of the solutions of Zn2+ (50 mM) and 1-VI (1.25 mM) in 20 mM Tris-HCl buffer (pH 9.0) was investigated. No significant activity was observed when the reaction was carried out with the mixture of Zn2+ and 1-VI. Although polymer beads without GO modification also showed the catalytic activity, the initial rate of hydrolysis of paraoxon was only two-third of the activity of polymer bead-GOs. A well-defined active site is required for catalysis, and hydrolysis occurs at the surface of the GO. The activity of polymer bead-GOs markedly increased as a function of temperature (Figure S6). The activity at 60 °C is almost 7.9 times higher than that at 25 °C (Figure S7). The steady-state kinetics of the catalytic hydrolysis was further studied by keeping a constant amount (2 mg) of polymer bead-GOs. In this case, a typical Michaelis−Menten plot was obtained when the concentration of paraoxon solution was increased from 1 mM to 7.5 mM (Figure 3c). Furthermore, the straight line in the Lineweaver−Burk plot (Figure 3d) suggests that the polymer bead-GOs follow enzyme-like kinetics. The kinetic parameters obtained from these plots show significantly high maximal initial rate of product formation (Vmax = 0.014 mM min−1) and Km (Km = 13.5 mM) values. Although a high value of Km indicates low affinity of polymer bead-GOs toward paraoxon, the weak binding of organophosphates may help in the easy elimination of the product from the reaction sites.6 To roughly evaluate the performance of the polymer bead-GOs, we assumed that the number of the active sites (Nactive) equals a quarter of the number of the 1-VI molecules used during polymerization based on four histidines in the active site of OPH. The turnover frequency (TOF) (Vmax/Nactive, product produced per second per site) for the hydrolysis of paraoxon was estimated as 0.65 s−1. Under similar conditions/concentrations, polymer beadGOs affords a 10-fold TOF when compared with the previously reported molecularly imprinted catalysts by copolymerization C

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such as paraoxon, with high efficiency and can be employed as a molecular recognition element to fabricate electrochemical sensors. A detailed mechanistic investigation revealed the synergistic effect among the clusters of imidazole, the presence of −COOH groups on the GO surface and Zn2+. This study is the first example of a hydrolysis reaction by the enzyme active site-engineered GO.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b07770. Materials and methods, Scheme S1, Table S1, and Figures S1−S8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yaodong Zhang: 0000-0003-0014-025X Notes

The authors declare the following competing financial interest(s): The methodology presented in this letter was included in a Chinese Patent Application (2016104968307) filed by Y.D.Z., X.J.M, L.Z., K.K.M., C.X.Z, and M.F.X. on June 29, 2016.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21275097), Fundamental Research Fund for the Central Universities (GK201602010), and the 111 Project(B14041).

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REFERENCES

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DOI: 10.1021/acsami.7b07770 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX