doi.org/10.26434/chemrxiv.11888022.v1

Fe-N-C Nanozyme with Both Accelerated and Inhibited BiocatalyticActivities Capable of Accessing Drug-Drug InteractionYuan Xu, Jing Xue, Qing Zhou, Yongjun ZHENG, Xinghua Chen, Songqin Liu, Yanfei Shen, Yuanjian Zhang

Submitted date: 22/02/2020 • Posted date: 24/02/2020Licence: CC BY-NC-ND 4.0Citation information: Xu, Yuan; Xue, Jing; Zhou, Qing; ZHENG, Yongjun; Chen, Xinghua; Liu, Songqin; et al.(2020): Fe-N-C Nanozyme with Both Accelerated and Inhibited Biocatalytic Activities Capable of AccessingDrug-Drug Interaction. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.11888022.v1

Emerged as a cost-effective and robust enzyme mimic, nanozymes have drawn increasing attention withbroad applications ranging from cancer therapy to biosensing. Developing nanozymes with both acceleratedand inhibited biocatalytic properties in a biological context is highly envisioned for perusing more advancedfunctions of natural enzymes, such as in drug-drug interaction, but remains challenging. By re-visiting thewell-known Fe-N-C electrocatalyst that has a heme-like Fe-Nx coordination active center, herein, we reportthat the Fe-N-C with a minimum graphitization had an even superior cytochrome P450 (CYP)-like biocatalyticactivity. Moreover, the drug metabolization by the Fe-N-C upon co-existence of other foods and drugsdemonstrated a trend of inhibition similar to CYP, indicating its great potential as a replacement for drugdosing guide and outcome prediction. Beyond boosting the enzyme-like activity, this work would open a newvista of nanozymes with inhibited behavior for keeping up more demanding applications, enabled by furthermimicking the molecular structure of enzymes.

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1

Fe-N-C Nanozyme with Both Accelerated and Inhibited Biocatalytic

Activities Capable of Accessing Drug-Drug Interaction

Yuan Xu, Jing Xue, Qing Zhou, Yongjun Zheng, Xinghua Chen, Songqin Liu, Yanfei Shen,

Yuanjian Zhang*

Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu

Province Hi-Tech Key Laboratory for Bio-Medical Research, School of Chemistry and

Chemical Engineering, Medical School, Southeast University, Nanjing 211189, China

E-mail: Yuanjian.Zhang@seu.edu.cn

Abstract

Emerged as a cost-effective and robust enzyme mimic, nanozymes have drawn

increasing attention with broad applications ranging from cancer therapy to biosensing.

Developing nanozymes with both accelerated and inhibited biocatalytic properties in

a biological context is highly envisioned for perusing more advanced functions of

natural enzymes, such as in drug-drug interaction, but remains challenging. By re-

visiting the well-known Fe-N-C electrocatalyst that has a heme-like Fe-Nx coordination

active center, herein, we report that the Fe-N-C with a minimum graphitization had an

even superior cytochrome P450 (CYP)-like biocatalytic activity. Moreover, the drug

metabolization by the Fe-N-C upon co-existence of other foods and drugs

demonstrated a trend of inhibition similar to CYP, indicating its great potential as a

replacement for drug dosing guide and outcome prediction. Beyond boosting the

enzyme-like activity, this work would open a new vista of nanozymes with inhibited

behavior for keeping up more demanding applications, enabled by further mimicking

the molecular structure of enzymes.

Keywords: Fe-N-C, Nanozymes, Inhibition, P450, Drug-drug interaction

2

Introduction

Due to unrivaled catalytic activity towards various reactions with high substrate

specificity, selectivity and yields, enzymes have attracted extensive attention from the

synthesis of high value-added chemicals to biosensing. Nonetheless, denaturation and

degradation under harsh conditions, difficulties in recycling, and high costs in

preparation and purification restrict their broad applications.1 In this context,

exploring enzyme mimics to overcome these limitations but have an uncompromised

biocatalytic activity is highly envisioned.2 To this end, many nanomaterials, such as

Fe3O4 nanoparticles,3 have recently emerged as “nanozymes” in mimicking biocatalytic

functions of enzymes, already demonstrating prospective applications in cancer

therapy4 and biosensing.5 However, compared to the vast majority of the studies

pursuing an improved biocatalytic function (“accelerator”),6 the mimicking of

enzymatic inhibition (“brake”), a parallelly important character of enzymatic reactions

that makes the in vivo signaling (“vehicle trail”) highly precise, has been rarely explored

in a biological context so far, except for a few works regarding “turn-off” colorimetric

sensors.7 From the viewpoint of potential applications, such as in new drug discovery,

the enzymatic inhibition (mostly of cytochrome P450, CYP) need a careful evaluation

to avoid the risk of an adverse drug interaction that would cause death at worst.8

Therefore, it is crucial both in fundamental and technical to explore nanozymes with

both accelerated and inhibited biocatalytic properties.

Transition metal-N-doped carbon (M-N-C) dates back to the first oxygen reduction

reaction (ORR) catalyst reported in 1964 with macrocycle and metal-nitrogen

coordination.9 Inspired by the similarity of oxygen-activation between ORR and

aerobic reaction in vivo, very recently, we and other groups independently explored

Fe−N−C as an oxidase-like catalyst in direct activation of O2 for dehydrogenation and

monoxygenation of a variety of interesting organic substrates.10 Compared to many

well-known nanozymes, such as noble metal, metal oxides, 2D matters, and

nanocarbon, Fe-N-C not only had some enzyme-like functions but also owned a unique

Fe−Nx active moiety that is very similar to heme cofactor in CYP. Such features offer us

3

a great opportunity to mimic more advanced behaviors of CYP using Fe-N-C.

Nonetheless, the prior synthesis of Fe-N-C nanozyme mostly adopted the same

strategies for ORR electrocatalysts, making the Fe-N-C far less than fully optimized as

a nanozyme.

Herein, we report a facile structure engineering of Fe-N-C nanozyme by tuning the

pyrolysis temperature and its CYP-like accelerated and inhibited biocatalytic behaviors.

It was observed that in contrast to ORR that requires highly graphitic carbon for fast

electron transfer through electrocatalysts, only a minimum carbonization temperature

for cross-linking molecular precursors was needed for Fe-N-C to drive (per)oxidase-like

reactions with even superior activities. Moreover, in a showcase catalytic oxidation of

1,4-dihydropyridine (1,4-DHP, a cardiovascular drug), Fe-N-C demonstrated a CYP-like

inhibition behavior upon several food/ drug inhibitors under different mechanisms. As

a mimic of CYP, Fe-N-C would be promising for drug-drug interaction study for drug

dosing guide and outcomes prediction in a more facile and low-cost way.

Briefly, Fe-N-C nanozymes were prepared by pyrolysis of 3-butyl-1-methyl-1H-

imidazol-3-ium iron(III) bromide trichloride ([BMIM]FeBrCl3), which simultaneously

contains C, N and Fe in a molecular level (Figure 1a). As known, the pyrolysis

temperature had a profound influence on the final structure of Fe-N-C, thus in the first

set of experiments, Fe-N-C under carbonization temperature of 400 and 850 oC were

synthesized. The transmission electron microscopy (TEM) images disclosed that onion-

like nanoparticles of tens of nanometers with multiple graphitic shells and void cores

were observed for Fe-N-C-850 (Figure 1b). In contrast, only an amorphous morphology

was found for Fe-N-C-400 (Figure 1c), indicative of a lower degree of graphitization. X-

ray powder diffraction (XRD) was further performed to give crystallinity information.

Figure 1c showed that both Fe-N-C-400 and Fe-N-C-850 show characteristic (002)

diffraction of the graphitic structure at 26.2°. Nevertheless, a peak at 42.9° that

indicated an even higher graphitization was exclusively observed for Fe-N-C-850,

consistent with the TEM results.

4

Figure 1. Structures of Fe-N-C. (a) General procedures for the synthesis of Fe-N-C.

SEM images of (b) Fe-N-C-400 and (c) Fe-N-C-850. (d) XRD patterns and (e) N 1s XPS

spectra of Fe-N-C-400 and Fe-N-C-850.

The chemical bonding information of Fe-N-C-400 and Fe-N-C-850 was further

identified by X-ray photoelectron spectroscopy (XPS) measurements. The N1s XPS

spectra in Figure 1d could be deconvoluted into five peaks, assigning to pyridinic N (N1,

397.7 eV), Nx−Fe (N2, 398.2 eV), pyrrolic N (N3, 399.8 eV), graphitic N (N4, 400.9 eV)

and oxidized N (N5, 401.8 eV)respectively.10a, 11 The quantitive analysis revealed that

except for N4, the atomic ratios of other types of N in Fe-N-C-400 were higher than

that in Fe-N-C-850 (Table S1). Consistently, the C1s XPS spectra in Figure S1

demonstrated a higher ratio of non-graphitized carbon in Fe-N-C-400.

These results jointly demonstrated the critical role of higher temperature in

enhancing the crystallinity and graphitization of Fe-N-C, in agreement with previous

reports. It is well-known that graphitic structures play a vital role in ORR applications

because the electrons transfer from the substrate electrode via a graphitic framework

5

are ready to reach the catalyst surface (Figure 2a). That is why the high-temperature

carbonization is generally adopted in the synthesis of M-N-C for ORR electrocatalyst.

However, from a kinetics point of view, different reaction processes generally request

catalysts with different structures. In thse case of CYP-like biocatalytic reactions, the

graphitic framework of Fe-N-C may not as important as that for ORR, because the

electron exchange is mainly confined at the catalyst surface only (Figure 2a). For

instance, Figure 2b depicts the ORR activity of different Fe-N-C obtained in rotating ring-

disk electrode (RRDE) voltammetric measurements, the onset potential (Eonset) and

limiting current at Fe-N-C-850 was much superior to that at Fe-N-C-400, indicating a

higher ORR catalytic activity.12 Nevertheless, to drive (per)oxidase-like reaction, the

situation was altered. 3,3’,5,5’-tetramethylbenzidine (TMB), a representative

peroxidase substrate, was used to evaluate the biocatalytic activity of Fe-N-C-400 and

Fe-N-C-850 in an acetate buffer solution (0.2 M, pH=3.6) containing H2O2 (1 mM). After

oxidation, the TMB aqueous solution turned from colorless into green-blue and deep-

blue, and the biocatalytic activity could be estimated using the maximum absorbance

at 651 nm (Figure 2c).3 In contrast to that for ORR, it was interestingly observed that

Fe-N-C-400 exhibited a much higher peroxidase-like activity than Fe-N-C-850. When

1,4-Dihydropyridine (1,4-DHP) was used as another showcase substrate, a similar

enhancement of oxidase-like activity for Fe-N-C-400 was observed (Figure 2d and S2).

The catalytic activities for the above reactions by Fe-N-C prepared at other

temperatures (i.e. 600 and 750 oC) were also evaluated and summarized in Figure 2e,

along with the catalyst’s synthesis yield. It was evident that distinct to that for ORR the

lowest carbonization temperature made Fe-N-C not only have a much higher synthesis

yield but also own a higher (per)oxidase-like activity. The TGA and DTG curves of

[BMIM]FeBrCl3, the Fe-N-C precursor, showed the carbonization reaction rate

achieved maximation at ca. 294 oC and reached a plateau at ca. 391 oC (Figure 2f). In

this context, the minimum carbonization temperature at ca. 400 oC (e.g. in case of Fe-

N-C-400) was sufficient to endow an overall-highly efficient biocatalytic activity, which

was quite different to M-N-C ORR electrocatalysts.

6

Figure 2. Structure and activity correlation of Fe-N-C. (a) Scheme of reaction

processes and electron-transfer for Fe-N-C in ORR and (per)oxidase-like reactions in

solution. (b) RRDE voltammograms of Fe-N-C-400 and Fe-N-C-850. (c) UV-vis

absorption spectra of TMBox in sodium acetate–acetic acid buffer (pH 3.6) containing

H2O2 catalyzed by Fe-N-C-400 or Fe-N-C-850. (d) High-Performance Liquid

Chromatography (HPLC) of 1,4-DHP oxidation product (DDPD) oxidized by using the

catalyst of Fe-N-C-400 or Fe-N-C-850. (e) Radar map for performance assessment of

various Fe-N-C catalysts. (f) TGA and DTG curves of [BMIM]FeBrCl3.

To get more insight into the origination of (per)oxidase-like activities for Fe-N-C,

electron spin resonance (ESR) and optical trapping experiments were carried out to

detect the possible intermediate reactive oxygen species (ROS). As shown in Figure 3a,

by using 2,2,6,6-Tetramethylpiperidine (TEMP) as spin trapping agent, the typical 3-

fold characteristic peak of TEMP-1O2 spin adduct with the relative signal intensity of

1:1:1 was observed for both Fe-N-C-400 and Fe-N-C-850 in air-saturated aqueous

solution, indicating the existence of singlet oxygen (1O2). The stronger signal intensity

by Fe-N-C-400 was noticed, suggesting a higher efficiency in 1O2 generation.

Complementarily, the UV-Vis kinetic experiments using 9,10-anthracenediyl-

bis(methylene) dimalonic acid (ADA) as the 1O2 scavenger were also carried out.13 It

was observed that the oxidation rate of TMB decreased sharply after adding ADA, re-

confirming the existence of 1O2 (Figure S4). •O2- species was monitored by superoxide

7

dismutase (SOD), an enzyme for the dismutation reaction of •O2- into O2 and H2O2.

Through the UV-visible kinetic test, an enhanced catalytic rate was observed after

adding SOD (Figure 3b), indicating the co-existence of •O2-.14 The potential •OH- was

also monitored by ESR as well, followed by the same experiment condition using

DMPO as a spin trapping agent to form DMPO-OH adduct. However, the distinct 4-fold

characteristic peaks were not clearly observed, depicting •OH- was not the major ROS

intermediates (Figure 3c).15 Hence, the remarkable oxidase-like activities of the as-

prepared Fe-N-C were mainly owing to the generation of 1O2 and •O2-.

Figure 3. ROS acquisition and active site exploration. (a) ESR spectra of TEMP-1O2

adduct generated by Fe-N-C nanozymes in the presence of TEMP. (b) Time-dependent

UV-vis absorption spectra of ADA catalyzed by Fe-N-C with or without SOD as •O2–

trapping agent. (c) ESR spectra of DMPO with or without H2O2, Fe-N-C or both of them.

UV-vis absorption spectra of TMBox catalyzed by (d) Fe-N-C-400, (e) Fe-N-C-600 and (f)

Fe-N-C-850 before and after SCN- poisoning.

To understand the possible active sites in Fe-N-C, sodium sulfite was firstly used to

inhibit the oxidation of TMB. Figure S5 showed the typical absorbance of TMBox

decrease gradually with an incremental concentration of sulfite. Nevertheless, the

original activity of Fe-N-C-400 could be recovered by washing, indicated that sodium

8

sulfite served as a competitive reagent with TMB by consumption of O2 in the solution,

rather than chemical bonding with Fe-N-C (Figure S5). In the next experiment, SCN-,

which was supposed to be able to coordinate with Fe, was used to inhibit the oxidation

of TMB. It was found that catalytic TMB oxidation activity of all Fe-N-C catalysts was

irreversibly inhibited by SCN- poison, indicating these catalysts shared the similar

Fe−Nx moieties.16 Moreover, such inhibiting by SCN- was enhanced as the

carbonization temperature of Fe-N-C increased (Figure 3d-f). It was supposed that

under higher pyrolyzed temperature, the more planar graphitic structure would make

the Fe-Nx coordination center easier to be exposed and attacked by the small molecule

like SCN-. From another point of view, the absolute amount and the exposure extent

of Fe-Nx moieties jointly dertermined the catalytic activity and the inhibition of Fe-N-

C nanozyme, offering a fine tuning way for task-specific applications.

In these regards, different to the criteria for the synthesis of highly efficient ORR

electrocatalysts,17 the graphitic carbon, graphitic N and pyridinic N of Fe-N-C was not

the indispensable structures to drive the (per)oxidase-like reactions. Instead, the

amount of the coordination site for transition metals (e.g. Fe) to form heme-like Fe-Nx

moieties and the exposure extent, became more important for mimicking the

(per)oxidase activity; meanwhile, in contrast, the conductivity was less vital. Hence,

for Fe-N-C nanozymes, only minimum carbonization was required to crosslink the

precursors. It strongly suggested that the well-developed synthesis strategies of Fe-N-

C for ORR in the past several decades should be re-visited for new applications.

The excellent (per)oxidase-like catalytic activity of Fe-N-C and the inhibition effects

motivated us to explore more challenging biomimetic reactions, except for extensively

studied TMB oxidation system in colormeric sensors. As known, 1,4-DHP is a calcium

channel blocker and has been widely used as a cardiovascular drug.18 The oxidation of

1,4-DHP into diethyl-2,6dimethyl-3,5-pyridine-dicarboxylate (DDPD) is a crucial

process in the metabolism that is catalyzed by cytochrome P450 3A419 (CYP3A4, Figure

4a). Notably, CYP3A4 is an isoform of the CYP superfamily of monooxygenases,

9

oxidizing a considerable number of drugs by a large variety of metabolic processes. At

least 50% of marketed drugs that are metabolized by P450s are metabolized by the

CYP3A4, which constitutes 30 % approximately of total CYP in the liver.8 Thus, CYP3A4

is widely used in drug-drug interaction studies for new drug discovery to avoid the risk

of an adverse drug interaction that even causes death at the worst. Nevertheless,

considering the high-cost and instability of CYP, it is highly envisioned to perform the

preliminary evaluation for massive potential chemicals, drug dosing guide and

outcome prediction by artificial enzymes, such as Fe-N-C in this work.

Figure 4. CYP3A4-like accelerated and inhibited biocatalytic behaviors of Fe-N-C. (a)

Scheme of dehydrogenation of 1,4-DHP catalyzed by CYP3A4 or Fe-N-C. (b) Catalytic

dehydrogenation activity for 1,4-DHP by Fe-N-C and that in the presence of

ketoconazole inhibitor with different concentrations. The inset shows the logarithmic

transformation in calculating IC50. (c) Normalized DDPD yield without/with inhibitors,

i.e. ketoconazole (50 M), grapefruit juice supernatant (a dose of 1 mL), erythromycin

(50 M) and enoxacin (50 M) for screening drug-drug interaction. Blank sample

stands for 250 M of 1,4-DHP catalyzed by 50 g of Fe-N-C-400 without any inhibitors

in 1 mL of an aqueous solution. For ketoconazole and grapefruit juice inhabitation, the

deactivated Fe-N-C-400 was washed thoroughly using water and re-tested in catalytic

dehydrogenation of 1,4-DHP.

10

Ketoconazole is known as a potent inhibitor of CYP3A4 aimed at the oxidation of

1,4-DHP.8 In the first set of experiments, HPLC was used to evaluate the yield of

DDPD in the presence of ketoconazole. As seen from Figure 4b, the increase of

ketoconazole amount resulted in a gradual decline of DDPD, which suggested that

ketoconazole could inhibit the oxidation of 1,4-DHP into DDPD. In addition, the half-

maximal inhibitory concentration (IC₅₀), universally used to evaluate the potency of

a substance in inhibiting function, was measured. Generally, the smaller the value of

IC50, the stronger the inhibitory ability. It was observed that IC50 was about 1.2 M

in the Fe-N-C nanozyme-Ketoconazole system, which is similar to the behavior of

CYP450 inhibited by Ketoconazole.20

To obtain a more comprehensive understanding of Fe-N-C’s CYP3A4 mimicking

activity, different inhibitors under the same experimental condition were investigated.

As known, grapefruit juice has strong interactions with 1,4-DHP in the CYP3A4

metabolic processes, e.g., severe clinical consequence including death was reported

when a patient takes 1,4-DHP drugs and consumes grapefruit juice at the same time.8

To evaluate the influence of grapefruit juice, experiments were conducted with 1 mL

supernatant liquid centrifugal separated from fresh grapefruit juice, using Fe-N-C-400

as a CYP3A4 mimic and 1,4-DHP as a drug substrate. Figure 4c demonstrates an evident

inhibition effect of grapefruit juice after 30 min incubation. Erythromycin and enoxacin

were also investigated as controls, because the former, which is widely used in

dentistry, has a typical drug interaction with benzodiazepine agents,21 or 3-hydroxy-3-

methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor,22 rather than aiming at

1,4-DHPs, while the latter was reported as potent CYP1A2 inhibitor, not CYP3A4.23

Interestingly, their inhibition was much less than ketoconazole and grapefruit juice.

The reason for the specific inhibition of ketoconazole and grapefruit juice was also

investigated by washing the Fe-N-C catalyst sufficiently after the inhibition

experiments. As shown in Figure 4c, the activity that was originally inhibited by

ketoconazole was almost recovered, while that inhibited by grapefruit juice was not.

11

The ESR spectra showed that the introduction of ketoconazole had no evident

influence on the generation of ROS (1O2) by Fe-N-C catalyst (Figure S7). In these regards,

the ketoconazole inhibition was most presumably caused by a reversible absorption

on the Fe-N-C catalyst blocking the active sites. In contrast, the ingredients of

furanocoumarins, bergamottin, and dihydroxybergamottin in grapefruit juice were

supposed to act as substrate analogs, which use heme prosthetic group in CYP3A4 to

form a stable inactive complex, thus leading to overdosing and adverse drug

interaction.824

It is interesting that Fe-N-C not only demonstrated the similar function of CYP3A4 in

the metabolization of 1,4-DHP but also had a very high level of similarity in inhibiting

interactions with other drugs. It is strongly assumed that the Fe-Nx active site in Fe-N-

C that mimics the heme structure in CYP should be responsible for this new

observation. Referring to many other nanozymes, they have enzyme-like functions,

but few of them also have enzyme-like molecular structures. In this sense, Fe-N-C

combined the advantages of both heterogeneous nanozymes and homogeneous

molecular artificial enzymes. As more patients take two or more drugs and a diversity

of foods, the even increased risk of drug-drug interaction appeals for rapid and low-

cost checking approaches. Nevertheless, owing to time-consuming and high cost, the

traditional cell culture route by evaluation of drug-metabolizing CYP are challenged,

particularly for small molecules.25 The unique feature of Fe-N-C with both accelerated

and inhibited biocatalytic CYP-like behaviors in a biological context would provide an

alternative potential solution for drug-drug interaction studies. Rather than actually

replacing a clinical trial, this method would replace a study that was never going to

happen, which can guide dosing and predict outcomes.

Conclusion

In summary, we discovered that Fe-N-C can drive accelerated and inhibited biocatalytic

reactions like CYP in a biological context, owing to the similar Fe-Nx coordination active

center. Such a unique feature of Fe-N-C enabled more intriguing applications of

12

nanozymes that require more advanced functions of natural enzymes, e.g. as a low-

cost alternative of CYP for drug-drug interaction study. Interestingly, the showcase 1,4-

DHP metabolization by the as-prepared Fe-N-C upon co-existence of other common

foods and drugs demonstrated a trend of inhabitation very similar to CYP, indicating

its great potential as a facile tool kit in drug dosing guide and outcomes prediction. In

addition, distinct to the Fe-N-C widely used as electrocatalysts, only a minimum

graphitization was needed to promote an even superior nanozymatic activity,

appealing for a more comprehensive study of task-specific structure-activity

correlation in the future.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21775018,

21675022), the Natural Science Foundation of Jiangsu Province (BK20160028), the Open

Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201909), and the

Fundamental Research Funds for the Central Universities.

References

1. (a) Sun, H. J.; Zhou, Y.; Ren, J. S.; Qu, X. G., Carbon Nanozymes: Enzymatic Properties, Catalytic

Mechanism, and Applications. Angew. Chem. Int. Ed. 2018, 57 (30), 9224-9237; (b) Yan, X. Y.,

Nanozyme: a New Type of Artificial Enzyme. Prog. Biochem. Biophys. 2018, 45 (2), 101-104.

2. Murakami., Y.; Kikuchi., J.-i.; Hisaeda., Y.; Hayashida., O., Artificial Enzymes. Chem. Rev. 1996,

96 (2), 721-758.

3. Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.;

Perrett, S.; Yan, X., Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat.

Nanotechnol. 2007, 2 (9), 577-583.

4. (a) Fan, K. L.; Xi, J. Q.; Fan, L.; Wang, P. X.; Zhu, C. H.; Tang, Y.; Xu, X. D.; Liang, M. M.; Jiang, B.;

Yan, X. Y.; Gao, L. Z., In vivo guiding nitrogen-doped carbon nanozyme for tumor catalytic therapy.

Nat. Commun. 2018, 9, 1440-1450; (b) Huo, M. F.; Wang, L. Y.; Chen, Y.; Shi, J. L., Tumor-selective

catalytic nanomedicine by nanocatalyst delivery. Nat. Commun. 2017, 8 (1), 357-368; (c) Singh, N.;

Savanur, M. A.; Srivastava, S.; D'Silva, P.; Mugesh, G., A Redox Modulatory Mn3O4 Nanozyme with

Multi-Enzyme Activity Provides Efficient Cytoprotection to Human Cells in a Parkinson's Disease

Model. Angew. Chem. Int. Ed. 2017, 56 (45), 14267-14271; (d) Zhang, W.; Hu, S. L.; Yin, J. J.; He,

W. W.; Lu, W.; Ma, M.; Gu, N.; Zhang, Y., Prussian Blue Nanoparticles as Multienzyme Mimetics and

13

Reactive Oxygen Species Scavengers. J. Am. Chem. Soc. 2016, 138 (18), 5860-5865; (e) Xi, J.; Wei,

G.; An, L.; Xu, Z.; Xu, Z.; Fan, L.; Gao, L., Copper/Carbon Hybrid Nanozyme: Tuning Catalytic Activity

by the Copper State for Antibacterial Therapy. Nano. Lett. 2019, 19 (11), 7645-7654.

5. (a) Huang, Y. Y.; Ren, J. S.; Qu, X. G., Nanozymes: Classification, Catalytic Mechanisms, Activity

Regulation, and Applications. Chem. Rev. 2019, 119 (6), 4357-4412; (b) Manea, F.; Houillon, F. B.;

Pasquato, L.; Scrimin, P., Nanozymes: Gold-nanoparticle-based transphosphorylation catalysts.

Angew. Chem. Int. Ed. 2004, 43 (45), 6165-6169; (c) Jiang, D.; Ni, D.; Rosenkrans, Z. T.; Huang, P.;

Yan, X.; Cai, W., Nanozyme: new horizons for responsive biomedical applications. Chem. Soc. Rev.

2019, 48 (14), 3683-3704; (d) Wang, X. Y.; Hu, Y. H.; Wei, H., Nanozymes in bionanotechnology:

from sensing to therapeutics and beyond. Inorg. Chem. Front. 2016, 3 (1), 41-60.

6. (a) Fan, K. L.; Wang, H.; Xi, J. Q.; Liu, Q.; Meng, X. Q.; Duan, D. M.; Gao, L. Z.; Yan, X. Y.,

Optimization of Fe3O4 nanozyme activity via single amino acid modification mimicking an enzyme

active site. Chem. Commun. 2017, 53 (2), 424-427; (b) Hu, Y. H.; Gao, X. J. J.; Zhu, Y. Y.; Muhammad,

F.; Tan, S. H.; Cao, W.; Lin, S. C.; Jin, Z.; Gao, X. F.; Wei, H., Nitrogen-Doped Carbon Nanomaterials

as Highly Active and Specific Peroxidase Mimics. Chem. Commun. 2018, 30 (18), 6431-6439; (c)

Wang, Q. Q.; Zhang, X. P.; Huang, L.; Zhang, Z. Q.; Dong, S. J., One-Pot Synthesis of Fe3O4

Nanoparticle Loaded 3D Porous Graphene Nanocomposites with Enhanced Nanozyme Activity for

Glucose Detection. ACS Appl. Mater. Interfaces 2017, 9 (8), 7465-7471; (d) Zhang, Y.; Wang, F. M.;

Liu, C. Q.; Wang, Z. Z.; Kang, L. H.; Huang, Y. Y.; Dong, K.; Ren, J. S.; Qu, X. G., Nanozyme Decorated

Metal-Organic Frameworks for Enhanced Photodynamic Therapy. Acs Nano 2018, 12 (1), 651-

661.

7. (a) Wu, C. T.; Fan, D. Q.; Zhou, C. Y.; Liu, Y. Q.; Wang, E. K., Colorimetric Strategy for Highly

Sensitive and Selective Simultaneous Detection of Histidine and Cysteine Based on G-Quadruplex-

Cu(II) Metalloenzyme. Anal. Chem. 2016, 88 (5), 2899-2903; (b) Singh, M.; Weerathunge, P.;

Liyanage, P. D.; Mayes, E.; Ramanathan, R.; Bansal, V., Competitive Inhibition of the Enzyme-Mimic

Activity of Gd-Based Nanorods toward Highly Specific Colorimetric Sensing of L-Cysteine.

Langmuir 2017, 33 (38), 10006-10015.

8. Dresser, G. K.; Spence, J. D.; Bailey, D. G., Pharmacokinetic-pharmacodynamic consequences

and clinical relevance of cytochrome P450 3A4 inhibition. Clin. Pharmacokinet. 2000, 38 (1), 41-

57.

9. (a) Jasinski, R., A new fuel cell cathode catalyst. Nature 1964, 201 (4925), 1212-1213; (b)

Gupta, S.; Tryk, D.; Bae, I.; Aldred, W.; Yeager, E., Heat-treated polyacrylonitrile-based catalysts for

oxygen electroreduction. J. Appl. Electrochem. 1989, 19 (1), 19-27.

10. (a) He, F.; Mi, L.; Shen, Y.; Mori, T.; Liu, S.; Zhang, Y., Fe-N-C Artificial Enzyme: Activation of

Oxygen for Dehydrogenation and Monoxygenation of Organic Substrates under Mild Condition

and Cancer Therapeutic Application. ACS Appl. Mater. Interfaces 2018, 10 (41), 35327-35333; (b)

Cheng, N.; Li, J. C.; Liu, D.; Lin, Y. H.; Du, D., Single-Atom Nanozyme Based on Nanoengineered

Fe-N-C Catalyst with Superior Peroxidase-Like Activity for Ultrasensitive Bioassays. Small 2019, 15

(48), 1901485; (c) Huang, L.; Chen, J. X.; Gan, L. F.; Wang, J.; Dong, S. J., Single-atom nanozymes.

Sci. Adv. 2019, 5 (5), eaav5490; (d) Kim, M. S.; Lee, J.; Kim, H. S.; Cho, A.; Shim, K. H.; Le, T. N.; An,

S. S. A.; Han, J. W.; Kim, M. I.; Lee, J., Heme Cofactor‐Resembling Fe–N Single Site Embedded

Graphene as Nanozymes to Selectively Detect H2O2 with High Sensitivity. Adv. Funct. Mater. 2019,

30 (1); (e) Ma, W.; Mao, J.; Yang, X.; Pan, C.; Chen, W.; Wang, M.; Yu, P.; Mao, L.; Li, Y., A single-

atom Fe–N4 catalytic site mimicking bifunctional antioxidative enzymes for oxidative stress

14

cytoprotection. Chem. Commun. 2019, 55 (2), 159-162.

11. Jiao, L.; Wan, G.; Zhang, R.; Zhou, H.; Yu, S. H.; Jiang, H. L., From Metal-Organic Frameworks

to Single-Atom Fe Implanted N-doped Porous Carbons: Efficient Oxygen Reduction in Both

Alkaline and Acidic Media. Angew. Chem. Int. Ed. 2018, 57 (28), 8525-8529.

12. Sa, Y. J.; Park, C.; Jeong, H. Y.; Park, S.-H.; Lee, Z.; Kim, K. T.; Park, G.-G.; Joo, S. H., Carbon

Nanotubes/Heteroatom-Doped Carbon Core-Sheath Nanostructures as Highly Active, Metal-

Free Oxygen Reduction Electrocatalysts for Alkaline Fuel Cells. Angew. Chem. Int. Ed. 2014, 53 (16),

4102-4106.

13. Entradas, T.; Waldron, S.; Volk, M., The detection sensitivity of commonly used singlet oxygen

probes in aqueous environments. J. Photochem. and Photobio. B: Bio. 2020, 204, 111787.

14. MARKLUND, S.; MARKLUND, G., Involvement of the Superoxide Anion Radical in the

Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase. Eur. J. Biochem.

1974, 47, 469-474

15. Jeong, M. S.; Yu, K. N.; Chung, H. H.; Park, S. J.; Lee, A. Y.; Song, M. R.; Cho, M. H.; Kim, J. S.,

Methodological considerations of electron spin resonance spin trapping techniques for measuring

reactive oxygen species generated from metal oxide nanomaterials. Sci. Rep. 2016, 6, 26347.

16. Liang, H.-W.; Brüller, S.; Dong, R.; Zhang, J.; Feng, X.; Müllen, K., Molecular metal–Nx centres

in porous carbon for electrocatalytic hydrogen evolution. Nat. Commun. 2015, 6 (1).

17. (a) Guo, D.; Shibuya, R.; Akiba, C.; Saji, S.; Kondo, T.; Nakamura, J., Active sites of nitrogen-

doped carbon materials for oxygen reduction reaction clarified using model catalysts. Science

2016, 351 (6271), 361-365; (b) Wu, G.; More, K. L.; Johnston, C. M.; Zelenay, P., High-Performance

Electrocatalysts for Oxygen Reduction Derived from Polyaniline, Iron, and Cobalt. Science 2011,

332 (6028), 443-447.

18. Filipan-Litvić , M.; Litvić , M.; Vinković , V., An efficient, metal-free, room temperature

aromatization of Hantzsch-1,4-dihydropyridines with urea–hydrogen peroxide adduct, catalyzed

by molecular iodine. Tetrahedron 2008, 64 (24), 5649-5656.

19. Bocker, R. H.; Guengerich, F. P., Oxidation of 4-Aryl- and 4-Alkyl-Substituted 2,6-Dimethyl-

3,5-bis(alkoxycarbonyl)-l,4-dihydropyridines by Human Liver Microsomes and Immunochemical

Evidence for the Involvement of a Form of Cytochrome P-450. J. Med. Chem. 1986, 29 (9), 1596-

1603.

20. (a) Eagling, V. A.; Tjia, J. F.; Back, D. J., Differential selectivity of cytochrome P450 inhibitors

against probe substrates in human and rat liver microsomes. Br. J. Clin. Pharmacol. 1998, 45 (2),

107-114; (b) Zhou, S. F., Drugs behave as substrates, inhibitors and inducers of human cytochrome

P450 3A4. Curr. Drug Metab. 2008, 9 (4), 310-322.

21. Hersh, E. V.; Moore, P. A., Drug interactions in dentistry: the importance of knowing your CYPs.

J. Am. Dent. Assoc. 2004, 135 (3), 298-311.

22. Cooper, K. J.; Martin, P. D.; Dane, A. L.; Warwick, M. J.; Raza, A.; Schneck, D. W., The effect of

erythromycin on the pharmacokinetics of rosuvastatin. Eur. J. Clin. Pharmacol. 2003, 59 (1), 51-56.

23. (a) Kinzig-Schippers, M.; Fuhr, U.; Zaigler, M.; Dammeyer, J.; Rusing, G.; Labedzki, A.; Bulitta,

J.; Sorgel, F., Interaction of pefloxacin and enoxacin with the human cytochrome P450 enzyme

CYP1A2. Clin. Pharmacol. Ther. 1999, 65 (3), 262-274; (b) Lahu, G.; Nassr, N.; Herzog, R.; Elmlinger,

M.; Ruth, P.; Hinder, M.; Huennemeyer, A., Effect of Steady-State Enoxacin on Single-Dose

Pharmacokinetics of Roflumilast and Roflumilast N-Oxide. J. Clin. Pharmacol. 2011, 51 (4), 586-

593.

15

24. Fontana, E.; Dansette, P. M.; Poli, S. M., Cytochrome P450 enzymes mechanism based

inhibitors: Common sub-structures and reactivity. Curr. Drug Metab. 2005, 6 (5), 413-454.

25. Barzi, M.; Pankowicz, F. P.; Zorman, B.; Liu, X.; Legras, X.; Yang, D.; Borowiak, M.; Bissig-Choisat,

B.; Sumazin, P.; Li, F.; Bissig, K.-D., A novel humanized mouse lacking murine P450 oxidoreductase

for studying human drug metabolism. Nat. Comm. 2017, 8 (1), 39-47.

download fileview on ChemRxivManuscript.pdf (834.99 KiB)

S-1

Supporting information

Fe-N-C Nanozyme with Both Accelerated and Inhibited Biocatalytic

Activities Capable of Accessing Drug-Drug Interaction

Yuan Xu, Jing Xue, Qing Zhou, Yongjun Zheng, Xinghua Chen, Songqin Liu, Yanfei

Shen, Yuanjian Zhang*

Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu

Province Hi-Tech Key Laboratory for Bio-Medical Research, School of Chemistry and

Chemical Engineering, Medical School, Southeast University, Nanjing 211189, China,

E-mail: Yuanjian.Zhang@seu.edu.cn

S-2

Table of Contents

No. Name Page

1 Experimental Procedures 3

2 Figure S1. C1s XPS spectra of (a) Fe-N-C-400 and (b) Fe-N-C-850. 5

3 Figure S2. Catalytic activity evaluated of Fe-N-C-400-X using HPLC. X

indicates the molar ratio of FeCl3 to BMIMBr in the precursor

optimization.

6

4 Figure S3. Recovery and recyclability of Fe−N−C-400. 7

5 Figure S4. Time-dependent UV-vis absorption spectra of the Fe-N-C-400-

catalyzed TMB oxidation at 651nm in the absence and presence of 1O2

scavenger ADA.

8

6 Figure S5. TMB-oxidation Inhibition mechanism investigation using

Na2SO3. (a) UV-vis absorption spectra of the Fe-N-C-catalyzed TMB

oxidation inhibited by successive increase of the concentration of

Na2SO3.(b) UV-vis absorption spectra and (c) time-dependent UV-vis

absorbance at 651 nm of the TMB oxidation product catalyzed by Fe-N-

C-400 in the absence and presence of Na2SO3 or that with thorough wash

using water after the inhibition.

9

7 Figure S6. Scheme of Fe-N-C–catalyzed TMB oxidation inhibited by (a)

SO32- and (b)SCN- in a reversible and irreversible manner, respectively.

10

8 Figure S7. Electron spin resonance (ESR) spectra of 1O2 in various reaction

systems. 11

9 Table S1. Atomic ratio of different N species in Fe-N-C-400 and Fe-N-C-

850 from the analysis of the XPS spectra. 12

10 Table S2. Atomic ratio of different C species in Fe-N-C-400 and Fe-N-C-

850 from the analysis of the XPS spectra. 12

14 Supporting references 12

S-3

Experimental

Chemicals. 1-butyl-3-methylimidazolium bromide and TMB was purchased from

Aladdin Chemistry Co., Ltd., China. Superoxide dismutase, 5,5-Dimethyl-1-pyrroline N-

oxide (DMPO), 2,2,6,6-Tetramethylpiperidine (TEMP), 9,10-anthracenediyl-

bis(methylene) dimalonic acid（ABDA）were purchased from Sigma-Aldrich. Ultrapure

water (18.2 MΩ cm) was obtained from a Direct-Q 3 UV pure water purification system

(Millipore, USA). Unless otherwise specified, all other chemicals and solvents in this

work were of analytical grade, and used without further purification.

Characterization. Transmission electron microscopy (TEM) was performed on a JEOL

2100F (Japan) at an accelerating voltage of 200 kV. X-Ray diffraction (XRD) was carried

out by using Ultima IV (Rigaku, Japan). X-ray photoelectron spectroscopy (XPS) was

taken on an ESCALAB 250XI electron spectrometer (Thermo, USA) with

monochromatic Al Kα X-rays (h = 1486.6 eV) as the excitation source, and the binding

energy were corrected by reference C1s level to 284.6 eV to compensate for the

specimen charging. UV-Vis absorption spectra were measured on an Agilent Cary 100

UV-Vis spectrophotometer (USA). HPLC analysis was performed on a Shimadzu LC-

2010C system (Japan) equipped with an Agilent ZOBAX NH2 column (5 m, 250 × 4.6

mm, USA). Electron spin resonance (ESR) evaluation was conducted on a Bruker

microESR.

Preparation of Fe-N-C precursor. Fe-N-C precursor, i.e., 3-butyl-1-methyl-1H-imidazol-

3-ium iron(III) bromide trichloride ([BMIM]FeBrCl3), was first prepared by following

previous method.[ref] Briefly, [BMIM]FeBrCl3 was obtained by a quaternization

reaction between 1-butyl-3-methylimidazolium bromide (21.91 g, 100 mmol) and

iron(III) chloride hexahydrate (2.7 g, 10 mmol) in anhydrous ethanol (100 mL)at RT. The

final product was obtained by the removal of ethanol solvent via rotary evaporator,

rinsing using anhydrous ether, and drying at 80°C overnight under vacuum.

Preparation of Fe-N-C-X. (X = pyrolysis temperature). Fe-N-C-X was prepared by

heating [BMIM]FeBrCl3 at different temperatures (X=400 °C, 600 °C, 750° C, and 850 °C)

in a furnace using a ceramic crucible of 30 mL as the reactor in nitrogen atmosphere

S-4

with a ramp rate of 10 °C/min. The excessive iron species in the raw Fe-N-C-X was

leached out using HCl (37 wt. %, 20 mL) at RT for 9 h. Then, after centrifugation and

wash with water, absolute ethyl alcohol and anhydrous ether, the final dark black

powder was obtained by successive centrifugation, washing with water, absolute ethyl

alcohol and anhydrous ether, and drying at 80 °C overnight under vacuum.

Electrochemical measurements. ORR activity assessment of Fe-N-C-X was measured

by rotating ring-disk electrode (RRDE) voltammetry in a standard three-electrode glass

cell (RRDE-3A, BAS, Japan). A Ag/AgCl, Pt wire, and the Fe-N-C-X modified rotating ring-

disk electrode were used as reference, counter, and working electrodes, respectively.

The procedure of the working electrode modification was described as follows. 10 L

of the Fe-N-C-X ethanol suspension (4 mg mL-1) was cast on the pre-polished surface

of the rotating ring-disk electrode (4 mm diameter). The electrode was firstly dried at

RT, and then heated in air at 60 oC for 15 min. After that, 5 L of Nafion (0.05 wt. %)

was further cast on the surface of the electrode. Lastly, the electrode was dried at RT

and heated in air at 60 oC for 15 min again. The electrolyte (0.1 M KOH, pH=13) was

purged thoroughly with O2 for 30 min and was protected by O2 flow during the ORR

measurements. CV curves were recorded with the scan rate of 10 mV s-1. The onset

potential (Eonset) was defined as the potential at which the current density reached 5%

of the diffusion-limiting current density in the cathodic scan.

Oxidation of 1,4-Dihydropyridine. 1,4-Dihydropyridine (1,4-DHP, 250 μM) and Fe-N-

C-X catalyst (50 μg) were mixed in 1 mL of air-saturated phosphate buffer solution (pH

= 7.4) and vibrated at RT in a 2 mL centrifuge tube for 30 min. The reaction mixture

was then extracted with CH2Cl2 (1 mL). The organic layer of DDPD was analyzed by

HPLC at 30 °C. A mixture of n-hexene and isopropyl alcohol (20:1, v/v) was used as a

mobile phase at a flow rate of 1 mL min−1.

Fe-Nx active site poisoned by SCN-. 50 g of Fe-N-C-X (X=400 oC, 600 oC and 850 oC)

and 500 M of SCN- were added into 1mL of HAc/NaAc buffer solution (pH = 3.6). After

30 min shaking reaction, the mixture was washed thoroughly to remove free SCN-.

After that, TMB (500 M) was added into 1 mL buffer and kept for reaction for 3 min.

Lastly, the absorbance in 651 nm was examined using the UV-Vis spectroscopy.

S-5

Figure S1. C1s XPS spectra of (a) Fe-N-C-400 and (b) Fe-N-C-850.

The C1s XPS spectra in Figure S1 could be deconvoluted into four peaks, assigning

to C-C sp2 (C1, ~284.5 eV), C-C sp3 (C2, ~285.5 eV), C-N (C3, ~286.5 eV) and C-O (C4,

~288.5 eV), respectively.1 It was noted that the atomic ratios of C1, which generally

represent graphitized carbon depicted that Fe-N-C-850 have a distinct higher ratio of

graphitized carbon compared to Fe-N-C-400.

S-6

4 6 8 10

86.8%

91.3 %

81.7%

82.7 % Fe-N-C-400-0.05

Fe-N-C-400-1

Fe-N-C-400-0.25

Fe-N-C-400-0.1

Inte

nsi

ty (

a.u

)

Time (min)

without Fe-N-C

Fe-N-C-400-0.0179.1 %

Figure S2. Catalytic activity evaluated of Fe-N-C-400-X using HPLC. X indicates the

molar ratio of FeCl3 to BMIMBr in the precursor optimization.

The oxidation of 1,4-DHP into (DDPD) catalyzed by different Fe-N-C-400 was carried

out in an air-saturated phosphate buffer solution (pH = 7.4) at room temperature, and

assessed by HPLC through referring to the standard peak of DDPD that appeared at

4.1 min. Figure S2 showed the oxidation of 1,4-DHP reached a maximum yield of 91.3%

by using Fe-N-C-400-0.25, which was used as the default Fe-N-C-400 in this study,

unless otherwise specified.

S-7

1 2 3 4 5 6 70

50

100

Rel

ativ

e ac

tivi

ty (

%)

Recycling times

Figure S3. Recovery and recyclability of Fe−N−C-400.

Cycling test was conducted to detect the stability and recyclability of Fe-N-C-400. As

shown in Figure 3b, Fe-N-C-400 retained 90+% activity after 7 cycling tests,

outperforming natural enzymes which is homogeneous and usually hard to be

separated or recycled.

S-8

0 300 600 9000.0

0.3

0.6

Ab

s@6

51

nm

Time (sec)

Fe-N-C-400

Fe-N-C-400 + ADA

Figure. S4. Time-dependent UV-vis absorption spectra of the Fe-N-C-400-catalyzed

TMB oxidation at 651 nm in the absence and presence of 1O2 scavenger ADA.

The UV-Vis kinetic experiments using 9,10-anthracenediyl-bis(methylene) dimalonic

acid (ADA) as the 1O2 scavenger were carried out in a sodium acetate–acetic acid buffer

(1 mL, 0.2M, pH 3.6) contained TMB (500 M), ADA (300 M), and Fe-N-C-400 (50

g/mL). It was observed that the oxidation rate of TMB decreased sharply after adding

ADA.

S-9

Figure. S5. TMB-oxidation Inhibition mechanism investigation using Na2SO3. (a) UV-

vis absorption spectra of the Fe-N-C-catalyzed TMB oxidation inhibited by successive

increase of the concentration of Na2SO3.(b) UV-vis absorption spectra and (c) time-

dependent UV-vis absorbance at 651 nm of the TMB oxidation product catalyzed by

Fe-N-C-400 in the absence and presence of Na2SO3 or that with thorough wash using

water after the inhibition.

As shown in Figure S5a, different concentration of sodium sulfite was added

successively into 1 mL of sodium acetate–acetic acid buffer (pH 3.6) containing TMB

(500 M) and Fe-N-C-400 (50 g/mL). It can be seen that the typical absorbance of the

TMB oxidation product decrease gradually with an incremental concentration of sulfite,

and the totally inhibited the TMB oxidation was observed by 120 M sodium sulfite.

Nevertheless, when washed thoroughly with water, the original activity of Fe-N-C-400

could be recovered, as Figure S5b depicted. The kinetic study using the time-

dependent UV-vis absorbance at 651 nm revealed that the oxidation of TMB inhibited

by sodium sulfite was spontaneously recovered at 800 s (Figure S5c). This set of

experiments jointly indicated that the inhibition behavior of sodium sulfite most

possibly caused by the consumption of O2 that was dissolved in the aqueous solution,

rather than a strong chemical bonding with Fe-N-C like SCN- (Figure 3d-f).

S-10

(a)

(b)

Figure. S6. Scheme of Fe-N-C–catalyzed TMB oxidation inhibited by (a) SO32- and

(b)SCN- in a reversible and irreversible manner, respectively.

S-11

3350 3450 3550

ESR

am

plit

ud

e

Fe-N-C

1,4-DHP+Fe-N-C

Inhibitor+Fe-N-C

Magnetic Field (G)

1,4-DHP+inhibitor+Fe-N-C

Figure S7. Electron spin resonance (ESR) spectra of 1O2 in various reaction systems.

Monitoring 1O2 production was achieved by ESR spectroscopy. For this purpose,

TEMP (20 mM) and Fe-N-C-400 (50 g/mL) were added into an air-saturated

phosphate buffer solution (1 mL, pH 7.4) and incubate for 5 min. Then, the mixture

was transferred into a capillary tube for ESR measurements. To further explain the

inhibition behavior of ketoconazole to the catalytic oxidation by Fe-N-C-400, 1,4-DHP

(250 M) and ketoconazole (50 M) were added respectively into the above-

mentioned system. It was observed that the introduction of ketoconazole had no

evident influence on the generation of 1O2 by Fe-N-C catalyst both in the absence and

presence of substrate (black and red line). While a significant decrease of 1O2

generation could be noted in the presence of substrate (blue and green line),

suggesting that the inhibition behavior of ketoconazole have a negligible influence on

the 1O2 generation pathway. Thus, the ketoconazole inhibition was most presumably

caused by a reversible absorption on the Fe-N-C catalyst, blocking the active sites.

S-12

Table S1. Atomic ratio of different N species in Fe-N-C-400 and Fe-N-C-850 from the

analysis of the XPS spectra.

Entry Pyridinic N Nx−Fe Pyrrolic N Graphitic N Oxidized N Total N

Fe-N-C-400 2.74 % 3.45 % 8.03 % 0.28 % 0.36 % 14.86 %

Fe-N-C-850 0.14 % 0.53 % 0.45 % 0.62 % 0.12 % 1.86 %

Table S2. Atomic ratio of different C species in Fe-N-C-400 and Fe-N-C-850 from the

analysis of the XPS spectra.

Entry C-C sp2 C-C sp3 C-N C-O Total C

Fe-N-C-400 52.45 % 15.76 % 4.06 % 4.07 % 76.34 %

Fe-N-C-850 74.87 % 12.27 % 4.31 % 1.89 % 93.34 %

Reference

1. He, W.; Jiang, C.; Wang, J.; Lu, L., High-Rate Oxygen Electroreduction over Graphitic-N

Species Exposed on 3D Hierarchically Porous Nitrogen-Doped Carbons. Angew. Chem. Int. Ed.

2014, 53 (36), 9503-9507.

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