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Nitrogen-Centered Lactate Oxidase Nanozyme for Tumor Lactate Modulation and Microenvironment RemodelingClick to copy article linkArticle link copied!
氮中心乳酸氧化酶纳米酶在肿瘤乳酸调控和微环境重塑中的应用点击复制文章链接
- Senfeng ZhaoSenfeng ZhaoHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaCollege of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou, Zhejiang 311121, ChinaMore by Senfeng Zhao
- Huihuang LiHuihuang LiDepartment of Urology, Xiangya Hospital, Central South University, Changsha, Hunan 410083, ChinaMore by Huihuang Li
- Renyu LiuRenyu LiuDepartment of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan 410083, ChinaMore by Renyu Liu
- Na TaoNa TaoHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaMore by Na Tao
- Liu Deng*Liu Deng*Email: dengliu@csu.edu.cnHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaMore by Liu Deng
- Qianqian XuQianqian XuState Key Laboratory for Powder Metallurgy, Central South University, Changsha, Hunan 410083, ChinaMore by Qianqian Xu
- Jianing HouJianing HouHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaMore by Jianing Hou
- Jianping ShengJianping ShengSchool of Resources and Environment, University of Electronic Science and Technology of China, Chengdu, Sichuan 611731, ChinaMore by Jianping Sheng
- Jia ZhengJia ZhengHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaMore by Jia Zheng
- Liqiang WangLiqiang WangHenan Province Industrial Technology Research Institute of Resources and Materials, School of Material Science and Engineering, Zhengzhou University, Zhengzhou, Henan 450001, ChinaMore by Liqiang Wang
- Wansong ChenWansong ChenHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaMore by Wansong Chen
- Shaojun Guo*Shaojun Guo*Email: guosj@pku.edu.cnSchool of Materials Science and Engineering, Peking University, Beijing 100871, ChinaMore by Shaojun Guo
- You-Nian Liu*You-Nian Liu*Email: liuyounian@csu.edu.cnHunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, ChinaCollege of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou, Zhejiang 311121, ChinaMore by You-Nian Liu
Abstract
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摘要点击复制章节链接
Designing nanozymes that match natural enzymes have always been an attractive and challenging goal. In general, researchers mainly focus on the construction of metal centers and the control of non-metallic ligands of nanozyme to regulate their activities. However, this is not applicable to lactate oxidase, i.e., flavoenzymes with flavin mononucleotide (FMN)-dependent pathways. Herein, we propose a coordination strategy to mimic lactate oxidase based on engineering the electronic properties at the N center by modulating the Co number near N in the Cox–N nanocomposite. Benefitting from the manipulated coordination fields and electronic structure around the electron-rich N sites, Co4N/C possesses a precise recognition site for lactate and intermediate organization and optimizes the absorption energies for intermediates, leading to superior oxidation of the lactate α-C–sp(3)–H bond toward ketone. The optimized nanozyme delivers much improved anticancer efficacy by reversing the high lactate and the immunosuppressive state of the tumor microenvironment, subsequently achieving excellent tumor growth and distant metastasis inhibition. The developed Co4N/C NEs open a new window for building a bridge between chemical catalysis and biocatalysis.
设计与天然酶相匹配的纳米酶一直是一个有吸引力且具有挑战性的目标。总体而言，研究人员主要关注金属中心的构建和控制纳米酶的非金属配体以调节其活性。然而，这不适用于乳酸氧化酶，即具有黄素单核苷酸 （FMN） 依赖性通路的黄素酶。在此，我们提出了一种配位策略，基于通过调节Cox-N纳米复合材料中N附近的Co数来设计N中心的电子性质，模拟乳酸氧化酶。得益于富电子N位点周围的调控配位场和电子结构，Co4N/C对乳酸和中间体组织具有精确的识别位点，并优化了中间体的吸收能，导致乳酸α-C–sp（3）–H键向酮的氧化性能更好。优化的纳米酶通过逆转肿瘤微环境的高乳酸和免疫抑制状态，大大提高了抗癌功效，从而实现了出色的肿瘤生长和远处转移抑制。开发的Co4N/C NEs为搭建化学催化与生物催化之间的桥梁打开了新的窗口。
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Copyright © 2023 American Chemical Society
版权所有 © 2023 美国化学学会
版权所有 © 2023 美国化学学会
1. Introduction
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1. 简介点击复制版块链接
The ever-increasing pursuit of biological context regulation platforms and the fallibility of enzymes’ nature propel the exploration of applicable alternatives to long-term biological activity. (1) Recently, nanomaterials with artificial enzyme-mimicking capability (nanozymes) have demonstrated potential for improving anti-cancer therapeutic efficacy via catalytic conversion of small metabolic molecules, such as glucose, glutathione, NADH and H2O2. (1−3) However, the design of nanozymes for the catalytic conversion of physiological molecules, especially key signaling metabolites in tumor progression, is still highly desirable but challenging.
对生物环境调节平台的日益追求和酶性质的易错性推动了对长期生物活性的适用替代品的探索。（1）最近，具有人工酶模拟能力的纳米材料（纳米酶）已显示出通过催化葡萄糖、谷胱甘肽、NADH和H2O2等小代谢分子来提高抗癌治疗效果的潜力。（1−3） 然而，用于催化生理分子转化的纳米酶的设计，尤其是肿瘤进展中的关键信号代谢物，仍然非常可取，但具有挑战性。
对生物环境调节平台的日益追求和酶性质的易错性推动了对长期生物活性的适用替代品的探索。（1）最近，具有人工酶模拟能力的纳米材料（纳米酶）已显示出通过催化葡萄糖、谷胱甘肽、NADH和H2O2等小代谢分子来提高抗癌治疗效果的潜力。（1−3） 然而，用于催化生理分子转化的纳米酶的设计，尤其是肿瘤进展中的关键信号代谢物，仍然非常可取，但具有挑战性。
Tumor lactate acts as a vital signaling molecule and plays an important role in tumor malignant progression, angiogenesis, immunity inhibition, and therapeutic resistance. (4,5) The modulation of lactate has been proposed as a promising strategy for enhanced cancer therapy. (6) Rational design of nanozymes with satisfactory lactate oxidase (LOX)-mimicking activities can encourage lactate-responsive tumor therapeutic strategies. However, the oxidation of the α-C–sp(3)–H bond of lactate to pyruvate is extremely challenging for conventional chemical catalysts at mild conditions due to the high bond energy of the C–H bond. (7) Traditionally, oxidative dehydrogenation of lactate to pyruvate is unavoidable under high temperatures (typically over 200 °C) or pressure (e.g., over 1 MPa), greatly hindering the exploitation of nanozymes with lactate management ability. (8) LOX, a member of flavoenzyme, can specifically catalyze the oxidation of lactate to pyruvate. (9,10) LOX is characteristic of a particular flavin mononucleotide (FMN)–histidine residue (His) pair as a catalytic pocket (Figure 1a), (11−13) where proton abstraction of lactate and deprotonation of intermediate toward pyruvate subsequently occurred at imidazole N(1) of His and flavin N(5) of oxidized FMN (Figure 1b and Figure S1). (14,15) The strong electronegativity of N at the FMN-His catalytic pocket serves as a key factor for the breakage of the α-C–H bond. (16) Inspired by nature, the breakthrough of nanozyme in LOX-mimicking efficiency should be considered from an optimized electronic configuration around the electron-rich N site.
乳酸肿瘤是一种重要的信号分子，在肿瘤恶性进展、血管生成、免疫抑制和治疗耐药性中起重要作用。（4,5） 乳酸的调节已被提出为增强癌症治疗的有前途的策略。（6）合理设计具有令人满意的乳酸氧化酶（LOX）模拟活性的纳米酶可以促进乳酸反应性肿瘤治疗策略。然而，由于C-H键的高键能，乳酸的α-C-sp（3）-H键在温和条件下氧化为丙酮酸对于传统的化学催化剂极具挑战性。（7）传统上，在高温（通常超过200°C）或压力（例如，超过1 MPa）下，乳酸氧化脱氢为丙酮酸是不可避免的，这极大地阻碍了具有乳酸管理能力的纳米酶的开发。（8）LOX是黄酮酶的成员，能特异性催化乳酸氧化为丙酮酸。（9,10） LOX 是特定黄素单核苷酸 （FMN）-组氨酸残基 （His） 对作为催化口袋的特征（图 1a），（11−13），其中乳酸的质子提取和中间体向丙酮酸的去质子化随后发生在 His 的咪唑 N（1） 和氧化 FMN 的黄素 N（5） 处（图 1b 和图 S1）。（14,15） N在FMN-His催化口袋处的强电负性是α-C-H键断裂的关键因素。（16）受自然界的启发，纳米酶在LOX模拟效率方面的突破应该从富电子N位点周围的优化电子构型来考虑。
乳酸肿瘤是一种重要的信号分子，在肿瘤恶性进展、血管生成、免疫抑制和治疗耐药性中起重要作用。（4,5） 乳酸的调节已被提出为增强癌症治疗的有前途的策略。（6）合理设计具有令人满意的乳酸氧化酶（LOX）模拟活性的纳米酶可以促进乳酸反应性肿瘤治疗策略。然而，由于C-H键的高键能，乳酸的α-C-sp（3）-H键在温和条件下氧化为丙酮酸对于传统的化学催化剂极具挑战性。（7）传统上，在高温（通常超过200°C）或压力（例如，超过1 MPa）下，乳酸氧化脱氢为丙酮酸是不可避免的，这极大地阻碍了具有乳酸管理能力的纳米酶的开发。（8）LOX是黄酮酶的成员，能特异性催化乳酸氧化为丙酮酸。（9,10） LOX 是特定黄素单核苷酸 （FMN）-组氨酸残基 （His） 对作为催化口袋的特征（图 1a），（11−13），其中乳酸的质子提取和中间体向丙酮酸的去质子化随后发生在 His 的咪唑 N（1） 和氧化 FMN 的黄素 N（5） 处（图 1b 和图 S1）。（14,15） N在FMN-His催化口袋处的强电负性是α-C-H键断裂的关键因素。（16）受自然界的启发，纳米酶在LOX模拟效率方面的突破应该从富电子N位点周围的优化电子构型来考虑。
Figure 1 图1
Figure 1. Enzyme-mimetic approach and characterization. (a) Natural LOX catalysis model and (b) proton abstraction from lactate toward natural LOX based on carbanion formation mechanism (PDB code: 5EBU). (c) N-centered nanozyme catalysis model. (d) Crystal structures of Co2N, Co3N, and Co4N. (e) N 1s XPS of the Co4N/C NEs. (f) Co 2p (sat. stands for satellite peak) XPS of Co3O4/C and Co4N/C NEs. (g) TEM images of Co4N/C NEs (inset: TEM image of Co4N/C NEs under higher magnification). (h) HRTEM image of Co4N/C NE. (i) HADDF image and element mapping of Co4N/C NEs (scale bar = 100 nm). (j) XRD patterns of Co2N/C, Co3N/C, and Co4N/C NEs (bottom: standard pattern for Co2N: 72-1368, Co3N: 06-0691, and Co4N: 41-0943).
图 1.酶模拟方法和表征。（a）天然LOX催化模型和（b）基于碳离子形成机理的质子从乳酸向天然LOX的提取（PDB代码：5EBU）。（c） N中心纳米酶催化模型。（d） Co2N、Co3N和Co4N的晶体结构。（e） Co4N/C NEs的N 1s XPS。（f） Co3O4/C和Co4N/C NEs的Co 2p（卫星峰值代表）XPS。（g） Co4N/C NEs的TEM图像（插图：高倍率下Co4N/C NEs的TEM图像）。（h） Co4N/C NE 的 HRTEM 图像。 （i） Co4N/C NEs 的 HADDF 图像和元素映射（比例尺 = 100 nm）。（j） Co2N/C、Co3N/C 和 Co4N/C NEs 的 XRD 图谱（下图：Co2N：72-1368、Co3N：06-0691 和 Co4N：41-0943 的标准图谱）。
Coordination engineering has been proposed to be an effective way to modulate the local electronic structure by precisely controlling the number or species of ligand atoms. (2,17) Recent studies demonstrated that the coordination of heterocyclic N with the metal can improve the catalytic activity in carbon-supported metalx–Ny (MxNy) materials. (18,19) Various strategies focused mainly on manipulating the charge density of metal centers via controlling the number of N coordinates to metal, for instance, M–N2, M–N3, and M–N4, to mimic the active sites similar to natural metalloenzyme. (2) However, different from the metalloenzymes, LOX is a flavoenzyme that is involved in the FMN-His pair pathway, where the non-metal N occupies a significant center. (20) Due to the strong electronegativity of N, the modulation of electron density for nonmetal N atoms in active sites could offer a great opportunity to construct flavoenzyme-mimic nanozymes. However, identifying the effects of the metal number to modulation on the local coordination environment and electronic configuration at the N-active sites as well as the resulting regulation of the lactate catalysis reaction is still a formable challenge.
配位工程被认为是通过精确控制配体原子的数量或种类来调制局部电子结构的有效方法。（2,17） 最近的研究表明，杂环N与金属的配位可以提高碳负载金属-钪（MxNy）材料的催化活性。（18,19） 各种策略主要集中在通过控制金属的 N 坐标数量来操纵金属中心的电荷密度，例如 M-N2、M-N3 和 M-N4，以模拟类似于天然金属酶的活性位点。（2）然而，与金属酶不同，LOX是一种黄素酶，参与FMN-His对途径，其中非金属N占据重要中心。（20）由于N的强电负性，活性位点中非金属N原子的电子密度的调制为构建黄素酶模拟纳米酶提供了很好的机会。然而，确定金属数对调节对N活性位点的局部配位环境和电子构型的影响，以及由此产生的乳酸催化反应的调节仍然是一个可形成的挑战。
配位工程被认为是通过精确控制配体原子的数量或种类来调制局部电子结构的有效方法。（2,17） 最近的研究表明，杂环N与金属的配位可以提高碳负载金属-钪（MxNy）材料的催化活性。（18,19） 各种策略主要集中在通过控制金属的 N 坐标数量来操纵金属中心的电荷密度，例如 M-N2、M-N3 和 M-N4，以模拟类似于天然金属酶的活性位点。（2）然而，与金属酶不同，LOX是一种黄素酶，参与FMN-His对途径，其中非金属N占据重要中心。（20）由于N的强电负性，活性位点中非金属N原子的电子密度的调制为构建黄素酶模拟纳米酶提供了很好的机会。然而，确定金属数对调节对N活性位点的局部配位环境和电子构型的影响，以及由此产生的乳酸催化反应的调节仍然是一个可形成的挑战。
In this work, we designed a LOX-mimic-based Co4N/C nanozymes (Co4N/C NEs) to realize the catalytic oxidation of lactate to pyruvate, via manipulating coordination fields and electronic structure by tuning the coordination shell of N with different numbers of metals (M2–N, M3–N, and M4–N). The experimental and theoretical calculations demonstrate that the N at the Mx–N sites dominated the catalytic process, and the improved electron density at N sites can promote the breakage of the C–H bond of lactate at room temperature and facilitates the abstraction of the α-C–H proton and α-C–OH proton from lactate (Figure 1c,d) as well as favors the O2 abstraction of H atoms from the Co4N/C into H2O2. Furthermore, the in vitro and in vivo experiments manifest that Co4N/C NEs can reverse the high-lactate tumor microenvironments (TME) and activate its immunosuppressive state. The significant augmented anti-tumor immunity is employed to defend against subcutaneous tumors and metastatic lymphatic tumors. This strategy of designing such nanozymes contributes to the understanding of nanozyme evolution and inspires the construction of the next generation of artificial enzymes.
在这项工作中，我们设计了一种基于LOX模拟的Co4N/C纳米酶（Co4N/C NEs），通过调控配位场和电子结构，通过调控不同数量的金属（M2-N、M3-N和M4-N）的N配位壳，实现乳酸催化氧化为丙酮酸。实验和理论计算表明，Mx–N位点的N在催化过程中占主导地位，N位点电子密度的提高可以促进室温下乳酸C-H键的断裂，有利于乳酸中α-C–H质子和α-C–OH质子的提取（图1c，d） 以及有利于 O2 从 Co4N/C 中提取 H 原子转化为 H2O2。此外，体外和体内实验表明，Co4N/C NEs可以逆转高乳酸肿瘤微环境（TME）并激活其免疫抑制状态。显着增强的抗肿瘤免疫力用于防御皮下肿瘤和转移性淋巴瘤。这种设计这种纳米酶的策略有助于理解纳米酶的进化，并激发了下一代人工酶的构建。
在这项工作中，我们设计了一种基于LOX模拟的Co4N/C纳米酶（Co4N/C NEs），通过调控配位场和电子结构，通过调控不同数量的金属（M2-N、M3-N和M4-N）的N配位壳，实现乳酸催化氧化为丙酮酸。实验和理论计算表明，Mx–N位点的N在催化过程中占主导地位，N位点电子密度的提高可以促进室温下乳酸C-H键的断裂，有利于乳酸中α-C–H质子和α-C–OH质子的提取（图1c，d） 以及有利于 O2 从 Co4N/C 中提取 H 原子转化为 H2O2。此外，体外和体内实验表明，Co4N/C NEs可以逆转高乳酸肿瘤微环境（TME）并激活其免疫抑制状态。显着增强的抗肿瘤免疫力用于防御皮下肿瘤和转移性淋巴瘤。这种设计这种纳米酶的策略有助于理解纳米酶的进化，并激发了下一代人工酶的构建。
2. Results and Discussion
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2. 结果与讨论点击复制章节链接
2.1. Material Synthesis and Characterization
2.1. 材料合成与表征
To obtain the N-centered nanozyme, Co3O4/C was first synthesized through the pyrolysis of Co-MOF and further transformed into CoxN phases after high-temperature nitridation using NH3 as a nitrogen source (Figures S2 and S3). (21) Taking Co4N as an example, the full X-ray photoelectron spectroscopy (XPS) spectrum of Co3O4/C shows the presence of Co, C, and O, and an exclusive N peak appeared after its nitridation (Figure S4). In the high-resolution XPS spectrum of the N 1s spectra of as-synthesized Co4N/C, the characteristic peak at 398.9 eV is attributed to N–Co in the Co4N phase (Figure 1e). The peak that appears at 400.7 eV stands for the N–H surface terminal group after ammonia treatments, and the peak at 403.6 eV could be assigned to N–O as Co4N would passivate to form a thin surface layer of cobalt oxide when exposed to air. (22) In the Co 2p spectra, characteristic Co–N peaks of Co4N/C appear at 796.3 and 780.8 eV, indicating the successful permeation of N atoms into the Co lattice after nitridation (Figure 1f). Moreover, the peaks of Co4N/C show a positive chemical shift compared with Co3O4/C, suggesting electron transfer from Co to N atoms. (23,24) The C 1s peaks at 293.1, 288.4, 285.3, and 284.7 eV belong to C═O, C–N, C–C, and C–Co, respectively (Figure S5a). From the O 1s spectra, a clear peak at 531.2 eV means that hydroxy or oxygen is adsorbed on the surface of Co4N/C NEs (Figure S5b). The transmission electron microscopy (TEM) image reveals that Co4N/C NE possesses a cubic morphology with an average diameter of about 160 nm (Figure 1g). The corrosion of NH3 endows Co4N/C NEs with hollow structures, while obvious mesopores and micropores formed on the surface (Figure 1g, inset). In the high-resolution TEM (HRTEM) image, the lattice fringes with spacings of 0.205 and 0.172 nm correspond to (010) crystal facets and (200) planes of Co4N (Figure 1h). HADDF-element mapping of Co4N/C NEs shows that elements N, Co, C, and O homogeneously distribute on the Co4N/C NEs matrix (Figure 1i). The selected-area electron diffraction (SAED) pattern further confirms that the Co4N/C NEs are polycrystalline (Figure S6). As a comparison, different proportions of Co atoms were gradually introduced to coordinate N atoms (Co2N/C, Co3N/C, and Co4N/C) to study N atoms with favorable local coordination environments and electronic configurations for lactate oxidation. With the pyrolysis and nitridation temperature increasing, the Cox–N species transformed from the low symmetric orthorhombic structure (Co2N) at 360 °C to a hexagonal structure (Co3N) at 460 °C and transformed to high symmetric cubic Co4N after 500 °C (Figure 1d). (22) The X-ray powder diffraction (XRD) patterns of Co3O4/C, Co2N/C, Co3N/C, and Co4N/C are well matched with their respective standard JCPDS cards, indicating the successful synthesis of single-phase cobalt nitrides with different crystal structures (Figure S7 and Figure 1j).
为了获得以N为中心的纳米酶，首先通过Co-MOF热解合成Co3O4/C，并以NH3为氮源，经过高温氮化后进一步转化为CoxN相（图S2和S3）。（21）以Co4N为例，Co3O4/C的全X射线光电子能谱（XPS）光谱显示Co、C、O的存在，氮化后出现排他性的N峰（图S4）。在合成的Co4N/C的N 1s光谱的高分辨率XPS光谱中，398.9 eV处的特征峰归因于Co4N相中的N-Co（图1e）。在400.7 eV处出现的峰值代表氨处理后的N-H表面末端组，在403.6 eV处的峰值可以分配给N-O，因为Co4N在暴露于空气时会钝化形成一层薄薄的氧化钴表面层。（22） 在Co 2p光谱中，Co4N/C的特征Co-N峰出现在796.3和780.8 eV处，表明氮化后N原子成功渗透到Co晶格中（图1f）。此外，与Co3O4/C相比，Co4N/C的峰显示出正的化学位移，表明电子从Co转移到N原子。（23,24） C 1s 在 293.1、288.4、285.3 和 284.7 eV 处的峰值分别属于 C═O、C-N、C-C 和 C-Co（图 S5a）。从 O 1s 光谱来看，531.2 eV 处的清晰峰值意味着羟基或氧被吸附在 Co4N/C NE 表面（图 S5b）。透射电子显微镜（TEM）图像显示，Co4N/C NE具有立方形貌，平均直径约为160 nm（图1g）。NH3的腐蚀赋予Co4N/C NEs中空结构，表面形成明显的介孔和微孔（图1g，插图）。 在高分辨率透射电镜（HRTEM）图像中，间距为0.205和0.172 nm的晶格条纹对应于（010）个晶面和（200）个Co4N平面（图1h）。Co4N/C NEs 的 HADDF 元素映射表明，元素 N、Co、C 和 O 均匀分布在 Co4N/C NEs 基质上（图 1i）。选定区域电子衍射（SAED）图谱进一步证实了Co4N/C NEs是多晶的（图S6）。作为比较，逐渐引入不同比例的Co原子来配位N原子（Co2N/C、Co3N/C和Co4N/C），以研究具有有利局部配位环境和乳酸氧化电子构型的N原子。随着热解和氮化温度的升高，Cox-N物种从360 °C的低对称正交结构（Co2N）转变为460 °C的六方结构（Co3N），并在500 °C后转变为高对称的立方Co4N（图1d）。（22）Co3O4/C、Co2N/C、Co3N/C和Co4N/C的X射线粉末衍射（XRD）图谱与各自的标准JCPDS卡非常匹配，表明成功合成了具有不同晶体结构的单相氮化钴（图S7和图1j）。
为了获得以N为中心的纳米酶，首先通过Co-MOF热解合成Co3O4/C，并以NH3为氮源，经过高温氮化后进一步转化为CoxN相（图S2和S3）。（21）以Co4N为例，Co3O4/C的全X射线光电子能谱（XPS）光谱显示Co、C、O的存在，氮化后出现排他性的N峰（图S4）。在合成的Co4N/C的N 1s光谱的高分辨率XPS光谱中，398.9 eV处的特征峰归因于Co4N相中的N-Co（图1e）。在400.7 eV处出现的峰值代表氨处理后的N-H表面末端组，在403.6 eV处的峰值可以分配给N-O，因为Co4N在暴露于空气时会钝化形成一层薄薄的氧化钴表面层。（22） 在Co 2p光谱中，Co4N/C的特征Co-N峰出现在796.3和780.8 eV处，表明氮化后N原子成功渗透到Co晶格中（图1f）。此外，与Co3O4/C相比，Co4N/C的峰显示出正的化学位移，表明电子从Co转移到N原子。（23,24） C 1s 在 293.1、288.4、285.3 和 284.7 eV 处的峰值分别属于 C═O、C-N、C-C 和 C-Co（图 S5a）。从 O 1s 光谱来看，531.2 eV 处的清晰峰值意味着羟基或氧被吸附在 Co4N/C NE 表面（图 S5b）。透射电子显微镜（TEM）图像显示，Co4N/C NE具有立方形貌，平均直径约为160 nm（图1g）。NH3的腐蚀赋予Co4N/C NEs中空结构，表面形成明显的介孔和微孔（图1g，插图）。 在高分辨率透射电镜（HRTEM）图像中，间距为0.205和0.172 nm的晶格条纹对应于（010）个晶面和（200）个Co4N平面（图1h）。Co4N/C NEs 的 HADDF 元素映射表明，元素 N、Co、C 和 O 均匀分布在 Co4N/C NEs 基质上（图 1i）。选定区域电子衍射（SAED）图谱进一步证实了Co4N/C NEs是多晶的（图S6）。作为比较，逐渐引入不同比例的Co原子来配位N原子（Co2N/C、Co3N/C和Co4N/C），以研究具有有利局部配位环境和乳酸氧化电子构型的N原子。随着热解和氮化温度的升高，Cox-N物种从360 °C的低对称正交结构（Co2N）转变为460 °C的六方结构（Co3N），并在500 °C后转变为高对称的立方Co4N（图1d）。（22）Co3O4/C、Co2N/C、Co3N/C和Co4N/C的X射线粉末衍射（XRD）图谱与各自的标准JCPDS卡非常匹配，表明成功合成了具有不同晶体结构的单相氮化钴（图S7和图1j）。
2.2. LOX-Mimicking Activity of the Co4N/C NEs
2.2. Co4N/C NEs的LOX模拟活性
To study the role of the modulated metal number at the coordination shell of N on the catalytic process, the catalytic oxidation properties of lactate of the as-obtained N/C, Co2N/C, Co3N/C, and Co4N/C were evaluated. To exclude the influence of metallic Co, Co3O4/C was set as a control (Figure 2a). Co3O4/C, N/C, Co2N/C, Co3N/C, and Co4N/C were reacted with 5 mM of lactate, and the lactate consumptions were detected by a lactic acid assay kit. As expected, Co3O4/C induced a negligible lactate concentration change, and a slight lactate consumption was observed in the presence of N/C. Meanwhile, an obvious lactate consumption of approximately 0.43 mM was found in the Co2N/C solution, 4.3-fold higher than that of the N/C sample, which indicates that the electron-rich site at the N-heterocycle after the coordination of Co to N can realize the catalytic capacity for lactate oxidation. Moreover, Co3N/C and Co4N/C achieved significant lactate consumption values of 0.53 and 0.86 mM, representing 5.3-fold and 8.6-fold that of N/C, respectively. These results demonstrate that the increase of the metal number coordinates to the N can facilitate lactate oxidation via the enhancement of electron density for nonmetal-N-atoms. To further confirm the role of the N center in lactate catalysis, Co was inhibited by SCN–, a Co complexing agent. Compared with Co inhibition before, lactate consumption of Co2N/C and Co3N/C was slightly decreased to 86.2 and 87.1% after KSCN addition, respectively. Inspiringly, Co4N/C was just decreased to 95.2%, indicating that N plays an essential role in the process of lactate consumption (Figure 2b). Moreover, Co4N/C NEs show lower activation energy (7.77 kJ mol–1) than those of Co2N/C (13.89 kJ mol–1) and Co3N (13.79 kJ mol–1), indicating that the energy barrier of lactate oxidation can be reduced with the increase of the Co coordination number (Figure 2c).
为研究N配位壳层调制金属数对催化过程的影响，评价了N/C、Co2N/C、Co3N/C和Co4N/C乳酸的催化氧化性能。为了排除金属Co的影响，将Co3O4 / C设置为对照（图2a）。将Co3O4/C、N/C、Co2N/C、Co3N/C和Co4N/C与5 mM乳酸反应，用乳酸检测试剂盒检测乳酸消耗量。正如预期的那样，Co3O4/C 诱导的乳酸浓度变化可以忽略不计，并且在 N/C 存在下观察到轻微的乳酸消耗。同时，Co2N/C溶液的乳酸消耗量明显约为0.43 mM，比N/C样品高4.3倍，表明Co与N配位后N杂环的富电子位点可以实现乳酸氧化的催化能力。此外，Co3N/C和Co4N/C的乳酸消耗量分别为0.53和0.86 mM，分别是N/C的5.3倍和8.6倍。这些结果表明，金属数坐标向N的增加可以通过提高非金属N原子的电子密度来促进乳酸氧化。为了进一步证实N中心在乳酸催化中的作用，Co被Co络合剂SCN–抑制。与之前的Co抑制相比，添加KSCN后Co2N/C和Co3N/C的乳酸消耗量分别略微下降至86.2%和87.1%。令人鼓舞的是，Co4N/C刚刚下降到95.2%，表明N在乳酸消耗过程中起着至关重要的作用（图2b）。此外，Co4N/C NEs的活化能（7.77 kJ mol–1）低于Co2N/C（13.89 kJ mol–1）和Co3N（13.79 kJ mol–1），表明乳酸氧化的能量势垒可以随着协配位数的增加而降低（图2c）。
为研究N配位壳层调制金属数对催化过程的影响，评价了N/C、Co2N/C、Co3N/C和Co4N/C乳酸的催化氧化性能。为了排除金属Co的影响，将Co3O4 / C设置为对照（图2a）。将Co3O4/C、N/C、Co2N/C、Co3N/C和Co4N/C与5 mM乳酸反应，用乳酸检测试剂盒检测乳酸消耗量。正如预期的那样，Co3O4/C 诱导的乳酸浓度变化可以忽略不计，并且在 N/C 存在下观察到轻微的乳酸消耗。同时，Co2N/C溶液的乳酸消耗量明显约为0.43 mM，比N/C样品高4.3倍，表明Co与N配位后N杂环的富电子位点可以实现乳酸氧化的催化能力。此外，Co3N/C和Co4N/C的乳酸消耗量分别为0.53和0.86 mM，分别是N/C的5.3倍和8.6倍。这些结果表明，金属数坐标向N的增加可以通过提高非金属N原子的电子密度来促进乳酸氧化。为了进一步证实N中心在乳酸催化中的作用，Co被Co络合剂SCN–抑制。与之前的Co抑制相比，添加KSCN后Co2N/C和Co3N/C的乳酸消耗量分别略微下降至86.2%和87.1%。令人鼓舞的是，Co4N/C刚刚下降到95.2%，表明N在乳酸消耗过程中起着至关重要的作用（图2b）。此外，Co4N/C NEs的活化能（7.77 kJ mol–1）低于Co2N/C（13.89 kJ mol–1）和Co3N（13.79 kJ mol–1），表明乳酸氧化的能量势垒可以随着协配位数的增加而降低（图2c）。
Figure 2 图2
Figure 2. LOX-mimicking activities of Co4N/C NEs. (a) Lactate consumption of Co3O4/C, N/C, Co2N/C, Co3N/C, and Co4N/C. (b) Influence of KSCN (a Co complexing agent) during the oxidation of lactate in the presence of Co2N/C, Co3N/C, and Co4N/C NEs. (c) Activation energy (Ea) for the catalytic reaction of the LOX-mimicking activity of Co2N/C, Co3N/C, and Co4N/C. (d) Relative activity of lactate oxidation catalyzed by Co4N/C NEs and natural LOX (100 ng mL–1) with various pH values. (e) Lactate consumption curves at different concentrations of Co4N/C NEs. (f) Michaelis–Menten kinetic curves and (g) Lineweaver–Burk plot for the Co4N/C NEs (LOX-mimicking activity). (h) Lactate consumption effect of Co4N/C NEs under air or N2 conditions. The stability of (i) lactate consumption and pyruvate generation and (j) H2O2 generation in four cycles. (k) Selectivity of Co4N/C NEs toward some cellular reductive biomolecules under US. (l) Schematic illustration of the LOX-mimic Ping-Pong mechanism of Co4N/C NEs.
图2.Co4N/C NEs的LOX模拟活性。（a） 乳酸消耗Co3O4/C、N/C、Co2N/C、Co3N/C和Co4N/C。 （b） KSCN（一种Co络合剂）在Co2N/C、Co3N/C和Co4N/C NEs存在下对乳酸氧化的影响。（c） Co2N/C、Co3N/C和Co4N/C的LOX模拟活性催化反应的活化能（Ea） （d） Co4N/C NEs和天然LOX（100 ng mL–1）在不同pH值下催化的乳酸氧化相对活性。（e） 不同浓度Co4N/C NEs下的乳酸消耗曲线。（f） Co4N/C NEs的Michaelis-Menten动力学曲线和（g） Lineweaver-Burk图（LOX模拟活动）。（h） 空气或N2条件下Co4N/C NEs的乳酸消耗效应。（i） 乳酸消耗和丙酮酸生成以及 （j） H2O2 生成在四个循环中的稳定性。（k） US下Co4N/C NEs对某些细胞还原生物分子的选择性。（l） Co4N/C NEs的LOX模拟Ping-Pong机理示意图。
The reaction parameters of the Co4N/C NEs were investigated for better imitation of LOX. The activity of the Co4N/C NEs is enhanced gradually with increasing pH and temperature (Figure 2d and Figure S8). Notably, the Co4N/C NEs show improved catalytic performance when the pH increases from 5.0 to 9.0, which can further confirm the decrease of positive charge, and the relatively enhanced negatively charged environment around the N site can facilitate lactate oxidation. (25) However, the lactate catalytic efficiency of natural LOX was significantly reduced over pH 6.5 and at 50 °C, demonstrating the superior performance of the Co4N/C NEs over natural LOX at harsh environments. The kinetics of the oxidation of lactate is mainly dependent on the concentration of the Co4N/C NEs (Figure 2e), which is also well fitted to Michaelis–Menten kinetics and the Lineweaver–Burk plot of enzyme reactions (Figure 2f,g). The maximum initial velocity (Vmax) and Menten constant (Km) were calculated to be 8.98 ± 0.605 μM s–1 and 10.5 ± 1.14 mM, respectively. The catalytic efficiency for the LOX-mimicking activity of Co4N/C NEs was determined to be (0.349 ± 0.0609) × 106 mM–1 s–1. Moreover, it is worth mentioning that the catalytic reaction only occurs in the presence of O2. The catalytic activity of the Co4N/C NEs shows an oxygen-dependent manner (Figure 2h), while the Co4N/C NEs failed to induce obvious changes in lactate concentration under a N2 atmosphere. Co4N/C NEs maintain stable crystal structures during the process of lactate oxidation, indicative of the catalyst role of the nanozyme (Figure S9). Co4N/C NEs display similar LOX activity when lactate was consumed to about 80% of its original level; thus, significant H2O2 was generated. In addition, the LOX-like activity of the Co4N/C NEs maintains high stability after 4 cycles (Figure 2i,j). Moreover, the selectivity of the nanozyme was studied in some cellular reductive biomolecules. Excitingly, lactate can be significantly consumed by Co4N/C NEs while few (less than 10%) other biomolecules were influenced (Figure 2k).
研究了Co4N/C NEs的反应参数，以更好地模拟LOX。随着pH值和温度的升高，Co4N/C NEs的活性逐渐增强（图2d和图S8）。值得注意的是，当pH从5.0提高到9.0时，Co4N/C NEs表现出更好的催化性能，这可以进一步证实正电荷的减少，并且N位点周围相对增强的负电荷环境可以促进乳酸氧化。（25）然而，在pH 6.5和50 °C下，天然LOX的乳酸催化效率显著降低，表明Co4N/C NEs在恶劣环境下的性能优于天然LOX。乳酸氧化的动力学主要取决于Co4N / C NEs的浓度（图2e），这也非常适合Michaelis-Menten动力学和酶反应的Lineweaver-Burk图（图2f，g）。计算出的最大初始速度 （Vmax） 和 Menten 常数 （Km） 分别为 8.98 ± 0.605 μM s–1 和 10.5 ± 1.14 mM。Co4N/C NEs模拟LOX活性的催化效率确定为（0.349 ± 0.0609）×106 mM–1 s–1。此外，值得一提的是，催化反应仅在O2存在下发生。Co4N/C NEs的催化活性呈氧依赖性（图2h），而Co4N/C NEs在N2气氛下未能引起乳酸浓度的明显变化。Co4N/C NEs在乳酸氧化过程中保持稳定的晶体结构，表明纳米酶的催化剂作用（图S9）。当乳酸消耗至其原始水平的80%左右时，Co4N/C NEs表现出相似的LOX活性;因此，产生了显着的H2O2。 此外，Co4N/C NEs的LOX样活性在4个循环后仍保持高稳定性（图2i，j）。此外，还研究了纳米酶在一些细胞还原性生物分子中的选择性。令人兴奋的是，乳酸可以被Co4N/C NEs显着消耗，而其他生物分子很少（不到10%）受到影响（图2k）。
研究了Co4N/C NEs的反应参数，以更好地模拟LOX。随着pH值和温度的升高，Co4N/C NEs的活性逐渐增强（图2d和图S8）。值得注意的是，当pH从5.0提高到9.0时，Co4N/C NEs表现出更好的催化性能，这可以进一步证实正电荷的减少，并且N位点周围相对增强的负电荷环境可以促进乳酸氧化。（25）然而，在pH 6.5和50 °C下，天然LOX的乳酸催化效率显著降低，表明Co4N/C NEs在恶劣环境下的性能优于天然LOX。乳酸氧化的动力学主要取决于Co4N / C NEs的浓度（图2e），这也非常适合Michaelis-Menten动力学和酶反应的Lineweaver-Burk图（图2f，g）。计算出的最大初始速度 （Vmax） 和 Menten 常数 （Km） 分别为 8.98 ± 0.605 μM s–1 和 10.5 ± 1.14 mM。Co4N/C NEs模拟LOX活性的催化效率确定为（0.349 ± 0.0609）×106 mM–1 s–1。此外，值得一提的是，催化反应仅在O2存在下发生。Co4N/C NEs的催化活性呈氧依赖性（图2h），而Co4N/C NEs在N2气氛下未能引起乳酸浓度的明显变化。Co4N/C NEs在乳酸氧化过程中保持稳定的晶体结构，表明纳米酶的催化剂作用（图S9）。当乳酸消耗至其原始水平的80%左右时，Co4N/C NEs表现出相似的LOX活性;因此，产生了显着的H2O2。 此外，Co4N/C NEs的LOX样活性在4个循环后仍保持高稳定性（图2i，j）。此外，还研究了纳米酶在一些细胞还原性生物分子中的选择性。令人兴奋的是，乳酸可以被Co4N/C NEs显着消耗，而其他生物分子很少（不到10%）受到影响（图2k）。
The LOX-mimic performance of Co4N/C NEs can be further regulated by ultrasound (US) irradiation. Under US treatment (1.75 W cm–2, 1 MHz, 40% duty cycle), the lactate consumption ability of the Co4N/C NEs increased by 1.68-fold, H2O2 generation increased by 1.33-fold (Figure S10), the Vmax was raised to 13.3 ± 0.915 μM s–1, and the Km dropped to 9.08 ± 0.950 mM with a catalytic efficiency of (0.597 ± 0.103) × 106 mM–1 s–1 (Figure S11 and Table S1). Quasi in situ ATR-FTIR was applied to understand the status of Co4N/C NEs during the catalytic oxidation of lactate (Figure S12). The spectrum of Co3O4/C after treatment with lactate under US irradiation exhibits no obvious peaks. However, a peak at 3236 cm–1 was found in the spectrum of the Co4N/C NEs, which originates from the stretching vibration of N–H, signaling the formation of Co4NHx. (23) Therefore, a Ping-Pong mechanism of the Co4N/C NEs can be proposed: the α-C–H and α-C–OH protons are dehydrogenated from lactate and transferred to Co4N, accompanied by the generation of pyruvate and Co4NH2. Subsequently, the H protons from Co4NH2 are reduced from O2 to H2O2 and turned back to Co4N (Figure 2l). These results demonstrate that Co4N/C NEs can be a promising LOX candidate for tumor lactate modulation.
Co4N/C NEs的LOX模拟性能可以通过超声（US）辐照进一步调节。在美片处理（1.75 W cm–2,1 MHz，40%占空比）下，Co4N/C NEs的乳酸消耗能力增加了1.68倍，H2O2的生成量增加了1.33倍（图S10），Vmax提高到13.3±0.915 μM s–1，Km下降到9.08±0.950 mM，催化效率为（0.597 ± 0.103）×106 mM–1 s–1（图S11和表S1）。应用准原位ATR-FTIR来了解乳酸催化氧化过程中Co4N/C NEs的状态（图S12）。在美声照射下用乳酸处理后的Co3O4/C光谱没有明显的峰值。然而，在Co4N/C NEs的光谱中发现了3236 cm–1的峰值，该峰值源于N-H的拉伸振动，标志着Co4NHx的形成。（23）因此，可以提出Co4N/C NEs的乒乓机制：α-C-H和α-C-OH质子从乳酸脱氢并转移到Co4N中，同时生成丙酮酸和Co4NH2。随后，来自 Co4NH2 的 H 质子从 O2 还原为 H2O2 并变回 Co4N（图 2l）。这些结果表明，Co4N/C NEs可以成为肿瘤乳酸调节的有前途的LOX候选者。
Co4N/C NEs的LOX模拟性能可以通过超声（US）辐照进一步调节。在美片处理（1.75 W cm–2,1 MHz，40%占空比）下，Co4N/C NEs的乳酸消耗能力增加了1.68倍，H2O2的生成量增加了1.33倍（图S10），Vmax提高到13.3±0.915 μM s–1，Km下降到9.08±0.950 mM，催化效率为（0.597 ± 0.103）×106 mM–1 s–1（图S11和表S1）。应用准原位ATR-FTIR来了解乳酸催化氧化过程中Co4N/C NEs的状态（图S12）。在美声照射下用乳酸处理后的Co3O4/C光谱没有明显的峰值。然而，在Co4N/C NEs的光谱中发现了3236 cm–1的峰值，该峰值源于N-H的拉伸振动，标志着Co4NHx的形成。（23）因此，可以提出Co4N/C NEs的乒乓机制：α-C-H和α-C-OH质子从乳酸脱氢并转移到Co4N中，同时生成丙酮酸和Co4NH2。随后，来自 Co4NH2 的 H 质子从 O2 还原为 H2O2 并变回 Co4N（图 2l）。这些结果表明，Co4N/C NEs可以成为肿瘤乳酸调节的有前途的LOX候选者。
2.3. DFT Calculations 2.3. DFT计算
To further understand the LOX-mimicking activities of Co4N/C NEs, density functional theory (DFT) calculations were carried out based on first principles. As shown in Figure 3a, the deformation charge density is calculated to visualize the lone pair electrons on the N atom. The blue-to-red transformation stands for the gradually increased charge transfer amount. When Co bonded with N, the electron density of Co is directional (green non-circular area), meaning that electrons transfer from Co to the N atom and the part closer to N shows a denser electron density. Most importantly, the red area of N gradually increases with more Co atom coordination, which indicates the increased electron density around the N center in the sequence of Co2N–Co3N–Co4N. An electron-rich N center gradually forms with Co coordination, which can facilitate the abstraction on the α-C–OH and α-C–H protons from lactate. Furthermore, the d-band center of Co was calculated according to the density of states (DOS) of Co2N, Co3N, and Co4N (Figure 3b). Compared with the Co2N model, Co-doping enhancement causes a distinct downshift of the d-band center of the Co orbital. Particularly, the Co4N model achieves the lowest band center position at −1.49 eV, which is the state farthest from the Fermi level compared with Co2N (−1.40 eV) and Co3N (−1.45 eV). Distance from the Fermi level contributes to weaker chemical bonding with intermediate species and accelerates the rate-limiting step. (3) Thus, we propose the following catalytic pathway of Co4N, mimicking the Ping-Pong mechanism based on the carbanion formation of natural LOX thermodynamically (Figure 3c and Figure S13a). The Co2N and Co3N models were set as a control (Figures S14 and S15). The reactions start at the (111) crystal facets of Co4N (step 1), and O2 enters and pre-adsorbs on the Co site of the Co4N model (step 2). Then, the O of C═O in the carboxyl of lactate links with Co and locates on Co4N like the binding pocket of natural LOX (step 3). Profiting from electron-richest N from the coordination of Co, the N of Co4N abstracts the α-C–H proton, forming the transition state 1 (TS1). After finishing the abstraction (step 4), the other N on the parallel site of Co4N abstracts the other α-C–OH proton, forming the TS2. The two H atoms captured from lactate are located at the top sites of the N atoms (step 5). The TS1 and TS2 of Co2N, Co3N, and Co4N are shown in Figure 3d, respectively. The corresponding barriers of TS1 are calculated as 2.45, 1.15, and 0.96 eV, respectively. The corresponding barriers of TS2 are 0.61, 0.35, and 0.22 eV. In the process of H proton abstraction, the barrier of Co4N is significantly lower than that of Co2N and Co3N, suggesting that Co4N most easily completes the abstraction from lactate. Followed by the desorption steps, pyruvate is released from the model after the completion of the H proton abstraction (step 6). This model can correspond to the structure in Figure S12 and Figure 2l. Subsequently, O2 moves to the H-binding site and oxidizes H into H2O2 (steps 7 and 8). Finally, Co4N returns to its initial state after the desorption of H2O2. The calculation results show that Co4N is theoretically a more efficient mimic of LOX compared with Co2N and Co3N (Figure 3e), which forms the most harmonized electronic configuration around the electron-rich N sites and electron-deficient Co sites for a more precise recognition site for lactate and intermediate organization and optimizes the absorption energies for intermediates. The DFT calculations of another possible hydride transfer mechanism similar to natural LOX were also performed (Figures S16a and S13b). Different from the carbanion formation mechanism that abstracts protons in order of α-C–H to α-C–OH, Co4N first abstracts an α-C–OH proton from lactate, forming TS1 with a barrier of 1.35 eV (Figure S16b). The α-C–H of hydride is then transferred to Co4N to form TS2 with a barrier of 1.09 eV. The barriers of TS1 and TS2 of hydride transfer are 0.39 and 0.87 eV higher than carbanion formation in the dehydrogenation progress, respectively (Figure S16c).
为了进一步了解Co4N/C NEs的LOX模拟活性，基于第一性原理进行了密度泛函理论（DFT）计算。如图3a所示，计算变形电荷密度以可视化N原子上的孤对电子。蓝色到红色的转变代表逐渐增加的电荷转移量。当 Co 与 N 键合时，Co 的电子密度是定向的（绿色非圆形区域），这意味着电子从 Co 转移到 N 原子，靠近 N 的部分显示出更致密的电子密度。最重要的是，随着Co原子配位的增加，N的红色区域逐渐增大，这表明在Co2N-Co3N-Co4N的序列中，N中心周围的电子密度增加。富电子的N中心在Co配位下逐渐形成，可以促进乳酸中α-C-OH和α-C-H质子的提取。此外，根据 Co2N、Co3N 和 Co4N 的态密度 （DOS） 计算 Co 的 d 波段中心（图 3b）。与Co2N模型相比，共掺杂增强导致Co轨道的d波段中心明显下移。特别是，Co4N模型在−1.49 eV处达到最低能带中心位置，这是与Co2N（−1.40 eV）和Co3N（−1.45 eV）相比离费米能级最远的状态。与费米能级的距离有助于与中间物质的化学键变弱，并加速限速步骤。（3）因此，我们提出了以下Co4N的催化途径，模拟了基于天然LOX热力学碳离子形成的乒乓球机制（图3c和图S13a）。将 Co2N 和 Co3N 模型设置为对照（图 S14 和 S15）。 反应从 Co4N 的 （111） 晶面开始（步骤 1），O2 进入 Co4N 模型的 Co 位点并预吸附（步骤 2）。然后，乳酸羧基中 C═O 的 O 与 Co 连接并位于 Co4N 上，就像天然 LOX 的结合口袋一样（步骤 3）。从 Co 配位中获益于最富含电子的 N，Co4N 的 N 抽象出 α-C-H 质子，形成过渡态 1 （TS1）。完成提取（步骤4）后，Co4N平行位点上的另一个N抽象另一个α-C-OH质子，形成TS2。从乳酸捕获的两个H原子位于N原子的顶部位点（步骤5）。Co2N、Co3N和Co4N的TS1和TS2分别如图3d所示。TS1的相应势垒分别计算为2.45、1.15和0.96 eV。TS2 的相应势垒为 0.61、0.35 和 0.22 eV。在H质子提取过程中，Co4N的势垒显著低于Co2N和Co3N，表明Co4N最容易完成乳酸的提取。在解吸步骤之后，丙酮酸在H质子提取完成后从模型中释放出来（步骤6）。该模型可以对应于图S12和图2l中的结构。随后，O2 移动到 H 结合位点并将 H 氧化成 H2O2（步骤 7 和 8）。最后，Co4N在H2O2解吸后恢复到初始状态。计算结果表明，与Co2N和Co3N相比，Co4N在理论上是LOX更有效的模拟物（图3e），在富电子N位点和缺电子Co位点周围形成最协调的电子构型，为乳酸和中间体组织提供更精确的识别位点，并优化了中间体的吸收能。 还对另一种可能的氢化物转移机制进行了类似于天然LOX的DFT计算（图S16a和S13b）。与以 α-C-H 到 α-C-OH 的顺序提取质子的碳离子形成机制不同，Co4N 首先从乳酸中提取一个 α-C-OH 质子，形成具有 1.35 eV 势垒的 TS1（图 S16b）。然后将氢化物的 α-C-H 转移到 Co4N 中形成势垒为 1.09 eV 的 TS2。在脱氢过程中，TS1和TS2的氢化物转移障碍分别比碳离子形成高0.39和0.87 eV（图S16c）。
为了进一步了解Co4N/C NEs的LOX模拟活性，基于第一性原理进行了密度泛函理论（DFT）计算。如图3a所示，计算变形电荷密度以可视化N原子上的孤对电子。蓝色到红色的转变代表逐渐增加的电荷转移量。当 Co 与 N 键合时，Co 的电子密度是定向的（绿色非圆形区域），这意味着电子从 Co 转移到 N 原子，靠近 N 的部分显示出更致密的电子密度。最重要的是，随着Co原子配位的增加，N的红色区域逐渐增大，这表明在Co2N-Co3N-Co4N的序列中，N中心周围的电子密度增加。富电子的N中心在Co配位下逐渐形成，可以促进乳酸中α-C-OH和α-C-H质子的提取。此外，根据 Co2N、Co3N 和 Co4N 的态密度 （DOS） 计算 Co 的 d 波段中心（图 3b）。与Co2N模型相比，共掺杂增强导致Co轨道的d波段中心明显下移。特别是，Co4N模型在−1.49 eV处达到最低能带中心位置，这是与Co2N（−1.40 eV）和Co3N（−1.45 eV）相比离费米能级最远的状态。与费米能级的距离有助于与中间物质的化学键变弱，并加速限速步骤。（3）因此，我们提出了以下Co4N的催化途径，模拟了基于天然LOX热力学碳离子形成的乒乓球机制（图3c和图S13a）。将 Co2N 和 Co3N 模型设置为对照（图 S14 和 S15）。 反应从 Co4N 的 （111） 晶面开始（步骤 1），O2 进入 Co4N 模型的 Co 位点并预吸附（步骤 2）。然后，乳酸羧基中 C═O 的 O 与 Co 连接并位于 Co4N 上，就像天然 LOX 的结合口袋一样（步骤 3）。从 Co 配位中获益于最富含电子的 N，Co4N 的 N 抽象出 α-C-H 质子，形成过渡态 1 （TS1）。完成提取（步骤4）后，Co4N平行位点上的另一个N抽象另一个α-C-OH质子，形成TS2。从乳酸捕获的两个H原子位于N原子的顶部位点（步骤5）。Co2N、Co3N和Co4N的TS1和TS2分别如图3d所示。TS1的相应势垒分别计算为2.45、1.15和0.96 eV。TS2 的相应势垒为 0.61、0.35 和 0.22 eV。在H质子提取过程中，Co4N的势垒显著低于Co2N和Co3N，表明Co4N最容易完成乳酸的提取。在解吸步骤之后，丙酮酸在H质子提取完成后从模型中释放出来（步骤6）。该模型可以对应于图S12和图2l中的结构。随后，O2 移动到 H 结合位点并将 H 氧化成 H2O2（步骤 7 和 8）。最后，Co4N在H2O2解吸后恢复到初始状态。计算结果表明，与Co2N和Co3N相比，Co4N在理论上是LOX更有效的模拟物（图3e），在富电子N位点和缺电子Co位点周围形成最协调的电子构型，为乳酸和中间体组织提供更精确的识别位点，并优化了中间体的吸收能。 还对另一种可能的氢化物转移机制进行了类似于天然LOX的DFT计算（图S16a和S13b）。与以 α-C-H 到 α-C-OH 的顺序提取质子的碳离子形成机制不同，Co4N 首先从乳酸中提取一个 α-C-OH 质子，形成具有 1.35 eV 势垒的 TS1（图 S16b）。然后将氢化物的 α-C-H 转移到 Co4N 中形成势垒为 1.09 eV 的 TS2。在脱氢过程中，TS1和TS2的氢化物转移障碍分别比碳离子形成高0.39和0.87 eV（图S16c）。
Figure 3 图3
Figure 3. DFT calculations and LOX-mimicking catalysis mechanism. (a) Calculated deformation charge density of Co2N, Co3N, and Co4N. (b) Calculated Co 3d density of states (DOS) of the Co2N, Co3N, and Co4N models. (c) Proposed reaction pathway of the Co4N model based on carbanion formation. (d) Transient state (TS) and corresponding barriers during lactate oxidation. (e) Free energy of the catalytic oxidation of lactate toward pyruvate on the Co2N, Co3N, and Co4N models. The yellow, blue, red, purple, and gray balls in (c) and (d) represent the N, Co, H, O, and C atoms, separately.
图3.DFT计算和LOX模拟催化机理。（a） 计算的Co2N、Co3N和Co4N的变形电荷密度。（b） 计算的Co2N、Co3N和Co4N模型的Co三维态密度（DOS）。（c） 基于碳离子形成的Co4N模型反应路径。（d） 乳酸氧化过程中的瞬态 （TS） 和相应的屏障。（e） 在Co2N、Co3N和Co4N模型上乳酸催化氧化向丙酮酸的自由能。（c） 和 （d） 中的黄色、蓝色、红色、紫色和灰色球分别代表 N、Co、H、O 和 C 原子。
2.4. Enzyme-like Activities of the Co4N/C NEs In Vitro
2.4. Co4N/C NEs在体外的酶样活性
Except for LOX-mimicking activity, Co4N/C NEs exhibit multiple enzyme-mimicking activities, including CAT (catalase), POD (peroxidase), and OXD (oxidase). The Co4N/C NEs can catalyze the decomposition of H2O2 to O2 (Figure S17a) and H2O2 to ·OH (Figure S18a) as well as O2 into ·O2– (Figure S19a). All these catalytic behaviors follow typical Michaelis–Menten kinetics (Figures S17b, S18b, and S19b), and the kinetic parameters are shown in Tables S2, S3, and S4. Therefore, the Co4N/C NEs can utilize H2O2 generated by lactate oxidation as substrate and further trigger a cascade reaction to generate more ·OH and ·O2– in a H2O2-recyclable manner (Figure S20).
除LOX模拟活性外，Co4N/C NEs还具有多种酶模拟活性，包括CAT（过氧化氢酶）、POD（过氧化物酶）和OXD（氧化酶）。Co4N/C NEs可以催化H2O2分解为O2（图S17a）和H2O2分解为·OH（图S18a）以及O2进入·O2– （图S19a）。所有这些催化行为都遵循典型的Michaelis-Menten动力学（图S17b、S18b和S19b），动力学参数如表S2、S3和S4所示。因此，Co4N/C NEs可以利用乳酸氧化生成的H2O2作为底物，进一步触发级联反应，生成更多的 ·OH 和 ·O2– 以 H2O2 可回收的方式（图 S20）。
除LOX模拟活性外，Co4N/C NEs还具有多种酶模拟活性，包括CAT（过氧化氢酶）、POD（过氧化物酶）和OXD（氧化酶）。Co4N/C NEs可以催化H2O2分解为O2（图S17a）和H2O2分解为·OH（图S18a）以及O2进入·O2– （图S19a）。所有这些催化行为都遵循典型的Michaelis-Menten动力学（图S17b、S18b和S19b），动力学参数如表S2、S3和S4所示。因此，Co4N/C NEs可以利用乳酸氧化生成的H2O2作为底物，进一步触发级联反应，生成更多的 ·OH 和 ·O2– 以 H2O2 可回收的方式（图 S20）。
To improve the stability of the Co4N/C NEs, the Co4N/C NEs were modified with NH2–PEG–NH2, which was confirmed by Fourier transform infrared spectra (FT-IR) and zeta potential (Figure S21). Before and after modification, the hydrodynamic diameters of Co4N/C NEs are 164 and 190 nm, respectively. The modified Co4N/C NEs are stable in water and cell culture medium within 72 h (Figure S22). The lactate modulation performance of the Co4N/C NEs in vitro was then investigated. Primarily, the Co4N/C NEs can be effectively internalized by 4T1 cells (Figure 4a). After co-incubation of the 4T1 cells and Co4N/C NEs, lactate concentration was reduced obviously in the extracellular supernatant and stronger consumption occurred under US treatment (Figure 4b). Meanwhile, it was found that the extracellular pH increased from 5.7 to 7.2 accompanied by a decrease in lactate content (Figure 4c). Furthermore, the fluorescence of BCECF-AM in the Co4N/C NEs + US-treated group is stronger than the control groups, suggesting the positive function of Co4N/C NEs in increasing the intracellular pH (Figure 4d). In conclusion, the LOX-mimicking activity of Co4N/C NEs can reverse the accumulation of extracellular lactate and acidosis.
为了提高Co4N/C NEs的稳定性，用NH2-PEG-NH2对Co4N/C NEs进行了修饰，并通过傅里叶变换红外光谱（FT-IR）和zeta电位证实了这一点（图S21）。改性前后Co4N/C NEs的流体动力学直径分别为164 nm和190 nm。修饰的Co4N / C NEs在水和细胞培养基中稳定72小时（图S22）。然后研究了Co4N/C NEs在体外的乳酸调节性能。首先，Co4N/C NEs可以被4T1细胞有效地内化（图4a）。4T1细胞和Co4N/C NEs共孵育后，细胞外上清液中的乳酸浓度明显降低，超声处理下消耗量更强（图4b）。同时，发现细胞外pH值从5.7增加到7.2，同时乳酸含量降低（图4c）。此外，Co4N/C NEs + US处理组BCECF-AM的荧光强于对照组，表明Co4N/C NEs在增加细胞内pH值方面具有正功能（图4d）。综上所述，Co4N/C NEs的LOX模拟活性可以逆转细胞外乳酸的积累和酸中毒。
为了提高Co4N/C NEs的稳定性，用NH2-PEG-NH2对Co4N/C NEs进行了修饰，并通过傅里叶变换红外光谱（FT-IR）和zeta电位证实了这一点（图S21）。改性前后Co4N/C NEs的流体动力学直径分别为164 nm和190 nm。修饰的Co4N / C NEs在水和细胞培养基中稳定72小时（图S22）。然后研究了Co4N/C NEs在体外的乳酸调节性能。首先，Co4N/C NEs可以被4T1细胞有效地内化（图4a）。4T1细胞和Co4N/C NEs共孵育后，细胞外上清液中的乳酸浓度明显降低，超声处理下消耗量更强（图4b）。同时，发现细胞外pH值从5.7增加到7.2，同时乳酸含量降低（图4c）。此外，Co4N/C NEs + US处理组BCECF-AM的荧光强于对照组，表明Co4N/C NEs在增加细胞内pH值方面具有正功能（图4d）。综上所述，Co4N/C NEs的LOX模拟活性可以逆转细胞外乳酸的积累和酸中毒。
Figure 4 图4
Figure 4. Enzyme-like activity of Co4N/C NEs in vitro. Cell experiments were divided into different groups: (1) Control, (2) US, (3) Co4N/C NEs, and (4) Co4N/C NEs + US. (a) Cellular uptake at different times was measured through Rhodamine B staining. (b) Lactate concentrations and (c) pH values of the supernatants of the culture media of 4T1 cells (n = 3). (d) Intracellular pH was detected through BCECF-AM staining. (e) The intracellular hypoxia level was measured using [Ru(dpp)3]2+Cl2 as a probe. (f) Intracellular ROS was detected through DCFH-DA staining. (g) Overall evaluation of the properties of US, Co4N/C NEs, and Co4N/C NEs + US during the anti-tumor process in vitro: (i) lactate consumption, (ii) intracellular pH elevation, (iii) hypoxia alleviation, (iv) ROS elevation, and (v) extracellular pH elevation. (h) Ratio (n = 4) and (i) populations of M1 (CD86high/CD206low) and M2 (CD86low/CD206high) macrophages after incubation with lactate and analysis by flow cytometry. Quantification of (j) TNF-α and (k) IL-10 (n = 3). (l) Cell viability of normal cells (NIH-3T3) and tumor cells (4T1) after different treatments. (m) Illustration of the immune modulation mechanism of Co4N/C NEs. The scale bars are 25 μm. Data are expressed as mean values ± standard deviations (*p < 0.05, **p < 0.01, ***p < 0.001).
图4.体外Co4N/C NEs的酶样活性。细胞实验分为不同的组：（1）对照组，（2）US，（3）Co4N/C NEs和（4）Co4N/C NEs + US。（a） 通过罗丹明 B 染色测量不同时间的细胞摄取。（b） 4T1 细胞培养基上清液的乳酸浓度和 （c） pH 值 （n = 3）。（d） 通过BCECF-AM染色检测细胞内pH值。（e） 使用 [Ru（dpp）3]2+Cl2 作为探针测量细胞内缺氧水平。（f） 通过DCFH-DA染色检测细胞内ROS。（g） 体外抗肿瘤过程中 US、Co4N/C NEs 和 Co4N/C NEs + US 特性的总体评价：（i） 乳酸消耗，（ii） 细胞内 pH 值升高，（iii） 缺氧缓解，（iv） ROS 升高，以及 （v） 细胞外 pH 值升高。（h） M1 （CD86high/CD206low） 和 M2 （CD86low/CD206high） 巨噬细胞与乳酸孵育并通过流式细胞术分析后的比率 （n = 4） 和 （i） 群体。（j） TNF-α 和 （k） IL-10 的定量 （n = 3）。（l）不同处理后正常细胞（NIH-3T3）和肿瘤细胞（4T1）的细胞活力。（m） Co4N/C NEs的免疫调节机制。比例尺为 25 μm。数据表示为标准差±均值（*p < 0.05，**p < 0.01，***p < 0.001）。
[Ru(dpp)3]2+Cl2 acts as a hypoxia probe to measure the intracellular O2 levels. The weaker red fluorescence in the Co4N/C NE-treated groups illustrates the capacity of hypoxia alleviation of Co4N/C NEs (Figure 4e). This is because the CAT-mimicking activity of Co4N/C NEs decomposes H2O2 into O2. Intracellular fluorescence of DCFH-DA increased, suggesting that POD and OXD-mimicking activity elevate the ROS level (Figure 4f), which can cause mitochondrial dysfunction and is beneficial to destroying the supporting effect of the TCA cycle on cancer cell metabolism. (3,26) Mitochondrial membrane potential was tested by a JC-1 probe, which can disaggregate into monomers in damaged mitochondria with green fluorescence (Figure S23a). After incubation of the Co4N/C NEs, the fluorescence ratio of green/red (G/R) raised from normal 0.36 to 0.53 and further to 0.76 with the US intervention, suggesting that the mitochondria were destroyed (Figure S23b). Additionally, a comprehensive evaluation of the enzyme-like properties shows that Co4N/C NEs + US has the best catalytic performance in lactic acid consumption, pH rise, hypoxia alleviation, and ROS compared with the use of US or Co4N/C NE alone (Figure 4g).
[Ru（dpp）3]2+Cl2 作为缺氧探针测量细胞内 O2 水平。Co4N/C NE处理组中较弱的红色荧光说明了Co4N / C NEs缺氧缓解的能力（图4e）。这是因为Co4N/C NEs的CAT模拟活性将H2O2分解成O2。DCFH-DA的细胞内荧光增加，表明POD和OXD模拟活性提高了ROS水平（图4f），这可导致线粒体功能障碍，有利于破坏TCA循环对癌细胞代谢的支持作用。（3,26） 通过 JC-1 探针测试线粒体膜电位，该探针可以分解成具有绿色荧光的受损线粒体中的单体（图 S23a）。孵育 Co4N/C NE 后，绿色/红色 （G/R） 的荧光比从正常值 0.36 提高到 0.53，并在 US 干预下进一步提高到 0.76，表明线粒体被破坏（图 S23b）。此外，对酶样特性的综合评价表明，与单独使用US或Co4N/C NE相比，Co4N/C NEs + US在乳酸消耗、pH升高、缺氧缓解和ROS方面具有最佳的催化性能（图4g）。
[Ru（dpp）3]2+Cl2 作为缺氧探针测量细胞内 O2 水平。Co4N/C NE处理组中较弱的红色荧光说明了Co4N / C NEs缺氧缓解的能力（图4e）。这是因为Co4N/C NEs的CAT模拟活性将H2O2分解成O2。DCFH-DA的细胞内荧光增加，表明POD和OXD模拟活性提高了ROS水平（图4f），这可导致线粒体功能障碍，有利于破坏TCA循环对癌细胞代谢的支持作用。（3,26） 通过 JC-1 探针测试线粒体膜电位，该探针可以分解成具有绿色荧光的受损线粒体中的单体（图 S23a）。孵育 Co4N/C NE 后，绿色/红色 （G/R） 的荧光比从正常值 0.36 提高到 0.53，并在 US 干预下进一步提高到 0.76，表明线粒体被破坏（图 S23b）。此外，对酶样特性的综合评价表明，与单独使用US或Co4N/C NE相比，Co4N/C NEs + US在乳酸消耗、pH升高、缺氧缓解和ROS方面具有最佳的催化性能（图4g）。
High lactate in TME causes the non-inflammatory polarization of tumor-associated macrophages. (27) As shown in Figure S24, lactate-induced macrophages polarize to the M2 phenotype. (28) After Co4N/C NE treatment, the population of the M1 phenotype increased from 26.5 to 34.0%, whereas the M2 phenotype decreased from 34.7 to 27.7% with the ratio of M1/M2 raised from 0.60 at high-lactate concentration conditions to 1.38 at lowered lactate conditions (Figure 4h,i and Figure S25). Moreover, Co4N/C NEs up-regulated the secretion of proinflammatory cytokine TNF-α from macrophages by 1.37-fold (Figure 4j) and down-regulated anti-inflammatory cytokine IL-10 by 1.35-fold (Figure 4k). The US treatment further increased the population of M1 macrophages to 46.0% and decreased M2 to just 13.7% with a M1/M2 ratio of 3.63 (Figure 4h,i, and Figure S25). The outcomes are even better than that of lipopolysaccharides (LPS, a widely used adjuvant, Figure S24). Moreover, the secretion of TNF-α increased by 1.52-fold, and that IL-10 decreased by 1.71-fold relative to the control group, which indicates the successful elevation of immune response by the US (Figure 4j,k). Compared with normal cells, tumor cells have a higher level of lactate and H2O2, which endows Co4N/C NEs with selective cytotoxicity to tumor cells. The Cell Counting Kit-8 (CCK-8) cytotoxicity test shows that the viability of the normal cells (NIH-3T3 and HUVEC) is much higher than that of the cancer cells (A549 cells and 4T1 cells) at various concentrations of Co4N/C NEs (Figure S26). Negligible differences in the antitumor effects of Co4N/C NEs are found in the 4T1 cell line in pH 6.0 and pH 7.4 cell culture media (Figure S27). Under the US treatment, the Co4N/C NEs further enhance the killing effect to cancer cells and the normal cells suffer negligible influences according to the cytotoxicity test (Figure 4l) and live-dead staining (Figure S28a). The hemolysis assay also shows that the hemolysis of red blood cells was lower than 5.0% (Figure S28b).
TME 中的高乳酸会导致肿瘤相关巨噬细胞的非炎症性极化。（27）如图S24所示，乳酸诱导的巨噬细胞极化为M2表型。（28）Co4N/C NE处理后，M1表型群体从26.5%增加到34.0%，而M2表型从34.7%下降到27.7%，M1/M2比值从高乳酸浓度条件下的0.60提高到低乳酸条件下的1.38（图4h，i和图S25）。此外，Co4N/C NEs将巨噬细胞中促炎细胞因子TNF-α的分泌上调了1.37倍（图4j），将抗炎细胞因子IL-10下调了1.35倍（图4k）。美国治疗进一步将 M1 巨噬细胞的数量增加到 46.0%，将 M2 减少到仅 13.7%，M1/M2 比值为 3.63（图 4h、i 和图 S25）。结果甚至比脂多糖（LPS，一种广泛使用的佐剂，图S24）更好。此外，与对照组相比，TNF-α的分泌增加了1.52倍，IL-10减少了1.71倍，这表明美国成功提高了免疫反应（图4j，k）。与正常细胞相比，肿瘤细胞具有更高的乳酸和H2O2水平，这赋予了Co4N/C NEs对肿瘤细胞的选择性细胞毒性。细胞计数试剂盒-8（CCK-8）细胞毒性试验表明，正常细胞（NIH-3T3和HUVEC）在不同浓度的Co4N/C NEs下远高于癌细胞（A549细胞和4T1细胞）的活力（图S26）。在 pH 6.0 和 pH 7.4 细胞培养基的 4T1 细胞系中，Co4N/C NE 的抗肿瘤作用差异可以忽略不计（图 S27）。 在美国处理下，Co4N/C NEs进一步增强了对癌细胞的杀伤作用，根据细胞毒性试验（图4l）和活死染色（图S28a），正常细胞受到的影响可以忽略不计。溶血试验还显示红细胞溶血率低于5.0%（图S28b）。
TME 中的高乳酸会导致肿瘤相关巨噬细胞的非炎症性极化。（27）如图S24所示，乳酸诱导的巨噬细胞极化为M2表型。（28）Co4N/C NE处理后，M1表型群体从26.5%增加到34.0%，而M2表型从34.7%下降到27.7%，M1/M2比值从高乳酸浓度条件下的0.60提高到低乳酸条件下的1.38（图4h，i和图S25）。此外，Co4N/C NEs将巨噬细胞中促炎细胞因子TNF-α的分泌上调了1.37倍（图4j），将抗炎细胞因子IL-10下调了1.35倍（图4k）。美国治疗进一步将 M1 巨噬细胞的数量增加到 46.0%，将 M2 减少到仅 13.7%，M1/M2 比值为 3.63（图 4h、i 和图 S25）。结果甚至比脂多糖（LPS，一种广泛使用的佐剂，图S24）更好。此外，与对照组相比，TNF-α的分泌增加了1.52倍，IL-10减少了1.71倍，这表明美国成功提高了免疫反应（图4j，k）。与正常细胞相比，肿瘤细胞具有更高的乳酸和H2O2水平，这赋予了Co4N/C NEs对肿瘤细胞的选择性细胞毒性。细胞计数试剂盒-8（CCK-8）细胞毒性试验表明，正常细胞（NIH-3T3和HUVEC）在不同浓度的Co4N/C NEs下远高于癌细胞（A549细胞和4T1细胞）的活力（图S26）。在 pH 6.0 和 pH 7.4 细胞培养基的 4T1 细胞系中，Co4N/C NE 的抗肿瘤作用差异可以忽略不计（图 S27）。 在美国处理下，Co4N/C NEs进一步增强了对癌细胞的杀伤作用，根据细胞毒性试验（图4l）和活死染色（图S28a），正常细胞受到的影响可以忽略不计。溶血试验还显示红细胞溶血率低于5.0%（图S28b）。
In conclusion, excess lactate can be consumed by Co4N/C NEs under US treatment, which can not only reduce the lactate secreted by tumor cells, leading to the reduction of H+ and recovery of pH in the TME and thus transforming the immunosuppressed TME into the immuno-promoting TME, but also greatly enhance the oxidative stress of tumor cells and destroy the mitochondrial function and TCA cycle (Figure 4m).
综上所述，超美处理下，过量的乳酸可被Co4N/C NEs消耗，不仅能减少肿瘤细胞分泌的乳酸，导致TME中H+的降低和pH值的恢复，从而将免疫抑制的TME转化为免疫促进的TME，而且大大增强了肿瘤细胞的氧化应激，破坏了线粒体功能和TCA循环（图4m）。
综上所述，超美处理下，过量的乳酸可被Co4N/C NEs消耗，不仅能减少肿瘤细胞分泌的乳酸，导致TME中H+的降低和pH值的恢复，从而将免疫抑制的TME转化为免疫促进的TME，而且大大增强了肿瘤细胞的氧化应激，破坏了线粒体功能和TCA循环（图4m）。
2.5. Immune Microenvironment Reprogramming and Anti-Tumor Performance
2.5. 免疫微环境重编程和抗肿瘤性能
Encouraged by the outcomes in vitro, the immune activation of Co4N/C NEs was further explored in vivo by establishing the subcutaneous tumor model (Figure 5a). Before that, the biodistribution of Co4N/C NEs was explored. From 4–12 h, the fluorescent signals tended to be stable, indicating that Co4N/C NEs can achieve effective accumulation in tumor sites after intravenous injections (Figure S29a). At 6 h, the major organs and tumors were harvested and about 8% of the fluorescence signal was observed at the tumor site (Figure S29b,c). Blood routine and blood biochemistry indexes suffer almost no influence after systematic administration (Figure S30). On day 8 after treatments, the tumor tissues were collected, and the lactate content was detected, which was significantly consumed by Co4N/C NEs (Figure 5b). Consistent with in vitro outcomes, the ratio of M1/M2 macrophages was upregulated with the decrease in lactate at the tumor site after the treatment of Co4N/C NEs plus US (Figure 5c). Specifically, the population of M1 macrophages was upregulated to 36.5%, and M2 macrophages were reduced to 15.4% (Figure 5d and Figure S31). According to the enzyme-linked immunosorbent assay (ELISA), the secretion of pro-inflammatory cytokines (TNF-α) increased, and the level of anti-inflammatory cytokines (IL-10) was reduced after Co4N/C NEs plus US (Figure S32). Similar results were also demonstrated by immunofluorescent staining of TNF-α and IL-10 of tumor slices (Figure S33). A high level of lactate inhibits the maturation of dendritic cells (DC) and the activation of cytotoxic T cells. (29,30) The outcomes in Figure S34 displayed the maturation of DC after Co4N/C NEs plus US treatment. The percentage of DC maturation was raised from 12.9 to 32.1% (Figure 5e). The infiltration of cytotoxic T cells was raised from 18.9 to 30.3% (Figure S35). Moreover, CD8+ cytotoxic T lymphocytes contribute to the immune system against cancer cells through the production of interferon-γ (IFN-γ). (31) The corresponding IFN-γ level was raised by 66.1% relative to the control group, confirming the successful activation of T cell-based anti-tumor immunity (Figure 5f).
在体外结果的鼓舞下，通过建立皮下肿瘤模型，在体内进一步探索了Co4N/C NEs的免疫激活（图5a）。在此之前，探索了Co4N/C NEs的生物分布。从4-12小时，荧光信号趋于稳定，表明Co4N/C NEs在静脉注射后可以在肿瘤部位实现有效积累（图S29a）。在6 h时，收获主要器官和肿瘤，并在肿瘤部位观察到约8%的荧光信号（图S29b，c）。系统给药后，血液常规和血液生化指标几乎没有影响（图S30）。治疗后第8天，收集肿瘤组织，检测乳酸含量，Co4N/C NEs显著消耗乳酸含量（图5b）。与体外结果一致，在Co4N / C NEs加US处理后，M1 / M2巨噬细胞的比率随着肿瘤部位乳酸的减少而上调（图5c）。具体而言，M1巨噬细胞群上调至36.5%，M2巨噬细胞群下调至15.4%（图5d和图S31）。根据酶联免疫吸附试验（ELISA），Co4N/C NEs加US后促炎细胞因子（TNF-α）分泌增加，抗炎细胞因子（IL-10）水平降低（图S32）。肿瘤切片的TNF-α和IL-10的免疫荧光染色也证明了类似的结果（图S33）。高水平的乳酸会抑制树突状细胞 （DC） 的成熟和细胞毒性 T 细胞的活化。（29,30） 图 S34 中的结果显示了 Co4N/C NE 加 US 处理后 DC 的成熟。直流成熟度的百分比从12.9%提高到32.1%（图5e）。 细胞毒性T细胞的浸润率从18.9%提高到30.3%（图S35）。此外，CD8+ 细胞毒性 T 淋巴细胞通过产生干扰素-γ （IFN-γ） 有助于免疫系统对抗癌细胞。（31）相对于对照组，相应的IFN-γ水平提高了66.1%，证实了基于T细胞的抗肿瘤免疫的成功激活（图5f）。
在体外结果的鼓舞下，通过建立皮下肿瘤模型，在体内进一步探索了Co4N/C NEs的免疫激活（图5a）。在此之前，探索了Co4N/C NEs的生物分布。从4-12小时，荧光信号趋于稳定，表明Co4N/C NEs在静脉注射后可以在肿瘤部位实现有效积累（图S29a）。在6 h时，收获主要器官和肿瘤，并在肿瘤部位观察到约8%的荧光信号（图S29b，c）。系统给药后，血液常规和血液生化指标几乎没有影响（图S30）。治疗后第8天，收集肿瘤组织，检测乳酸含量，Co4N/C NEs显著消耗乳酸含量（图5b）。与体外结果一致，在Co4N / C NEs加US处理后，M1 / M2巨噬细胞的比率随着肿瘤部位乳酸的减少而上调（图5c）。具体而言，M1巨噬细胞群上调至36.5%，M2巨噬细胞群下调至15.4%（图5d和图S31）。根据酶联免疫吸附试验（ELISA），Co4N/C NEs加US后促炎细胞因子（TNF-α）分泌增加，抗炎细胞因子（IL-10）水平降低（图S32）。肿瘤切片的TNF-α和IL-10的免疫荧光染色也证明了类似的结果（图S33）。高水平的乳酸会抑制树突状细胞 （DC） 的成熟和细胞毒性 T 细胞的活化。（29,30） 图 S34 中的结果显示了 Co4N/C NE 加 US 处理后 DC 的成熟。直流成熟度的百分比从12.9%提高到32.1%（图5e）。 细胞毒性T细胞的浸润率从18.9%提高到30.3%（图S35）。此外，CD8+ 细胞毒性 T 淋巴细胞通过产生干扰素-γ （IFN-γ） 有助于免疫系统对抗癌细胞。（31）相对于对照组，相应的IFN-γ水平提高了66.1%，证实了基于T细胞的抗肿瘤免疫的成功激活（图5f）。
Figure 5 图5
Figure 5. In vivo immune activation and transcriptomic analysis. The mice were divided into different groups: (1) Saline, (2) US, (3) Co4N/C NEs, and (4) Co4N/C NEs + US. (a) Treatment protocol for antitumor studies. (b) Lactate content in various groups. (c) Ratio (n = 4) and (d) Population of M1 (CD86high/CD206low) and M2 (CD86low/CD206high) macrophages (gating on F4/80) after different treatments. (e) Population of maturing dendritic cells (CD86high/CD80high) analyzed by flow cytometry (gating on CD11c). (f) INF-γ level in serum after different treatments (n = 3). (g) Tumor volume variation within 14 days (n = 5). (h) Volcano plot of up-regulated genes (red) and down-regulated genes (blue) after treatments. (i) GO enrichment and (j) KEGG enrichment analysis of pathways associated with immune activation. (k) Enrichment of immunotherapy-related gene signatures. Data are expressed as mean values ± standard deviations (*p < 0.05, **p < 0.01, ***p < 0.001).
图5.体内免疫激活和转录组学分析。将小鼠分为不同的组：（1）生理盐水，（2）US，（3）Co4N/C NEs和（4）Co4N/C NEs + US。（a） 抗肿瘤研究的治疗方案。（b） 各组的乳酸含量。（c） 不同处理后 M1 （CD86high/CD206low） 和 M2 （CD86low/CD206high） 巨噬细胞（F4/80 门控）的比率 （n = 4） 和 （d） 数量。（e） 通过流式细胞术（CD11c 上的门控）分析的成熟树突状细胞群 （CD86high/CD80high）。（f） 不同处理后血清中的 INF-γ 水平 （n = 3）。（g） 14 天内的肿瘤体积变化 （n = 5）。（h） 处理后上调基因（红色）和下调基因（蓝色）的火山图。（i） GO富集和（j）与免疫激活相关途径的KEGG富集分析。（k） 免疫治疗相关基因特征的富集。数据表示为标准差±均值（*p < 0.05，**p < 0.01，***p < 0.001）。
According to tumor growth curves during the therapeutic period, US alone exhibited negligible tumor inhibition effects, while Co4N/C NEs elevated the tumor growth inhibition (TGI) rate to 49.62%. Moreover, the TGI was significantly enhanced to 94.8% when treated with Co4N/C NEs plus US stimulation (Figure 5g and Figures S36, S37, and S38). The bioluminescence imaging of the 4T1 tumor acquired from the IVIS system showed the growth situation of cancer cells in vivo, and little bioluminescence signal was observed in Co4N/C NEs + US-treated mice (Figure S39). On day 14, the mice were sacrificed, and the tumors were extracted and weighed. The average weight of tumors in the Co4N/C NEs + US-treated group is only 12.2% of the control group (Figure S40a). It is worth noting that during the experimental period, the body weight was almost uninfluenced in all four groups (Figures S38 and S40b), and the lifespan of the treatment group was significantly prolonged (Figure S41). No apparent damage occurred in the main organs (heart, liver, spleen, lung, and kidney) during treatments (Figure S42). The histological staining of extracted tumors in Figures S43 and S44 reveal that the tumor cells underwent obvious apoptotic and necrotic damage accompanied by DNA breakage and almost lost proliferation ability after the catalytic immune therapy.
根据治疗期间的肿瘤生长曲线，仅超声的肿瘤抑制作用可以忽略不计，而Co4N/C NEs将肿瘤生长抑制（TGI）率提高到49.62%。此外，当用 Co4N/C NE 加 US 刺激处理时，TGI 显着提高至 94.8%（图 5g 和图 S36、S37 和 S38）。从IVIS系统获得的4T1肿瘤的生物发光成像显示了癌细胞在体内的生长情况，在Co4N/C NEs + US处理的小鼠中观察到的生物发光信号很少（图S39）。第14天，处死小鼠，取出肿瘤称重。Co4N/C NEs + US治疗组肿瘤的平均重量仅为对照组的12.2%（图S40a）。值得注意的是，在实验期间，所有四组的体重几乎没有受到影响（图S38和S40b），并且治疗组的寿命显着延长（图S41）。在治疗期间，主要器官（心脏、肝脏、脾脏、肺和肾脏）没有发生明显的损伤（图S42）。图S43和S44中提取的肿瘤的组织学染色表明，肿瘤细胞在催化免疫治疗后经历了明显的凋亡和坏死损伤，并伴有DNA断裂，几乎丧失了增殖能力。
根据治疗期间的肿瘤生长曲线，仅超声的肿瘤抑制作用可以忽略不计，而Co4N/C NEs将肿瘤生长抑制（TGI）率提高到49.62%。此外，当用 Co4N/C NE 加 US 刺激处理时，TGI 显着提高至 94.8%（图 5g 和图 S36、S37 和 S38）。从IVIS系统获得的4T1肿瘤的生物发光成像显示了癌细胞在体内的生长情况，在Co4N/C NEs + US处理的小鼠中观察到的生物发光信号很少（图S39）。第14天，处死小鼠，取出肿瘤称重。Co4N/C NEs + US治疗组肿瘤的平均重量仅为对照组的12.2%（图S40a）。值得注意的是，在实验期间，所有四组的体重几乎没有受到影响（图S38和S40b），并且治疗组的寿命显着延长（图S41）。在治疗期间，主要器官（心脏、肝脏、脾脏、肺和肾脏）没有发生明显的损伤（图S42）。图S43和S44中提取的肿瘤的组织学染色表明，肿瘤细胞在催化免疫治疗后经历了明显的凋亡和坏死损伤，并伴有DNA断裂，几乎丧失了增殖能力。
The potential biological process behind the therapeutic effect was explored via transcriptomic analysis. Tumor tissues of the control and Co4N/C NEs + US-treated groups were applied with the RNA sequencing, among which a total of 13,384 genes were analyzed. The differential expression genes were exhibited by the volcano plot (Figure 5h). A total of 34 genes were up-regulated, and 403 genes were down-regulated in the Co4N/C NEs + US-treated group. Among the up-regulated genes, GZMB, CCL3L1, CCL3, CCL3L3, and CCL18 are highly correlated to “cytotoxic T lymphocyte infiltration” and “immune chemokine enrichment”. (32,33) Gene ontology (GO) enrichment analysis exhibited that Co4N/C NEs + US significantly activated effector immune cell infiltration associated pathways (Figure 5i). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis reveals that antigen processing and representation, natural killer cell-mediated cytotoxicity, primary immunodeficiency, and the T cell receptor signaling pathway were up-regulated (Figure 5j). Moreover, Co4N/C NEs + US-activated immunotherapy-related gene signatures exhibit great potential to sensitize patients to immunotherapy (Figure 5k).
通过转录组学分析探索了治疗效果背后的潜在生物学过程。对照组和Co4N/C NEs+US处理组的肿瘤组织进行RNA测序，共分析13384个基因。差异表达基因由火山图展示（图5h）。Co4N/C NEs + US处理组共有34个基因上调，403个基因下调。在上调基因中，GZMB、CCL3L1、CCL3、CCL3L3和CCL18与“细胞毒性T淋巴细胞浸润”和“免疫趋化因子富集”高度相关。（32,33） 基因本体 （GO） 富集分析表明，Co4N/C NEs + US 显着激活了效应免疫细胞浸润相关通路（图 5i）。京都基因和基因组百科全书（KEGG）分析显示，抗原加工和表征、自然杀伤细胞介导的细胞毒性、原发性免疫缺陷和T细胞受体信号通路上调（图5j）。此外，Co4N/C NEs + 超声激活的免疫疗法相关基因特征在使患者对免疫疗法敏感方面表现出巨大的潜力（图5k）。
通过转录组学分析探索了治疗效果背后的潜在生物学过程。对照组和Co4N/C NEs+US处理组的肿瘤组织进行RNA测序，共分析13384个基因。差异表达基因由火山图展示（图5h）。Co4N/C NEs + US处理组共有34个基因上调，403个基因下调。在上调基因中，GZMB、CCL3L1、CCL3、CCL3L3和CCL18与“细胞毒性T淋巴细胞浸润”和“免疫趋化因子富集”高度相关。（32,33） 基因本体 （GO） 富集分析表明，Co4N/C NEs + US 显着激活了效应免疫细胞浸润相关通路（图 5i）。京都基因和基因组百科全书（KEGG）分析显示，抗原加工和表征、自然杀伤细胞介导的细胞毒性、原发性免疫缺陷和T细胞受体信号通路上调（图5j）。此外，Co4N/C NEs + 超声激活的免疫疗法相关基因特征在使患者对免疫疗法敏感方面表现出巨大的潜力（图5k）。
We next tried to establish a connection between the Co4N/C NEs and clinical patients in promoting the clinical application of Co4N/C NEs. The RNA sequencing data of 1069 breast cancer patients were downloaded from The Cancer Genome Atlas (TCGA) database. We analyzed the correlation between 437 differentially expressed genes in breast cancer prognosis and the tumor immune microenvironment. The expression of SRGAP1 and SEMA4G genes, which were significantly associated with a worse prognosis of breast cancer, was down-regulated after Co4N/C NEs + US (Figures S45a,b). Therefore, our strategy is promising in reducing the expression of SRGAP1 and SEMA4G and prolong the survival rate of breast cancer patients (P = 0.006 and P = 0.0077, respectively). We subsequently analyzed the relationship between the two genes and the immune microenvironment of breast cancer. Both SRGAP1 and SEMA4G are negatively correlated with cancer immune cycles (Figure S45c) and immune cell infiltration (Figures S45d and S45e). On the contrary, the PRF1 and GZMB genes were positively correlated with tumor immune responses, with which up-regulation provided a favorable outcome for the prognosis of cancer patients (Figure S46). Therefore, Co4N/C NEs could reprogram the immunosuppressive TME of breast cancer patients by down-regulating the transcription of SRGAP1 and SEMA4G and up-regulating PRF1 and GZMB.
接下来，我们试图在Co4N/C NEs和临床患者之间建立联系，以促进Co4N/C NEs的临床应用。1069名乳腺癌患者的RNA测序数据是从癌症基因组图谱（TCGA）数据库下载的。我们分析了437个差异表达基因在乳腺癌预后与肿瘤免疫微环境之间的相关性。SRGAP1 和 SEMA4G 基因的表达与乳腺癌预后较差显著相关，在 Co4N/C NEs + US 后下调（图 S45a，b）。因此，我们的策略在降低 SRGAP1 和 SEMA4G 的表达和延长乳腺癌患者的生存率方面具有希望（分别为 P = 0.006 和 P = 0.0077）。随后，我们分析了这两个基因与乳腺癌免疫微环境之间的关系。SRGAP1和SEMA4G都与癌症免疫周期（图S45c）和免疫细胞浸润（图S45d和S45e）呈负相关。相反，PRF1和GZMB基因与肿瘤免疫反应呈正相关，上调为癌症患者的预后提供了有利的结果（图S46）。因此，Co4N/C NEs可以通过下调SRGAP1和SEMA4G的转录以及上调PRF1和GZMB来重编程乳腺癌患者的免疫抑制TME。
接下来，我们试图在Co4N/C NEs和临床患者之间建立联系，以促进Co4N/C NEs的临床应用。1069名乳腺癌患者的RNA测序数据是从癌症基因组图谱（TCGA）数据库下载的。我们分析了437个差异表达基因在乳腺癌预后与肿瘤免疫微环境之间的相关性。SRGAP1 和 SEMA4G 基因的表达与乳腺癌预后较差显著相关，在 Co4N/C NEs + US 后下调（图 S45a，b）。因此，我们的策略在降低 SRGAP1 和 SEMA4G 的表达和延长乳腺癌患者的生存率方面具有希望（分别为 P = 0.006 和 P = 0.0077）。随后，我们分析了这两个基因与乳腺癌免疫微环境之间的关系。SRGAP1和SEMA4G都与癌症免疫周期（图S45c）和免疫细胞浸润（图S45d和S45e）呈负相关。相反，PRF1和GZMB基因与肿瘤免疫反应呈正相关，上调为癌症患者的预后提供了有利的结果（图S46）。因此，Co4N/C NEs可以通过下调SRGAP1和SEMA4G的转录以及上调PRF1和GZMB来重编程乳腺癌患者的免疫抑制TME。
Encouraged by remodeling the pathological microenvironment of Co4N/C NEs, the immune responses at the sentinel lymph nodes (SLN), which possess great tumor-immune tolerance, were investigated. (34,35) Herein, a metastatic SLN model was established by the direct injection of 4T1 cells on the right hind footpad of mice. (36) After about 17 days, a touchable spherical firm lump appeared over the inner knee of the mice, which represented the successful metastasis of the primary tumor to the lymph nodes (Figure 6a). After treatment, the lymph node slices were stained with an anti-CD3 antibody and the strong immunofluorescence indicated extensive T cell infiltration (Figure 6b). Specifically, the number of cytotoxic T cells (CD8a+ T cells) was upregulated and regulatory T cells (Foxp3+ T cells) were downregulated (Figure S47). This is due to lactate consumption by Co4N/C NEs reducing the metabolic support to T cells, and thus, the immune responses in SLN were reactivated. (37) Moreover, the Co4N/C NEs prevented the appearance of a heavy spleen (Figure 6c and Figure S48). The H&E staining and Tunel staining of metastasized lymphatic tumor slices revealed that the lymph cancer cells were confronted with significant apoptosis after treatments (Figure 6d and Figure S49). Only slight body weight changes could be found (Figure 6e and Figure S50), and the survival time of the lymphatic tumor-bearing mice was prolonged by over 66.7% (Figure 6f). To verify the reason, the lungs of the mice were harvested and stained with Indian ink. For the control groups, the tumor cells metastasized to the lungs, and metastatic nodules colonized the full lungs, which finally causes the death of the mice (Figure 6g,h). On the contrary, Co4N/C NEs with US stimulation-based catalytic therapy suppressed the metastatic nodules by 87.6%, and thus, most mice survived.
在重塑Co4N/C NEs病理微环境的启发下，研究了具有较强肿瘤免疫耐受性的前哨淋巴结（SLN）的免疫反应。（34,35） 本文通过在小鼠右后脚垫上直接注射 4T1 细胞来建立转移性 SLN 模型。（36）大约17天后，小鼠的膝盖内侧出现一个可触摸的球形坚硬肿块，这代表原发肿瘤成功转移到淋巴结（图6a）。处理后，淋巴结切片用抗CD3抗体染色，强免疫荧光表明T细胞广泛浸润（图6b）。具体来说，细胞毒性T细胞（CD8a + T细胞）的数量上调，调节性T细胞（Foxp3 + T细胞）下调（图S47）。这是由于 Co4N/C NE 消耗的乳酸减少了对 T 细胞的代谢支持，因此 SLN 中的免疫反应被重新激活。（37）此外，Co4N/C NEs防止了脾脏沉重的出现（图6c和图S48）。转移淋巴肿瘤切片的H&E染色和Tunel染色显示淋巴癌细胞在治疗后面临明显的凋亡（图6d和图S49）。仅发现轻微的体重变化（图6e和图S50），淋巴肿瘤小鼠的存活时间延长了66.7%以上（图6f）。为了验证原因，收获了小鼠的肺部并用印度墨水染色。对于对照组，肿瘤细胞转移到肺部，转移性结节定植于整个肺部，最终导致小鼠死亡（图6g，h）。 相反，基于超声刺激的催化疗法的Co4N/C NEs抑制了87.6%的转移性结节，因此大多数小鼠存活。
在重塑Co4N/C NEs病理微环境的启发下，研究了具有较强肿瘤免疫耐受性的前哨淋巴结（SLN）的免疫反应。（34,35） 本文通过在小鼠右后脚垫上直接注射 4T1 细胞来建立转移性 SLN 模型。（36）大约17天后，小鼠的膝盖内侧出现一个可触摸的球形坚硬肿块，这代表原发肿瘤成功转移到淋巴结（图6a）。处理后，淋巴结切片用抗CD3抗体染色，强免疫荧光表明T细胞广泛浸润（图6b）。具体来说，细胞毒性T细胞（CD8a + T细胞）的数量上调，调节性T细胞（Foxp3 + T细胞）下调（图S47）。这是由于 Co4N/C NE 消耗的乳酸减少了对 T 细胞的代谢支持，因此 SLN 中的免疫反应被重新激活。（37）此外，Co4N/C NEs防止了脾脏沉重的出现（图6c和图S48）。转移淋巴肿瘤切片的H&E染色和Tunel染色显示淋巴癌细胞在治疗后面临明显的凋亡（图6d和图S49）。仅发现轻微的体重变化（图6e和图S50），淋巴肿瘤小鼠的存活时间延长了66.7%以上（图6f）。为了验证原因，收获了小鼠的肺部并用印度墨水染色。对于对照组，肿瘤细胞转移到肺部，转移性结节定植于整个肺部，最终导致小鼠死亡（图6g，h）。 相反，基于超声刺激的催化疗法的Co4N/C NEs抑制了87.6%的转移性结节，因此大多数小鼠存活。
Figure 6 图6
Figure 6. Lymph node immune responses and lymphatic tumor therapies. The mice were placed in different groups: (1) Saline, (2) US, (3) Co4N/C NEs, and (4) Co4N/C NEs + US. (a) Treatment protocol for anti-lymphatic tumor studies. (b) Immunofluorescence staining of T cells in tumor slices with anti-CD3 antibody (red). (c) Weight of spleen extracted from mice. (d) H&E staining of lymph node metastasis tumors (scale bar = 50 μm). (e) Body weight of mice within 14 days. (f) Survival curves of different groups (n = 6). (g) Representative digital photos and (h) number of metastatic nodules in lungs after the treatments. Data are expressed as mean values ± standard deviations (n = 5, *p < 0.05).
图6.淋巴结免疫反应和淋巴肿瘤治疗。将小鼠分为不同的组：（1）盐水，（2）US，（3）Co4N / C NEs和（4）Co4N / C NEs + US。（a） 抗淋巴肿瘤研究的治疗方案。（b）用抗CD3抗体（红色）对肿瘤切片中的T细胞进行免疫荧光染色。（c）从小鼠中提取的脾脏重量。（d） 淋巴结转移肿瘤的 H&E 染色（比例尺 = 50 μm）。（e）小鼠14天内的体重。（f） 不同组的生存曲线（n = 6）。（g） 代表性数码照片和 （h） 治疗后肺部转移结节的数量。数据表示为标准差±均值 （n = 5， *p < 0.05）。
3. Conclusions
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In summary, we report a lactate oxidase nanozyme (Co4N/C NE) by mimicking the N-centered structural characteristics of natural enzymes. Through experimental data and DFT calculations, the optimal electronic configuration between the nonmetallic N atoms’ active center and metallic Co atom ligands was realized through the modulation of metallic Co atom coordination numbers, which boosted the precise recognition for lactate and capture of the α-C–OH proton and α-C–H proton from lactate by the strong electronegativity of N, and then prompted the generation of pyruvate and transformation of O2 to H2O2 at mild conditions. The high catalytic performance of Co4N/C NEs was also demonstrated in the catalytic oxidation of lactate in vitro and in vivo. LOX-mimicking activity acts on reversing the high lactate and remolding the immunosuppressive state of the tumor microenvironment. Meanwhile, the ultrasound amplification and multienzyme cascade abilities inside Co4N/C NEs further enhance the catalytic therapeutic efficiency. These features endow Co4N/C NEs with excellent anti-tumor growth and distant metastasis inhibition abilities. We believe that this study paves a new avenue forward to create nanozymes that truly rival their natural counterparts.
总之，我们通过模拟天然酶的N中心结构特征来报道乳酸氧化酶纳米酶（Co4N / C NE）。通过实验数据和DFT计算，通过调制金属Co原子配位数，实现了非金属N原子活性中心与金属Co原子配体之间的最佳电子构型，促进了对乳酸的精确识别和N的强电负性从乳酸中捕获α-C-OH质子和α-C-H质子， 然后在温和条件下促使丙酮酸的产生和O2转化为H2O2。Co4N/C NEs在体外和体内对乳酸的催化氧化中也表现出了较高的催化性能。LOX模拟活性作用于逆转高乳酸并重塑肿瘤微环境的免疫抑制状态。同时，Co4N/C NEs内部的超声扩增和多酶级联能力进一步提高了催化治疗效率。这些特性赋予Co4N/C NEs优异的抗肿瘤生长和远处转移抑制能力。我们相信，这项研究为创造真正与天然对应物相媲美的纳米酶铺平了一条新的道路。
总之，我们通过模拟天然酶的N中心结构特征来报道乳酸氧化酶纳米酶（Co4N / C NE）。通过实验数据和DFT计算，通过调制金属Co原子配位数，实现了非金属N原子活性中心与金属Co原子配体之间的最佳电子构型，促进了对乳酸的精确识别和N的强电负性从乳酸中捕获α-C-OH质子和α-C-H质子， 然后在温和条件下促使丙酮酸的产生和O2转化为H2O2。Co4N/C NEs在体外和体内对乳酸的催化氧化中也表现出了较高的催化性能。LOX模拟活性作用于逆转高乳酸并重塑肿瘤微环境的免疫抑制状态。同时，Co4N/C NEs内部的超声扩增和多酶级联能力进一步提高了催化治疗效率。这些特性赋予Co4N/C NEs优异的抗肿瘤生长和远处转移抑制能力。我们相信，这项研究为创造真正与天然对应物相媲美的纳米酶铺平了一条新的道路。
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其他实验细节，包括实验方法、材料表征、DFT 结果、多酶级联数据以及其他体外和体内数据 （PDF）
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Author Information
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- Liu Deng - Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China;
https://orcid.org/0000-0002-1392-6828;
刘邓 - 中南大学化学化工学院，湖南省微纳材料界面科学重点实验室，中国湖南410083长沙; https://orcid.org/0000-0002-1392-6828;电子邮件： dengliu@csu.edu.cn - Shaojun Guo - School of Materials Science and Engineering, Peking University, Beijing 100871, China;https://orcid.org/0000-0003-4427-6837;
郭少军 - 北京大学材料科学与工程学院，中国北京100871; https://orcid.org/0000-0003-4427-6837;电子邮件： guosj@pku.edu.cn - You-Nian Liu - Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China; College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China;https://orcid.org/0000-0002-7078-5937;
You-Nian Liu - 中南大学化学化工学院，湖南省微纳材料界面科学重点实验室，湖南410083长沙;杭州师范大学材料化学化工学院， 浙江杭州， 浙江311121; https://orcid.org/0000-0002-7078-5937;电子邮件： liuyounian@csu.edu.cn
- Na Tao - Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China;https://orcid.org/0000-0002-2349-5873
Na Tao - 中南大学化学化工学院，湖南省微纳材料界面科学重点实验室，湖南410083长沙; https://orcid.org/0000-0002-2349-5873 - Jianping Sheng - School of Resources and Environment, University of Electronic Science and Technology of China, Chengdu, Sichuan 611731, China;https://orcid.org/0000-0002-1794-9197
盛建平 - 中国电子科技大学资源与环境学院，中国四川611731成都; https://orcid.org/0000-0002-1794-9197 - Liqiang Wang - Henan Province Industrial Technology Research Institute of Resources and Materials, School of Material Science and Engineering, Zhengzhou University, Zhengzhou, Henan 450001, China;https://orcid.org/0000-0002-4579-7258
王立强 - 河南省资源与材料工业技术研究所，郑州大学材料科学与工程学院，河南450001郑州; https://orcid.org/0000-0002-4579-7258 - Wansong Chen - Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China;https://orcid.org/0000-0002-3426-2653
陈万松 - 中南大学化学化工学院，湖南省微纳材料界面科学重点实验室，中国湖南410083长沙; https://orcid.org/0000-0002-3426-2653
Acknowledgments
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This work was supported by the National Science Fund for Distinguished Young Scholars (no. 52025133), the National Natural Science Foundation of China (nos. 22238013, 21807117, and 22178393), the National Key R&D Program of China (no. 2017YFA0206701), Tencent Foundation through the XPLORER PRIZE, the Hunan Provincial Science and Technology Plan Project, China (nos. 2019TP1001 and 2020JJ3044), and the Fundamental Research Funds for the Central Universities of Central South University, Changsha, China (no. 2022ZZTS0397).
这项工作得到了国家杰出青年科学基金（第52025133项）、国家自然科学基金（第22238013、21807117和22178393项）、国家重点研发计划（第2017YFA0206701号）、腾讯基金会通过XPLORER PRIZE、湖南省科技计划项目（第2019TP1001号和2020JJ3044号）的支持。 中南大学中央高校基本科研业务费专项资金资助项目（编号：2022ZZTS0397）。
References
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37 other publications.
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- 1Huang, Y.; Ren, J.; Qu, X. Nanozymes: Classification, Catalytic Mechanisms, Activity Regulation, and Applications. Chem. Rev. 2019, 119, 4357– 4412, DOI: 10.1021/acs.chemrev.8b00672Google Scholar Google 学术搜索1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjsFSrsbo%253D&md5=bc90e3da0b0848b1abac07ca7916188fNanozymes: Classification, catalytic mechanisms, activity regulation, and applicationsHuang, Yanyan; Ren, Jinsong; Qu, XiaogangChemical Reviews (Washington, DC, United States) (2019), 119 (6), 4357-4412CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Because of the high catalytic activities and substrate specificity, natural enzymes have been widely used in industrial, medical, and biol. fields, etc. Although promising, they often suffer from intrinsic shortcomings such as high cost, low operational stability, and difficulties of recycling. To overcome these shortcomings, researchers have been devoted to the exploration of artificial enzyme mimics for a long time. Since the discovery of ferromagnetic nanoparticles with intrinsic horseradish peroxidase-like activity in 2007, a large amt. of studies on nanozymes have been constantly emerging in the next decade. Nanozymes are one kind of nanomaterials with enzymic catalytic properties. Compared with natural enzymes, nanozymes have the advantages such as low cost, high stability and durability, which have been widely used in industrial, medical, and biol. fields. A thorough understanding of the possible catalytic mechanisms will contribute to the development of novel and high-efficient nanozymes, and the rational regulations of the activities of nanozymes are of great significance. In this review, we systematically introduce the classification, catalytic mechanism, activity regulation as well as recent research progress of nanozymes in the field of biosensing, environmental protection, and disease treatments, etc. in the past years. We also propose the current challenges of nanozymes as well as their future research focus. We anticipate this review may be of significance for the field to understand the properties of nanozymes and the development of novel nanomaterials with enzyme mimicking activities.
1黄英;任，J.;Qu， X.纳米酶：分类、催化机制、活性调控和应用。Chem. Rev. 2019， 119， 4357– 4412， DOI： 10.1021/acs.chemrev.8b00672 - 2Ji, S.; Jiang, B.; Hao, H.; Chen, Y.; Dong, J.; Mao, Y.; Zhang, Z.; Gao, R.; Chen, W.; Zhang, R.; Liang, Q.; Li, H.; Liu, S.; Wang, Y.; Zhang, Q.; Gu, L.; Duan, D.; Liang, M.; Wang, D.; Yan, X.; Li, Y. Matching the Kinetics of Natural Enzymes with a Single-Atom Iron Nanozyme. Nat. Catal. 2021, 4, 407– 417, DOI: 10.1038/s41929-021-00609-xGoogle Scholar Google 学术搜索2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVOlt73K&md5=9eb2a22f5f1ce2ad43451fa042c878beMatching the kinetics of natural enzymes with a single-atom iron nanozymeJi, Shufang; Jiang, Bing; Hao, Haigang; Chen, Yuanjun; Dong, Juncai; Mao, Yu; Zhang, Zedong; Gao, Rui; Chen, Wenxing; Zhang, Ruofei; Liang, Qian; Li, Haijing; Liu, Shuhu; Wang, Yu; Zhang, Qinghua; Gu, Lin; Duan, Demin; Liang, Minmin; Wang, Dingsheng; Yan, Xiyun; Li, YadongNature Catalysis (2021), 4 (5), 407-417CODEN: NCAACP; ISSN:2520-1158. (Nature Portfolio)Abstr.: Developing artificial enzymes with the excellent catalytic performance of natural enzymes has been a long-standing goal for chemists. Single-atom catalysts with well-defined at. structure and electronic coordination environments can effectively mimic natural enzymes. Here, we report an engineered FeN3P-centered single-atom nanozyme (FeN3P-SAzyme) that exhibits comparable peroxidase-like catalytic activity and kinetics to natural enzymes, by controlling the electronic structure of the single-atom iron active center through the precise coordination of phosphorus and nitrogen. In particular, the engineered FeN3P-SAzyme, with well-defined geometric and electronic structures, displays catalytic performance that is consistent with Michaelis-Menten kinetics. We rationalize the origin of the high enzyme-like activity using d. functional theory calcns. Finally, we demonstrate that the developed FeN3P-SAzyme with superior peroxidase-like activity can be used as an effective therapeutic strategy for inhibiting tumor cell growth in vitro and in vivo. Therefore, SAzymes show promising potential for developing artificial enzymes that have the catalytic kinetics of natural enzymes. [graphic not available: see fulltext].
2季，S.;江， B.;郝，H.;陈英;董，J.;毛，Y.;张Z.;高，R.;陈伟;张，R.;梁，Q.;李，H.;刘，S.;王英;张琴;顾，L.;段，D.;梁，M.;王，D.;闫，X.;Li， Y.将天然酶的动力学与单原子铁纳米酶相匹配。国家加泰罗尼亚。2021， 4， 407– 417， DOI： 10.1038/s41929-021-00609-x - 3Yu, B.; Wang, W.; Sun, W.; Jiang, C.; Lu, L. Defect Engineering Enables Synergistic Action of Enzyme-Mimicking Active Centers for High-Efficiency Tumor Therapy. J. Am. Chem. Soc. 2021, 143, 8855– 8865, DOI: 10.1021/jacs.1c03510Google Scholar Google 学术搜索3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1aktb7F&md5=410b44c69b5f19461424b297ad0f4ab3Defect Engineering Enables Synergistic Action of Enzyme-Mimicking Active Centers for High-Efficiency Tumor TherapyYu, Bin; Wang, Wei; Sun, Wenbo; Jiang, Chunhuan; Lu, LehuiJournal of the American Chemical Society (2021), 143 (23), 8855-8865CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Perusing redox nanozymes capable of disrupting cellular homeostasis offers new opportunities to develop cancer-specific therapy, but remains challenging, because most artificial enzymes lack enzyme-like scale and configuration. Herein, for the first time, the authors leverage a defect engineering strategy to develop a simple yet efficient redox nanozyme by constructing enzyme-mimicking active centers and studied its formation and catalysis mechanism thoroughly. Specifically, the partial Fe doping in MoOx (donated as Fe-MoOv) activate structure reconstruction with abundant defect site generation, including Fe substitution and oxygen vacancy (OV) defects, which significantly enable the binding capacity and catalytic activity of Fe-MoOv nanozymes in a synergetic fashion. More intriguingly, plenty of delocalized electrons appear due to Fe-facilitated band structure reconstruction, directly contributing to the remarkable surface plasmon resonance effect in the near-IR (NIR) region. Under NIR-II laser irradn., the designed Fe-MoOv nanozymes are able to induce substantial disruption of redox and metab. homeostasis in the tumor region via enzyme-mimicking cascade reactions, thus significantly augmenting therapeutic effects. This study that takes advantage of defect engineering offers new insights into developing high-efficiency redox nanozymes.
3余，B.;王，W.;孙，W.;江，C.;Lu， L.Defect Engineering使酶模拟活性中心具有协同作用，以实现高效的肿瘤治疗。J. Am. Chem. Soc. 2021， 143， 8855– 8865， DOI： 10.1021/jacs.1c03510 - 4Colegio, O. R.; Chu, N. Q.; Szabo, A. L.; Chu, T.; Rhebergen, A. M.; Jairam, V.; Cyrus, N.; Brokowski, C. E.; Eisenbarth, S. C.; Phillips, G. M.; Cline, G. W.; Phillips, A. J.; Medzhitov, R. Functional Polarization of Tumour-Associated Macrophages by Tumour-Derived Lactic Acid. Nature 2014, 513, 559– 563, DOI: 10.1038/nature13490Google Scholar Google 学术搜索4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Wjs7nO&md5=19de6df0a2734ab8037ade191a02ed60Functional polarization of tumor-associated macrophages by tumor-derived lactic acidColegio, Oscar R.; Chu, Ngoc-Quynh; Szabo, Alison L.; Chu, Thach; Rhebergen, Anne Marie; Jairam, Vikram; Cyrus, Nika; Brokowski, Carolyn E.; Eisenbarth, Stephanie C.; Phillips, Gillian M.; Cline, Gary W.; Phillips, Andrew J.; Medzhitov, RuslanNature (London, United Kingdom) (2014), 513 (7519), 559-563CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Macrophages have an important role in the maintenance of tissue homeostasis. To perform this function, macrophages must have the capacity to monitor the functional states of their 'client cells': namely, the parenchymal cells in the various tissues in which macrophages reside. Tumors exhibit many features of abnormally developed organs, including tissue architecture and cellular compn. Similarly to macrophages in normal tissues and organs, macrophages in tumors (tumor-assocd. macrophages) perform some key homeostatic functions that allow tumor maintenance and growth. However, the signals involved in communication between tumors and macrophages are poorly defined. Here we show that lactic acid produced by tumor cells, as a byproduct of aerobic or anaerobic glycolysis, has a crit. function in signaling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumor-assocd. macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumor growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumor cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumors to promote their growth.
4Colegio， O.R.;朱，N.Q.;萨博，AL;朱，T.;雷伯根，AM睚兰，V.;赛勒斯，N.;布罗科夫斯基，CE;南卡罗来纳州艾森巴特;菲利普斯，GM;克莱恩，GW;菲利普斯，AJ;Medzhitov，R.肿瘤衍生乳酸对肿瘤相关巨噬细胞的功能极化。自然 2014， 513， 559– 563， DOI： 10.1038/nature13490 - 5Gatenby, R. A.; Gillies, R. J. Why Do Cancers Have High Aerobic Glycolysis?. Nat. Rev. Cancer 2004, 4, 891– 899, DOI: 10.1038/nrc1478Google Scholar Google 学术搜索5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXptFegt7w%253D&md5=0584954722468b0bfaa5821fa17f2db5Why do cancers have high aerobic glycolysis?Gatenby, Robert A.; Gillies, Robert J.Nature Reviews Cancer (2004), 4 (11), 891-899CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. If carcinogenesis occurs by somatic evolution, then common components of the cancer phenotype result from active selection and must, therefore, confer a significant growth advantage. A near-universal property of primary and metastatic cancers is upregulation of glycolysis, resulting in increased glucose consumption, which can be obsd. with clin. tumor imaging. We propose that persistent metab. of glucose to lactate even in aerobic conditions is an adaptation to intermittent hypoxia in pre-malignant lesions. However, upregulation of glycolysis leads to microenvironmental acidosis requiring evolution to phenotypes resistant to acid-induced cell toxicity. Subsequent cell populations with upregulated glycolysis and acid resistance have a powerful growth advantage, which promotes unconstrained proliferation and invasion.
5加滕比，RA;Gillies， RJ.为什么癌症有高需氧糖酵解？癌症 2004， 4， 891– 899， DOI： 10.1038/nrc1478 - 6Chen, J.; Zhu, Y.; Wu, C.; Shi, J. Engineering Lactate-Modulating Nanomedicines for Cancer Therapy. Chem. Soc. Rev. 2023, 52, 973– 1000, DOI: 10.1039/D2CS00479HGoogle Scholar Google 学术搜索6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjtFGjtw%253D%253D&md5=88669225ca43466a3842c5cdafb424c6Engineering lactate-modulating nanomedicines for cancer therapyChen, Jiajie; Zhu, Yufang; Wu, Chengtie; Shi, JianlinChemical Society Reviews (2023), 52 (3), 973-1000CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Lactate in tumors has long been considered "metabolic junk" derived from the glycolysis of cancer cells and utilized only as a biomarker of malignancy, but is presently believed to be a pivotal regulator of tumor development, maintenance and metastasis. Indeed, tumor lactate can be a "fuel" for energy supply and functions as a signaling mol., which actively contributes to tumor progression, angiogenesis, immunosuppression, therapeutic resistance, etc., thus providing promising opportunities for cancer treatment. However, the current approaches for regulating lactate homeostasis with available agents are still challenging, which is mainly due to the short half-life, low bioavailability and poor specificity of these agents and their unsatisfactory therapeutic outcomes. In recent years, lactate modulation nanomedicines have emerged as a charming and efficient strategy for fighting cancer, which play important roles in optimizing the delivery of lactate-modulating agents for more precise and effective modulation and treatment. Integrating specific lactate-modulating functions in diverse therapeutic nanomedicines may overcome the intrinsic restrictions of different therapeutic modalities by remodeling the pathol. microenvironment for achieving enhanced cancer therapy. In this review, the most recent advances in the engineering of functional nanomedicines that can modulate tumor lactate for cancer therapy are summarized and discussed, and the fundamental mechanisms by which lactate modulation benefits various therapeutics are elucidated. Finally, the challenges and perspectives of this emerging strategy in the anti-tumor field are highlighted.
6陈， J.;朱，Y.;吴，C.;Shi， J.工程乳酸调节纳米药物用于癌症治疗。化学学会修订版 2023， 52， 973– 1000， DOI： 10.1039/D2CS00479H - 7Liu, X. H.; Yu, H. Y.; Huang, J. Y.; Su, J. H.; Xue, C.; Zhou, X. T.; He, Y. R.; He, Q.; Xu, D. J.; Xiong, C.; Ji, H. B. Biomimetic Catalytic Aerobic Oxidation of C-sp(3)-H Bonds under Mild Conditions Using Galactose Oxidase Model Compound Cu(II)L. Chem. Sci. 2022, 13, 9560– 9568, DOI: 10.1039/D2SC02606FGoogle Scholar Google 学术搜索7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitVChurnL&md5=21634a94efb07971a68a15fa2c1301deBiomimetic catalytic aerobic oxidation of C-sp(3)-H bonds under mild conditions using galactose oxidase model compound CuIILLiu, Xiao-Hui; Yu, Hai-Yang; Huang, Jia-Ying; Su, Ji-Hu; Xue, Can; Zhou, Xian-Tai; He, Yao-Rong; He, Qian; Xu, De-Jing; Xiong, Chao; Ji, Hong-BingChemical Science (2022), 13 (33), 9560-9568CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Developed highly efficient catalytic protocols for C-sp(3)-H bond aerobic oxidn. under mild conditions was a long-desired goal of chemists. Inspired by nature, a biomimetic approach for the aerobic oxidn. of C-sp(3)-H by galactose oxidase model compd. CuIIL and NHPI (N-hydroxyphthalimide) were developed. The CuIIL-NHPI system exhibited excellent performance in the oxidn. of C-sp(3)-H bonds to ketones, esp. for light alkanes. The biomimetic catalytic protocol had a broad substrate scope. Mechanistic studies revealed that the CuI-radical intermediate species generated from the intramol. redox process of CuIILH2 was crit. for O2 activation. Kinetic expts. showed that the activation of NHPI were the rate-detg. step. Furthermore, activation of NHPI in the CuIIL-NHPI system were demonstrated by time-resolved EPR results. The persistent PINO (phthalimide-N-oxyl) radical mechanism for the aerobic oxidn. of C-sp(3)-H bond were demonstrated.
7刘旭华;俞禾晖;黄，J.Y.;苏建华;薛，C.;周， X. T.;他，Y.R.;他，Q.;徐， D. J.;熊，C.;Ji， H. B.在温和条件下使用半乳糖氧化酶模型化合物Cu（II）L对C-sp（3）-H键进行仿生催化有氧氧化。 Chem. Sci. 2022， 13， 9560– 9568， DOI： 10.1039/D2SC02606F - 8Sugiyama, S.; Kikumoto, T.; Tanaka, H.; Nakagawa, K.; Sotowa, K.-I.; Maehara, K.; Himeno, Y.; Ninomiya, W. Enhancement of Catalytic Activity on Pd/C and Te–Pd/C During the Oxidative Dehydrogenation of Sodium Lactate to Pyruvate in an Aqueous Phase Under Pressurized Oxygen. Catal. Lett. 2009, 131, 129– 134, DOI: 10.1007/s10562-009-9920-3Google Scholar Google 学术搜索8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjs1Kktrk%253D&md5=7801102ff9a117a70638e3e783433a19Enhancement of Catalytic Activity on Pd/C and Te-Pd/C During the Oxidative Dehydrogenation of Sodium Lactate to Pyruvate in an Aqueous Phase Under Pressurized OxygenSugiyama, Shigeru; Kikumoto, Tetsuo; Tanaka, Haruki; Nakagawa, Keizo; Sotowa, Ken-Ichiro; Maehara, Keiko; Himeno, Yoshiyuki; Ninomiya, WataruCatalysis Letters (2009), 131 (1-2), 129-134CODEN: CALEER; ISSN:1011-372X. (Springer)The oxidative dehydrogenation of sodium lactate to sodium pyruvate in an aq. phase proceeded favorably using a Pd/C catalyst and Pd/C with Te promoter at 358 K with no adjustment in soln. pH under pressurized oxygen, although previous reports had stated that this reaction would not proceed using Pd/C while Pd/C doped with either Pb, Bi or Te showed activity at atm. pressure, 363 K, and pH 8.
8杉山，S.;菊本，T.;田中，H.;中川，K.;索托瓦，K.-I.;前原，K.;姬野，Y.;Ninomiya， W.在加压氧下，乳酸钠在水相中氧化脱氢为丙酮酸过程中对Pd/C和Te-Pd/C的催化活性的增强。加泰罗尼亚。Lett. 2009， 131， 129– 134， DOI： 10.1007/s10562-009-9920-3 - 9Yorita, K.; Matsuoka, T.; Misaki, H.; Massey, V. Interaction of Two Arginine Residues in Lactate Oxidase with the Enzyme Flavin: Conversion of FMN to 8-Formyl-FMN. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13039– 13044, DOI: 10.1073/pnas.250472297Google Scholar Google 学术搜索9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXosVKqtL4%253D&md5=f5742e38bc30610f195f136a93cdac0aInteraction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMNYorita, Kazuko; Matsuoka, Takeshi; Misaki, Hideo; Massey, VincentProceedings of the National Academy of Sciences of the United States of America (2000), 97 (24), 13039-13044CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Two Arg residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-contg. enzymes that catalyze the oxidn. of α-hydroxy acids. In lactate oxidase from Aerococcus viridans, these residues were changed to Lys in 2 single mutations and in a double mutant form. In addn., Arg-181 was replaced by Met to det. the effect of removing the pos. charge on the residue. The effects of these replacements on the kinetic and thermodn. properties were detd. With all mutant forms, there were only small effects on the reactivity of the reduced flavin with O2. On the other hand, the efficiency of redn. of the oxidized flavin by L-lactate was greatly reduced, particularly with the R268K mutant forms. The results demonstrated the importance of the 2 Arg residues in the binding of the substrate and its interaction with the flavin, and were consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by Lys also resulted in a slow conversion of the 8-Me-substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different phys. and chem. properties from those of the FMN-enzyme.
9Yorita，K.;松冈，T.;美咲，H.;Massey， V.乳酸氧化酶中两个精氨酸残基与黄素酶的相互作用：FMN 向 8-甲酰基-FMN 的转化。美国科学院院刊 2000， 97， 13039– 13044， DOI： 10.1073/pnas.250472297 - 10Xiao, Y. P.; Chen, P. H.; Lei, S.; Bai, F.; Fu, L. H.; Lin, J.; Huang, P. Biocatalytic Depletion of Tumorigenic Energy Sources Driven by Cascade Reactions for Efficient Antitumor Therapy. Angew. Chem., Int. Ed. 2022, 61, e202204584 DOI: 10.1002/anie.202204584Google Scholar Google 学术搜索10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xitl2nsbnM&md5=1154551ae5fed8a9bf7c7a9284406854Biocatalytic Depletion of Tumorigenic Energy Sources Driven by Cascade Reactions for Efficient Antitumor TherapyXiao, Ya-Ping; Chen, Peng-Hang; Lei, Shan; Bai, Fang; Fu, Lian-Hua; Lin, Jing; Huang, PengAngewandte Chemie, International Edition (2022), 61 (42), e202204584CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Glucose and lactate play important roles for tumor growth. How to simultaneously deprive tumors of glucose and lactate is a big challenge. We have developed a cascade catalytic system (denoted as FPGLC) based on fluorinated polymer (FP) with co-loading of glucose oxidase (GOx), lactate oxidase (LOx), and catalase (CAT). GOx and LOx deprive glucose and lactate, resp., resulting in abundant hydrogen peroxide (H2O2) generation. Meanwhile, CAT catalyzes H2O2 into O2, which not only promotes catalytic reactions of GOx and LOx for consuming more glucose and lactate, but also alleviates tumor hypoxia. Benefiting from the excellent cross-membrane and transmucosal penetration capacities of FP, FPGLC rapidly accumulated in tumors and subsequently mediated enhanced cascade catalytic therapy under the guidance of photoacoustic imaging. These results demonstrate that the dual depletion of glucose and lactate with O2 supply is a promising strategy for efficient antitumor starvation therapy.
10肖英平;陈，P.H.;雷，S.;白，F.;傅，L.H.;林，J.;Huang， P.由级联反应驱动的致瘤能源的生物催化消耗，用于有效的抗肿瘤治疗。安琪。Chem.， Int. Ed. 2022， 61， e202204584 DOI： 10.1002/anie.202204584 - 11Falivene, L.; Cao, Z.; Petta, A.; Serra, L.; Poater, A.; Oliva, R.; Scarano, V.; Cavallo, L. Towards the Online Computer-Aided Design of Catalytic Pockets. Nat. Chem. 2019, 11, 872– 879, DOI: 10.1038/s41557-019-0319-5Google Scholar Google 学术搜索11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWktLrE&md5=071d5b625c560172fc6daa496afbf3f8Towards the online computer-aided design of catalytic pocketsFalivene, Laura; Cao, Zhen; Petta, Andrea; Serra, Luigi; Poater, Albert; Oliva, Romina; Scarano, Vittorio; Cavallo, LuigiNature Chemistry (2019), 11 (10), 872-879CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)A review. The engineering of catalysts with desirable properties can be accelerated by computer-aided design. To achieve this aim, features of mol. catalysts can be condensed into numerical descriptors that can then be used to correlate reactivity and structure. Based on such descriptors, we have introduced topog. steric maps that provide a three-dimensional image of the catalytic pocket-the region of the catalyst where the substrate binds and reacts-enabling it to be visualized and also reshaped by changing various parameters. These topog. steric maps, esp. when used in conjunction with d. functional theory calcns., enable catalyst structural modifications to be explored quickly, making the online design of new catalysts accessible to the wide chem. community. In this Perspective, we discuss the application of topog. steric maps either to rationalize the behavior of known catalysts-from synthetic mol. species to metalloenzymes-or to design improved catalysts.
11法利文，L.;曹志强;佩塔，A.;塞拉，L.;波特，A.;奥利瓦，R.;斯卡拉诺，V.;Cavallo， L.迈向催化口袋的在线计算机辅助设计。Nat. Chem. 2019， 11， 872– 879， DOI： 10.1038/s41557-019-0319-5 - 12Stoisser, T.; Brunsteiner, M.; Wilson, D. K.; Nidetzky, B. Conformational Flexibility Related to Enzyme Activity: Evidence for a Dynamic Active-Site Gatekeeper Function of Tyr(215) in Aerococcus Viridans Lactate Oxidase. Sci. Rep. 2016, 6, 27892, DOI: 10.1038/srep27892Google Scholar Google 学术搜索12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVWnsrrP&md5=b4bdc3706c8379a6ea7cc59344304a41Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidaseStoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, BerndScientific Reports (2016), 6 (), 27892CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidn. of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent Tyr residue in the substrate-binding pocket (Tyr-215 in A. viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr-215, the effects of substitutions of the Tyr residue (Y215F, Y215H) were analyzed kinetically, crystallog., and by mol. dynamics simulations. Enzyme variants showed slowed flavin redn. and oxidn. by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate showed that the H-bond between the phenolic OH group and the keto O atom in pyruvate was replaced with a potentially stronger hydrophobic interaction between the Phe residue and the Me group of pyruvate. Residues 200 through 215 or 216 appeared to be disordered in 2 of the 8 monomers in the asym. unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr-215 can thus lead to a kinetic bottleneck in product release.
12斯托伊瑟，T.;布伦斯坦纳，M.;威尔逊，DK;Nidetzky， B.与酶活性相关的构象灵活性：Tyr（215） 在草绿色气球菌乳酸氧化酶中的动态活性位点看门人功能的证据。Sci. Rep. 2016， 6， 27892， DOI： 10.1038/srep27892 - 13Noll, N.; Krause, A. M.; Beuerle, F.; Wurthner, F. Enzyme-Like Water Preorganization in a Synthetic Molecular Cleft for Homogeneous Water Oxidation Catalysis. Nat. Catal. 2022, 5, 867– 877, DOI: 10.1038/s41929-022-00843-xGoogle Scholar Google 学术搜索13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFCgtbnN&md5=ade688154ea42c10fda4b852484f1cf3Enzyme-like water preorganization in a synthetic molecular cleft for homogeneous water oxidation catalysisNoll, Niklas; Krause, Ana-Maria; Beuerle, Florian; Wuerthner, FrankNature Catalysis (2022), 5 (10), 867-877CODEN: NCAACP; ISSN:2520-1158. (Nature Portfolio)Abstr.: Inspired by the proficiency of natural enzymes, mimicking of nanoenvironments for precise substrate preorganization is a promising strategy in catalyst design. However, artificial examples of enzyme-like activation of H2O mols. for the challenging oxidative water splitting reaction are hardly explored. Here, we introduce a mononuclear Ru(bda) complex (M1, bda = 2,2'-bipyridine-6,6'-dicarboxylate) equipped with a bipyridine-functionalized ligand to preorganize H2O mols. in front of the metal center as in enzymic clefts. The confined pocket of M1 accelerates chem. driven water oxidn. at pH 1 by facilitating a water nucleophilic attack pathway with a remarkable turnover frequency of 140 s-1 that is comparable to the oxygen-evolving complex of photosystem II. Single crystal X-ray anal. of M1 under catalytic conditions allowed the observation of a seventh H2O ligand directly coordinated to a RuIII center. Another H2O substrate is preorganized via a well-defined hydrogen-bonding network for the crucial O-O bond formation by nucleophilic attack. [graphic not available: see fulltext].
13诺尔，N.;克劳斯，AM博尔勒，F.;Wurthner，F.合成分子裂隙中的酶样水预组织，用于均相水氧化催化。国家加泰罗尼亚。2022， 5， 867– 877， DOI： 10.1038/s41929-022-00843-x - 14Yorita, K.; Janko, K.; Aki, K.; Ghisla, S.; Palfey, B. A.; Massey, V. On the Reaction Mechanism of L-lactate Oxidase: Quantitative Structure-Activity Analysis of the Reaction with Para-Substituted L-Mandelates. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 9590– 9595, DOI: 10.1073/pnas.94.18.9590Google Scholar Google 学术搜索14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXlvVSqtLk%253D&md5=e3b8ccd251336c7d24bf943861f7119bOn the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelatesYorita, Kazuko; Janko, Karl; Aki, Kenji; Ghisla, Sandro; Palfey, Bruce A.; Massey, VincentProceedings of the National Academy of Sciences of the United States of America (1997), 94 (18), 9590-9595CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The rate consts. for redn. of the flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H=3-7) on the rate consts. of flavin redn. were found, indicating that flavin redn. is a direct measure of α-C-H bond breakage. The rate consts. show only a small influence of the electronic characteristic of the substituents, but show a good correlation when combined with some substituent vol. parameters. A surprisingly good correlation is found with the mol. mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.
14约里塔，K.;扬科，K.;阿奇，K.;吉斯拉，S.;帕尔菲，文学学士;Massey， V.关于L-乳酸氧化酶的反应机理：与对位取代的L-扁桃酸酯反应的定量构效分析。美国科学院院刊 1997， 94， 9590– 9595， DOI： 10.1073/pnas.94.18.9590 - 15Furuichi, M.; Suzuki, N.; Dhakshnamoorhty, B.; Minagawa, H.; Yamagishi, R.; Watanabe, Y.; Goto, Y.; Kaneko, H.; Yoshida, Y.; Yagi, H.; Waga, I.; Kumar, P. K.; Mizuno, H. X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with D-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation Mechanism. J. Mol. Biol. 2008, 378, 436– 446, DOI: 10.1016/j.jmb.2008.02.062Google Scholar Google 学术搜索15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlagtb4%253D&md5=7a686a4e8be9cce117861a5bebbde457X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with D-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation MechanismFuruichi, Makio; Suzuki, Nobuhiro; Dhakshnamoorhty, Balasundaresan; Minagawa, Hirotaka; Yamagishi, Ryosuke; Watanabe, Yuta; Goto, Yukari; Kaneko, Hiroki; Yoshida, Yoshihito; Yagi, Hirotaka; Waga, Iwao; Kumar, Penmetcha K. R.; Mizuno, HiroshiJournal of Molecular Biology (2008), 378 (2), 436-446CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Lactate oxidase (LOX) belongs to a family of FMN-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with D-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the D-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the active site, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystn. at pH 4.5, because the same flip is obsd. in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstrs. a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network; between His265-Asp174-Lys221 and, furthermore, brings mol. oxygen in between D-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O2 could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing mol. oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chem. steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.
15古一，M.;铃木，N.;达克什纳穆尔蒂，B.;Minagawa，H.;山岸，R.;渡边，Y.;后藤，Y.;金子，H.;吉田，Y.;八木，H.;瓦加，I.;库马尔，PK;Mizuno， H.X-ray 结构 Viridans Aerococcus viridans 乳酸氧化酶及其在 pH 4.5 下与 D-乳酸的复合物显示出α-羟基酸氧化机制。分子生物学杂志 2008， 378， 436– 446， DOI： 10.1016/j.jmb.2008.02.062 - 16Tabacchi, G.; Zucchini, D.; Caprini, G.; Gamba, A.; Lederer, F.; Vanoni, M. A.; Fois, E. L-lactate Dehydrogenation in Flavocytochrome b2: a First Principles Molecular Dynamics Study. FEBS J. 2009, 276, 2368– 2380, DOI: 10.1111/j.1742-4658.2009.06969.xGoogle Scholar Google 学术搜索16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVKnt78%253D&md5=30385feb5fecfe1fae13f2bf50d30aeeL-Lactate dehydrogenation in flavocytochrome b2: A first principles molecular dynamics studyTabacchi, Gloria; Zucchini, Daniela; Caprini, Gianluca; Gamba, Aldo; Lederer, Florence; Vanoni, Maria A.; Fois, EttoreFEBS Journal (2009), 276 (8), 2368-2380CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)First principles mol. dynamics (MD) studies on active-site models of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidn. reaction, a still-debated issue. In the calcd. enzyme-substrate model complex, the L-lactate α-OH hydrogen is hydrogen bonded to the active-site base H373 Nε, whereas the Hα is directed towards flavin N5, suggesting that the reaction is initiated by α-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidn. led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to FMN, without intermediates, but with α-OH proton abstraction preceding Hα transfer and a calcd. free energy barrier (12.1 kcal/mol-1) consistent with that detd. exptl. (13.5 kcal/mol-1). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a no. of flavoenzymes. Namely, they highlighted the role of: (a) the FMN-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin redn.; (b) an active site water mol. belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction.
16塔巴奇，G.;西葫芦，D.;卡普里尼，G.;甘巴，A.;莱德勒，F.;瓦诺尼，马萨诸塞州;Fois，黄细胞色素 b2 中的 E.L-乳酸脱氢：第一性原理分子动力学研究。FEBS J. 2009， 276， 2368– 2380， DOI： 10.1111/j.1742-4658.2009.06969.x - 17Feng, X.; Song, Y.; Chen, J. S.; Xu, Z.; Dunn, S. J.; Lin, W. Rational Construction of an Artificial Binuclear Copper Monooxygenase in a Metal-Organic Framework. J. Am. Chem. Soc. 2021, 143, 1107– 1118, DOI: 10.1021/jacs.0c11920Google Scholar Google 学术搜索17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlt1CgsQ%253D%253D&md5=aaf8240e547a92c664b50c6a2b2cc8c4Rational Construction of an Artificial Binuclear Copper Monooxygenase in a Metal-Organic FrameworkFeng, Xuanyu; Song, Yang; Chen, Justin S.; Xu, Ziwan; Dunn, Soren J.; Lin, WenbinJournal of the American Chemical Society (2021), 143 (2), 1107-1118CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Artificial enzymic systems are extensively studied to mimic the structures and functions of their natural counterparts. However, there remains a significant gap between structural modeling and catalytic activity in these artificial systems. Herein we report a novel strategy for the construction of an artificial binuclear copper monooxygenase starting from a Ti metal-org. framework (MOF). The deprotonation of the hydroxide groups on the secondary building units (SBUs) of MIL-125(Ti) (MIL = Mat´eriaux de l'Institut Lavoisier) allows for the metalation of the SBUs with closely spaced CuI pairs, which are oxidized by mol. O2 to afford the CuII2(μ2-OH)2 cofactor in the MOF-based artificial binuclear monooxygenase Ti8-Cu2. An artificial mononuclear Cu monooxygenase Ti8-Cu1 was also prepd. for comparison. The MOF-based monooxygenases were characterized by a combination of thermogravimetric anal., inductively coupled plasma-mass spectrometry, X-ray absorption spectroscopy, Fourier-transform IR spectroscopy, and UV-vis spectroscopy. In the presence of coreductants, Ti8-Cu2 exhibited outstanding catalytic activity toward a wide range of monooxygenation processes, including epoxidn., hydroxylation, Baeyer-Villiger oxidn., and sulfoxidn., with turnover nos. of up to 3450. Ti8-Cu2 showed a turnover frequency at least 17 times higher than that of Ti8-Cu1. D. functional theory calcns. revealed O2 activation as the rate-limiting step in the monooxygenation processes. Computational studies further showed that the Cu2 sites in Ti8-Cu2 cooperatively stabilized the Cu-O2 adduct for O-O bond cleavage with 6.6 kcal/mol smaller free energy increase than that of the mononuclear Cu sites in Ti8-Cu1, accounting for the significantly higher catalytic activity of Ti8-Cu2 over Ti8-Cu1.
17冯旭;宋，Y.;陈，J.S.;徐志明;邓恩，SJ;Lin， W.在金属有机框架下人工双核铜单加氧酶的合理构建.J. Am. Chem. Soc. 2021， 143， 1107– 1118， DOI： 10.1021/jacs.0c11920 - 18Zhang, S.; Li, Y.; Sun, S.; Liu, L.; Mu, X.; Liu, S.; Jiao, M.; Chen, X.; Chen, K.; Ma, H.; Li, T.; Liu, X.; Wang, H.; Zhang, J.; Yang, J.; Zhang, X. D. Single-Atom Nanozymes Catalytically Surpassing Naturally Occurring Enzymes as Sustained Stitching for Brain Trauma. Nat. Commun. 2022, 13, 4744, DOI: 10.1038/s41467-022-32411-zGoogle Scholar Google 学术搜索18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitFarurrM&md5=1d16487dc70034d2091853ab06b6e67bSingle-atom nanozymes catalytically surpassing naturally occurring enzymes as sustained stitching for brain traumaZhang, Shaofang; Li, Yonghui; Sun, Si; Liu, Ling; Mu, Xiaoyu; Liu, Shuhu; Jiao, Menglu; Chen, Xinzhu; Chen, Ke; Ma, Huizhen; Li, Tuo; Liu, Xiaoyu; Wang, Hao; Zhang, Jianning; Yang, Jiang; Zhang, Xiao-DongNature Communications (2022), 13 (1), 4744CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Regenerable nanozymes with high catalytic stability and sustainability are promising substitutes for naturally-occurring enzymes but are limited by insufficient and non-selective catalytic activities. Herein, we developed single-atom nanozymes of RhN4, VN4, and Fe-Cu-N6 with catalytic activities surpassing natural enzymes. Notably, Rh/VN4 preferably forms an Rh/V-O-N4 active center to decrease reaction energy barriers and mediates a "two-sided oxygen-linked" reaction path, showing 4 and 5-fold higher affinities in peroxidase-like activity than the FeN4 and natural horseradish peroxidase. Furthermore, RhN4 presents a 20-fold improved affinity in the catalase-like activity compared to the natural catalase; Fe-Cu-N6 displays selectivity towards the superoxide dismutase-like activity; VN4 favors a 7-fold higher glutathione peroxidase-like activity than the natural glutathione peroxidase. Bioactive sutures with Rh/VN4 show recyclable catalytic features without apparent decay in 1 mo and accelerate the scalp healing from brain trauma by promoting the vascular endothelial growth factor, regulating the immune cells like macrophages, and diminishing inflammation.
18张，S.;李英;孙，S.;刘，L.;穆，X.;刘，S.;焦，M.;陈旭;陈，K.;马，H.;李，T.;刘旭;王，H.;张，J.;杨，J.;Zhang， X. D.单原子纳米酶催化超越天然存在的酶，作为脑外伤的持续缝合。Nat. Commun.2022， 13， 4744， DOI： 10.1038/s41467-022-32411-z - 19Fan, K.; Xi, J.; Fan, L.; Wang, P.; Zhu, C.; Tang, Y.; Xu, X.; Liang, M.; Jiang, B.; Yan, X.; Gao, L. In Vivo Guiding Nitrogen-Doped Carbon Nanozyme for Tumor Catalytic Therapy. Nat. Commun. 2018, 9, 1440, DOI: 10.1038/s41467-018-03903-8Google Scholar Google 学术搜索19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mjit1Khtg%253D%253D&md5=c50850cc01e120026c745b573ebe9915In vivo guiding nitrogen-doped carbon nanozyme for tumor catalytic therapyFan Kelong; Wang Peixia; Liang Minmin; Jiang Bing; Yan Xiyun; Xi Juqun; Zhu Chunhua; Tang Yan; Gao Lizeng; Fan Lei; Xu Xiangdong; Wang Peixia; Jiang Bing; Yan XiyunNature communications (2018), 9 (1), 1440 ISSN:.Nanomaterials with intrinsic enzyme-like activities (nanozymes), have been widely used as artificial enzymes in biomedicine. However, how to control their in vivo performance in a target cell is still challenging. Here we report a strategy to coordinate nanozymes to target tumor cells and selectively perform their activity to destruct tumors. We develop a nanozyme using nitrogen-doped porous carbon nanospheres which possess four enzyme-like activities (oxidase, peroxidase, catalase and superoxide dismutase) responsible for reactive oxygen species regulation. We then introduce ferritin to guide nitrogen-doped porous carbon nanospheres into lysosomes and boost reactive oxygen species generation in a tumor-specific manner, resulting in significant tumor regression in human tumor xenograft mice models. Together, our study provides evidence that nitrogen-doped porous carbon nanospheres are powerful nanozymes capable of regulating intracellular reactive oxygen species, and ferritinylation is a promising strategy to render nanozymes to target tumor cells for in vivo tumor catalytic therapy.
19范，K.;习， J.;范，L.;王，P.;朱，C.;唐，Y.;徐， X.;梁，M.;江， B.;闫，X.;Gao， L.In 体内引导氮掺杂碳纳米酶用于肿瘤催化治疗。Nat. Commun.2018， 9， 1440， DOI： 10.1038/s41467-018-03903-8 - 20Matthews, A.; Saleem-Batcha, R.; Sanders, J. N.; Stull, F.; Houk, K. N.; Teufel, R. Aminoperoxide Adducts Expand the Catalytic Repertoire of Flavin Monooxygenases. Nat. Chem. Biol. 2020, 16, 556– 563, DOI: 10.1038/s41589-020-0476-2Google Scholar Google 学术搜索20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjtlanu7c%253D&md5=de80cfc4822f4e5ea788a87aa572e66cAminoperoxide adducts expand the catalytic repertoire of flavin monooxygenasesMatthews, Arne; Saleem-Batcha, Raspudin; Sanders, Jacob N.; Stull, Frederick; Houk, K. N.; Teufel, RobinNature Chemical Biology (2020), 16 (5), 556-563CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Abstr.: One of the hallmark reactions catalyzed by flavin-dependent enzymes is the incorporation of an oxygen atom derived from dioxygen into org. substrates. For many decades, these flavin monooxygenases were assumed to use exclusively the flavin-C4a-(hydro)peroxide as their oxygen-transferring intermediate. We demonstrate that flavoenzymes may instead employ a flavin-N5-peroxide as a soft α-nucleophile for catalysis, which enables chem. not accessible to canonical monooxygenases. This includes, for example, the redox-neutral cleavage of carbon-hetero bonds or the dehalogenation of inert environmental pollutants via atypical oxygenations. We furthermore identify a shared structural motif for dioxygen activation and N5-functionalization, suggesting a conserved pathway that may be operative in numerous characterized and uncharacterized flavoenzymes from diverse organisms. Our findings show that overlooked flavin-N5-oxygen adducts are more widespread and may facilitate versatile chem., thus upending the notion that flavin monooxygenases exclusively function as nature's equiv. to org. peroxides in synthetic chem.
20马修斯，A.;萨利姆-巴查，R.;桑德斯，J.N.;斯图尔，F.;霍克，K.N.;Teufel， R.氨基过氧化物加合物扩大了黄素单加氧酶的催化库。化学生物学 2020， 16， 556– 563， DOI： 10.1038/s41589-020-0476-2 - 21Sheng, J.; Wang, L.; Deng, L.; Zhang, M.; He, H.; Zeng, K.; Tang, F.; Liu, Y.-N. MOF-Templated Fabrication of Hollow Co4N@N-Doped Carbon Porous Nanocages with Superior Catalytic Activity. ACS Appl. Mater. Interfaces 2018, 10, 7191– 7200, DOI: 10.1021/acsami.8b00573Google Scholar Google 学术搜索21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVKku74%253D&md5=05bd112c36f451a274b597795f22c8f4MOF-Templated Fabrication of Hollow Co4N@N-Doped Carbon Porous Nanocages with Superior Catalytic ActivitySheng, Jianping; Wang, Liqiang; Deng, Liu; Zhang, Min; He, Haichuan; Zeng, Ke; Tang, Feiying; Liu, You-NianACS Applied Materials & Interfaces (2018), 10 (8), 7191-7200CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Metallic Co4N catalysts have been considered as one of the most promising non-noble materials for heterogeneous catalysis because of their high elec. cond., great magnetic property, and high intrinsic activity. However, the metastable properties seriously limit their applications for heterogeneous water phase catalysis. In this work, a novel Co-metal-org. framework (MOF)-derived hollow porous nanocages (PNCs) composed of metallic Co4N and N-doped carbon (NC) were synthesized for the first time. This hollow three-dimensional (3D) PNC catalyst was synthesized by taking advantage of Co-MOF as a precursor for fabricating 3D hollow Co3O4@C PNCs, along with the NH3 treatment of Co-oxide frames to promote the in situ conversion of Co-MOF to Co4N@NC PNCs, benefiting from the high intrinsic activity and electron cond. of the metallic Co4N phase and the good permeability of the hollow porous nanostructure as well as the efficient doping of N into the carbon layer. Besides, the covalent bridge between the active Co4N surface and PNC shells also provides facile pathways for electron and mass transport. The obtained Co4N@NC PNCs exhibit excellent catalytic activity and stability for 4-nitrophenol redn. in terms of low activation energy (Ea = 23.53 kJ mol-1), high turnover frequency (52.01 × 1020 mol. g-1 min-1), and high apparent rate const. (kapp = 2.106 min-1). Furthermore, its magnetic property and stable configuration account for the excellent recyclability of the catalyst. It is hoped that our finding could pave the way for the construction of other hollow transition metal-based nitride@NC PNC catalysts for wide applications.
21盛，J.;王，L.;邓，L.;张，M.;他，H.;曾国藩;唐，F.;Liu， Y.-N.MOF-Templated Fabriced of Hollow Co4N@N-Dopped Carbon Porous Nanocages with Superior CatalyActivity.ACS Appl. Mater.接口 2018， 10， 7191– 7200， DOI： 10.1021/acsami.8b00573 - 22Yang, Y.; Zeng, R.; Xiong, Y.; DiSalvo, F. J.; Abruna, H. D. Cobalt-Based Nitride-Core Oxide-Shell Oxygen Reduction Electrocatalysts. J. Am. Chem. Soc. 2019, 141, 19241– 19245, DOI: 10.1021/jacs.9b10809Google Scholar Google 学术搜索22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitF2nurvI&md5=83e9f48a566bee508eecc87e7eae0f2eCobalt-Based Nitride-Core Oxide-Shell Oxygen Reduction ElectrocatalystsYang, Yao; Zeng, Rui; Xiong, Yin; DiSalvo, Francis J.; Abruna, Hector D.Journal of the American Chemical Society (2019), 141 (49), 19241-19245CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Developing high-performance, low-cost, and conductive nonprecious electrocatalysts for the O redn. reaction (ORR) was a key challenge for advancing fuel cell technologies. Here, the authors report on a novel family of Co nitrides (CoxN/C, x = 2, 3, 4) as ORR electrocatalysts in alk. fuel cells. Co4N/C exhibited the highest ORR activity among the three types of Co nitrides studied, with a half-wave potential (E1/2) of 0.875 V vs. RHE in 1 M KOH, rivaling that of com. Pt/C (0.89 V). Also, Co4N/C showed an 8-fold improvement in mass activity at 0.85 V, when compared to Co oxide, Co3O4/C, and a negligible degrdn. (ΔE1/2 = 14 mV) after 10,000 potential cycles. The superior performance was ascribed to the formation of a conductive nitride core surrounded by a naturally formed thin oxide shell (∼2 nm). The conductive nitride core effectively mitigated the low cond. of the metal oxide, and the thin oxide shell on the surface provided the active sites for the ORR. Strategies developed herein represent a promising approach for the design of other novel metal nitrides as electrocatalysts for fuel cells.
22杨，Y.;曾，R.;熊英;迪萨尔沃，FJ;Abruna， H. D.钴基氮化物-核心氧化物-壳氧还原电催化剂。J. Am. Chem. Soc. 2019， 141， 19241– 19245， DOI： 10.1021/jacs.9b10809 - 23Wang, L.; Zhang, W.; Zheng, X.; Chen, Y.; Wu, W.; Qiu, J.; Zhao, X.; Zhao, X.; Dai, Y.; Zeng, J. Incorporating Nitrogen Atoms into Cobalt Nanosheets as a Strategy to Boost Catalytic Activity toward CO2 Hydrogenation. Nat. Energy 2017, 2, 869– 876, DOI: 10.1038/s41560-017-0015-xGoogle Scholar Google 学术搜索23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVehu74%253D&md5=ca887835cd0d140245e8726037aae2aeIncorporating nitrogen atoms into cobalt nanosheets as a strategy to boost catalytic activity toward CO2 hydrogenationWang, Liangbing; Zhang, Wenbo; Zheng, Xusheng; Chen, Yizhen; Wu, Wenlong; Qiu, Jianxiang; Zhao, Xiangchen; Zhao, Xiao; Dai, Yizhou; Zeng, JieNature Energy (2017), 2 (11), 869-876CODEN: NEANFD; ISSN:2058-7546. (Nature Research)Hydrogenation of CO2 into fuels and useful chems. could help to reduce reliance on fossil fuels. Although great progress has been made over the past decades to improve the activity of catalysts for CO2 hydrogenation, more efficient catalysts, esp. those based on non-noble metals, are desired. Here we incorporate N atoms into Co nanosheets to boost the catalytic activity toward CO2 hydrogenation. For the hydrogenation of CO2, Co4N nanosheets exhibited a turnover frequency of 25.6 h-1 in a slurry reactor under 32 bar pressure at 150 °C, which was 64 times that of Co nanosheets. The activation energy for Co4N nanosheets was 43.3 kJ mol-1, less than half of that for Co nanosheets. Mechanistic studies revealed that Co4N nanosheets were reconstructed into Co4NHx, wherein the amido-hydrogen atoms directly interacted with the CO2 to form HCOO* intermediates. In addn., the adsorbed H2O* activated amido-hydrogen atoms via the interaction of hydrogen bonds.
23王，L.;张伟;郑，X.;陈英;吴，W.;邱，J.;赵旭;赵旭;戴，Y.;Zeng， J.将氮原子掺入钴纳米片中作为提高 CO2 氢化催化活性的策略。国家能源 2017， 2， 869– 876， DOI： 10.1038/s41560-017-0015-x - 24Zhang, Y.; Ouyang, B.; Xu, J.; Jia, G.; Chen, S.; Rawat, R. S.; Fan, H. J. Rapid Synthesis of Cobalt Nitride Nanowires: Highly Efficient and Low-Cost Catalysts for Oxygen Evolution. Angew. Chem., Int. Ed. 2016, 55, 8670– 8674, DOI: 10.1002/anie.201604372Google Scholar Google 学术搜索24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptVyrtrs%253D&md5=0ed485fdb2c5a8a291fc2aef670672e7Rapid Synthesis of Cobalt Nitride Nanowires: Highly Efficient and Low-Cost Catalysts for Oxygen EvolutionZhang, Yongqi; Ouyang, Bo; Xu, Jing; Jia, Guichong; Chen, Shi; Rawat, Rajdeep Singh; Fan, Hong JinAngewandte Chemie, International Edition (2016), 55 (30), 8670-8674CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Electrochem. splitting of water to produce hydrogen and oxygen is an important process for many energy storage and conversion devices. Developing efficient, durable, low-cost, and earth-abundant electrocatalysts for the oxygen evolution reaction (OER) is of great urgency. To achieve the rapid synthesis of transition-metal nitride nanostructures and improve their electrocatalytic performance, a new strategy has been developed to convert cobalt oxide precursors into cobalt nitride nanowires through N2 radio frequency plasma treatment. This method requires significantly shorter reaction times (about 1 min) at room temp. compared to conventional high-temp. NH3 annealing which requires a few hours. The plasma treatment significantly enhances the OER activity, as evidenced by a low overpotential of 290 mV to reach a c.d. of 10 mA cm-2, a small Tafel slope, and long-term durability in an alk. electrolyte.
24张英;欧阳，B.;徐，J.;贾樟柯;陈，S.;拉瓦特，R.S.;Fan， H. J.氮化钴纳米线的快速合成：用于析氧的高效和低成本催化剂。安琪。Chem.， Int. Ed. 2016， 55， 8670– 8674， DOI： 10.1002/anie.201604372 - 25Chen, J.; Ma, Q.; Zheng, X.; Fang, Y.; Wang, J.; Dong, S. Kinetically Restrained Oxygen Reduction to Hydrogen Peroxide with Nearly 100% Selectivity. Nat. Commun. 2022, 13, 2808, DOI: 10.1038/s41467-022-30411-7Google Scholar Google 学术搜索25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVaqurnE&md5=7a4ed9c319e9ef813fb27a13419d0950Kinetically restrained oxygen reduction to hydrogen peroxide with nearly 100% selectivityChen, Jinxing; Ma, Qian; Zheng, Xiliang; Fang, Youxing; Wang, Jin; Dong, ShaojunNature Communications (2022), 13 (1), 2808CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Hydrogen peroxide has been synthesized mainly through the electrocatalytic and photocatalytic oxygen redn. reaction in recent years. Herein, we synthesize a single-atom rhodium catalyst (Rh1/NC) to mimic the properties of flavoenzymes for the synthesis of hydrogen peroxide under mild conditions. Rh1/NC dehydrogenates various substrates and catalyzes the redn. of oxygen to hydrogen peroxide. The max. hydrogen peroxide prodn. rate is 0.48 mol gcatalyst-1 h-1 in the phosphorus acid aerobic oxidn. reaction. We find that the selectivity of oxygen redn. to hydrogen peroxide can reach 100%. This is because a single catalytic site of Rh1/NC can only catalyze the removal of two electrons per substrate mol.; thus, the subsequent oxygen can only obtain two electrons to reduce to hydrogen peroxide through the typical two-electron pathway. Similarly, due to the restriction of substrate dehydrogenation, the hydrogen peroxide selectivity in com. Pt/C-catalyzed enzymic reactions can be found to reach 75%, which is 30 times higher than that in electrocatalytic oxygen redn. reactions.
25陈， J.;马，Q.;郑，X.;方，Y.;王，J.;Dong， S.动力学限制氧还原为过氧化氢，选择性接近100%。Nat. Commun.2022， 13， 2808， DOI： 10.1038/s41467-022-30411-7 - 26Hui, S.; Ghergurovich, J. M.; Morscher, R. J.; Jang, C.; Teng, X.; Lu, W.; Esparza, L. A.; Reya, T.; Le, Z.; Yanxiang Guo, J.; White, E.; Rabinowitz, J. D. Glucose Feeds the TCA Cycle via Circulating Lactate. Nature 2017, 551, 115– 118, DOI: 10.1038/nature24057Google Scholar Google 学术搜索26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M7itVeitA%253D%253D&md5=52d11bb82116a571b4bf551ecd2c4690Glucose feeds the TCA cycle via circulating lactateHui Sheng; Ghergurovich Jonathan M; Morscher Raphael J; Jang Cholsoon; Teng Xin; Lu Wenyun; Rabinowitz Joshua D; Hui Sheng; Morscher Raphael J; Jang Cholsoon; Teng Xin; Lu Wenyun; Rabinowitz Joshua D; Ghergurovich Jonathan M; Esparza Lourdes A; Reya Tannishtha; Le Zhan; Yanxiang Guo Jessie; White Eileen; Rabinowitz Joshua D; Le Zhan; White Eileen; Yanxiang Guo Jessie; Yanxiang Guo JessieNature (2017), 551 (7678), 115-118 ISSN:.Mammalian tissues are fuelled by circulating nutrients, including glucose, amino acids, and various intermediary metabolites. Under aerobic conditions, glucose is generally assumed to be burned fully by tissues via the tricarboxylic acid cycle (TCA cycle) to carbon dioxide. Alternatively, glucose can be catabolized anaerobically via glycolysis to lactate, which is itself also a potential nutrient for tissues and tumours. The quantitative relevance of circulating lactate or other metabolic intermediates as fuels remains unclear. Here we systematically examine the fluxes of circulating metabolites in mice, and find that lactate can be a primary source of carbon for the TCA cycle and thus of energy. Intravenous infusions of (13)C-labelled nutrients reveal that, on a molar basis, the circulatory turnover flux of lactate is the highest of all metabolites and exceeds that of glucose by 1.1-fold in fed mice and 2.5-fold in fasting mice; lactate is made primarily from glucose but also from other sources. In both fed and fasted mice, (13)C-lactate extensively labels TCA cycle intermediates in all tissues. Quantitative analysis reveals that during the fasted state, the contribution of glucose to tissue TCA metabolism is primarily indirect (via circulating lactate) in all tissues except the brain. In genetically engineered lung and pancreatic cancer tumours in fasted mice, the contribution of circulating lactate to TCA cycle intermediates exceeds that of glucose, with glutamine making a larger contribution than lactate in pancreatic cancer. Thus, glycolysis and the TCA cycle are uncoupled at the level of lactate, which is a primary circulating TCA substrate in most tissues and tumours.
26许，S.;盖尔古罗维奇，JM;莫舍尔，RJ;张，C.;滕，X.;卢，W.;洛杉矶埃斯帕扎;雷亚，T.;勒，Z.;郭燕翔，J.;怀特，E.;Rabinowitz， J. D.葡萄糖通过循环乳酸为 TCA 循环提供营养。自然 2017， 551， 115– 118， DOI： 10.1038/nature24057 - 27Sekine, H.; Yamamoto, M.; Motohashi, H. Tumors Sweeten Macrophages with Acids. Nat. Immunol. 2018, 19, 1281– 1283, DOI: 10.1038/s41590-018-0258-0Google Scholar Google 学术搜索27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2qtbfE&md5=6ad30efef43b5e3963a737bbbb0839c8Tumors sweeten macrophages with acidsSekine, Hiroki; Yamamoto, Masayuki; Motohashi, HozumiNature Immunology (2018), 19 (12), 1281-1283CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)Acidic microenvironments induced by highly glycolytic tumor cells promote the noninflammatory polarization of tumor-assocd. macrophages, which leads to immunoevasion.
27关根，H.;山本，M.;Motohashi， H.Tumors 用酸使巨噬细胞变甜。自然免疫学。2018， 19， 1281– 1283， DOI： 10.1038/s41590-018-0258-0 - 28Bohn, T.; Rapp, S.; Luther, N.; Klein, M.; Bruehl, T. J.; Kojima, N.; Aranda Lopez, P.; Hahlbrock, J.; Muth, S.; Endo, S.; Pektor, S.; Brand, A.; Renner, K.; Popp, V.; Gerlach, K.; Vogel, D.; Lueckel, C.; Arnold-Schild, D.; Pouyssegur, J.; Kreutz, M.; Huber, M.; Koenig, J.; Weigmann, B.; Probst, H. C.; von Stebut, E.; Becker, C.; Schild, H.; Schmitt, E.; Bopp, T. Tumor Immunoevasion via Acidosis-Dependent Induction of Regulatory Tumor-Associated Macrophages. Nat. Immunol. 2018, 19, 1319– 1329, DOI: 10.1038/s41590-018-0226-8Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2qur7M&md5=753dc4e3bb15b5d6f38c2825f4d78cd1Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophagesBohn, Toszka; Rapp, Steffen; Luther, Natascha; Klein, Matthias; Bruehl, Till-Julius; Kojima, Nobuhiko; Aranda Lopez, Pamela; Hahlbrock, Jennifer; Muth, Sabine; Endo, Shogo; Pektor, Stefanie; Brand, Almut; Renner, Kathrin; Popp, Vanessa; Gerlach, Katharina; Vogel, Dennis; Lueckel, Christina; Arnold-Schild, Danielle; Pouyssegur, Jacques; Kreutz, Marina; Huber, Magdalena; Koenig, Jochem; Weigmann, Benno; Probst, Hans-Christian; von Stebut, Esther; Becker, Christian; Schild, Hansjoerg; Schmitt, Edgar; Bopp, TobiasNature Immunology (2018), 19 (12), 1319-1329CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a mol. mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-assocd. macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a mol. mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.
- 29Certo, M.; Tsai, C. H.; Pucino, V.; Ho, P. C.; Mauro, C. Lactate Modulation of Immune Responses in Inflammatory Versus Tumour Microenvironments. Nat. Rev. Immunol. 2021, 21, 151– 161, DOI: 10.1038/s41577-020-0406-2Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1KhtL3P&md5=33df655fbc49326d6d7a4a03008f5f32Lactate modulation of immune responses in inflammatory versus tumour microenvironmentsCerto, Michelangelo; Tsai, Chin-Hsien; Pucino, Valentina; Ho, Ping-Chih; Mauro, ClaudioNature Reviews Immunology (2021), 21 (3), 151-161CODEN: NRIABX; ISSN:1474-1733. (Nature Research)A review. Abstr.: The microenvironment in cancerous tissues is immunosuppressive and pro-tumorigenic, whereas the microenvironment of tissues affected by chronic inflammatory disease is pro-inflammatory and anti-resoln. Despite these opposing immunol. states, the metabolic states in the tissue microenvironments of cancer and inflammatory diseases are similar: both are hypoxic, show elevated levels of lactate and other metabolic byproducts and have low levels of nutrients. In this Review, we describe how the bioavailability of lactate differs in the microenvironments of tumors and inflammatory diseases compared with normal tissues, thus contributing to the establishment of specific immunol. states in disease. A clear understanding of the metabolic signature of tumors and inflammatory diseases will enable therapeutic intervention aimed at resetting the bioavailability of metabolites and correcting the dysregulated immunol. state, triggering beneficial cytotoxic, inflammatory responses in tumors and immunosuppressive responses in chronic inflammation.
- 30Macintyre, A. N.; Gerriets, V. A.; Nichols, A. G.; Michalek, R. D.; Rudolph, M. C.; Deoliveira, D.; Anderson, S. M.; Abel, E. D.; Chen, B. J.; Hale, L. P.; Rathmell, J. C. The Glucose Transporter Glut1 is Selectively Essential for CD4 T Cell Activation and Effector Function. Cell Metab. 2014, 20, 61– 72, DOI: 10.1016/j.cmet.2014.05.004Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpvFWmsbw%253D&md5=5be0f77c6e21a18df8d94d80bafa8fe6The Glucose Transporter Glut1 Is Selectively Essential for CD4 T Cell Activation and Effector FunctionMacintyre, Andrew N.; Gerriets, Valerie A.; Nichols, Amanda G.; Michalek, Ryan D.; Rudolph, Michael C.; Deoliveira, Divino; Anderson, Steven M.; Abel, E. Dale; Chen, Benny J.; Hale, Laura P.; Rathmell, Jeffrey C.Cell Metabolism (2014), 20 (1), 61-72CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)CD4 T cell activation leads to proliferation and differentiation into effector (Teff) or regulatory (Treg) cells that mediate or control immunity. While each subset prefers distinct glycolytic or oxidative metabolic programs in vitro, requirements and mechanisms that control T cell glucose uptake and metab. in vivo are uncertain. Despite expression of multiple glucose transporters, Glut1 deficiency selectively impaired metab. and function of thymocytes and Teff. Resting T cells were normal until activated, when Glut1 deficiency prevented increased glucose uptake and glycolysis, growth, proliferation, and decreased Teff survival and differentiation. Importantly, Glut1 deficiency decreased Teff expansion and the ability to induce inflammatory disease in vivo. Treg cells, in contrast, were enriched in vivo and appeared functionally unaffected and able to suppress Teff, irresp. of Glut1 expression. These data show a selective in vivo requirement for Glut1 in metabolic reprogramming of CD4 T cell activation and Teff expansion and survival.
- 31Balin, S. J.; Pellegrini, M.; Klechevsky, E.; Won, S. T.; Weiss, D. I.; Choi, A. W.; Hakimian, J.; Lu, J.; Ochoa, M. T.; Bloom, B. R.; Lanier, L. L.; Stenger, S.; Modlin, R. L. Human Antimicrobial Cytotoxic T Lymphocytes, Defined by NK Receptors and Antimicrobial Proteins, Kill Intracellular Bacteria. Sci. Immunol. 2018, 3, eaat7668 DOI: 10.1126/sciimmunol.aat7668
- 32Liu, Y.; Liang, G.; Xu, H.; Dong, W.; Dong, Z.; Qiu, Z.; Zhang, Z.; Li, F.; Huang, Y.; Li, Y.; Wu, J.; Yin, S.; Zhang, Y.; Guo, P.; Liu, J.; Xi, J. J.; Jiang, P.; Han, D.; Yang, C. G.; Xu, M. M. Tumors Exploit FTO-Mediated Regulation of Glycolytic Metabolism to Evade Immune Surveillance. Cell Metab. 2021, 33, 1221– 1233.e11, DOI: 10.1016/j.cmet.2021.04.001Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVShs77E&md5=74422d1b32aa1faa1a04f0fd3a25eaceTumors exploit FTO-mediated regulation of glycolytic metabolism to evade immune surveillanceLiu, Yi; Liang, Guanghao; Xu, Hongjiao; Dong, Wenxin; Dong, Ze; Qiu, Zhiwei; Zhang, Zihao; Li, Fangle; Huang, Yue; Li, Yilin; Wu, Jun; Yin, Shenyi; Zhang, Yawei; Guo, Peijin; Liu, Jun; Xi, Jianzhong Jeff; Jiang, Peng; Han, Dali; Yang, Cai-Guang; Xu, Meng MichelleCell Metabolism (2021), 33 (6), 1221-1233.e11CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)The ever-increasing understanding of the complexity of factors and regulatory layers that contribute to immune evasion facilitates the development of immunotherapies. However, the diversity of malignant tumors limits many known mechanisms in specific genetic and epigenetic contexts, manifesting the need to discover general driver genes. Here, we have identified the m6A demethylase FTO as an essential epitranscriptomic regulator utilized by tumors to escape immune surveillance through regulation of glycolytic metab. We show that FTO-mediated m6A demethylation in tumor cells elevates the transcription factors c-Jun, JunB, and C/EBPβ, which allows the rewiring of glycolytic metab. Fto knockdown impairs the glycolytic activity of tumor cells, which restores the function of CD8+ T cells, thereby inhibiting tumor growth. Furthermore, we developed a small-mol. compd., Dac51, that can inhibit the activity of FTO, block FTO-mediated immune evasion, and synergize with checkpoint blockade for better tumor control, suggesting reprogramming RNA epitranscriptome as a potential strategy for immunotherapy.
- 33Adema, G. J.; Hartgers, F.; Verstraten, R.; de Vries, E.; Marland, G.; Menon, S.; Foster, J.; Xu, Y.; Nooyen, P.; McClanahan, T.; Bacon, K. B.; Figdor, C. G. A Dendritic-Cell-Derived C-C Chemokine That Preferentially Attracts Naive T Cells. Nature 1997, 387, 713– 717, DOI: 10.1038/42716Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktVWms7g%253D&md5=8be13f000a50bf8f569af22bb0a5e47eA dendritic-cell-derived C-C chemokine that preferentially attracts naive T cellsAdema, Gosse J.; Hartgers, Franca; Verstraten, Riet; de Vries, Edwin; Marland, Gill; Menon, Satish; Foster, Jessica; Xu, Yuming; Nooyan, Pete; McClanahan, Terrill; Bacon, Kevin B.; Figdor, Carl G.Nature (London) (1997), 387 (6634), 713-717CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)Dendritic cells form a system of highly efficient antigen-presenting cells. After capturing antigen in the periphery, they migrate to lymphoid organs where they present the antigen to T cells. Their seemingly unique ability to interact with the sensitized native T cells gives dendritic cells a central role in the initiation of immune responses and allows them to be used in therapeutic strategics against cancer, viral infection and other diseases. How they interact preferentially with naive rather than activated T lymphocytes is still poorly understood. Chemokines direct the transport of white blood cells in immune surveillance. Here the authors report the identification and characterization of a C-C chemokine (DC-CK1) that is specifically expressed by human dendritic cells at high levels. Tissue distribution anal. demonstrates that dendritic cells present in germinal centers and T-cell areas of secondary lymphoid organs express this chemokine. The authors show that DC-CK1, in contrast to RANTES, MIP-1α and interleukin-8, preferentially attracts naive T cells (CD45RA+). The specific expression of DC-CK1 by dendritic cells at the site of initiation of an immune response, combined with its chemotactic activity for native T cells, suggests that CD-CK1 has an important rule in the induction of immune responses.
- 34Chaffer, C. L.; Weinberg, R. A. A Perspective on Cancer Cell Metastasis. Science 2011, 331, 1559– 1564, DOI: 10.1126/science.1203543Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjs1Cgtbo%253D&md5=a361a1380ba9a72d974189059ebd9210A Perspective on Cancer Cell MetastasisChaffer, Christine L.; Weinberg, Robert A.Science (Washington, DC, United States) (2011), 331 (6024), 1559-1564CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Metastasis causes most cancer deaths, yet this process remains one of the most enigmatic aspects of the disease. Building on new mechanistic insights emerging from recent research, we offer our perspective on the metastatic process and reflect on possible paths of future exploration. We suggest that metastasis can be portrayed as a 2-phase process: The first phase involves the phys. translocation of a cancer cell to a distant organ, whereas the second encompasses the ability of the cancer cell to develop into a metastatic lesion at that distant site. Although much remains to be learned about the second phase, we feel that an understanding of the first phase is now within sight, due in part to a better understanding of how cancer cell behavior can be modified by a cell-biol. program called the epithelial-to-mesenchymal transition.
- 35Reticker-Flynn, N. E.; Zhang, W.; Belk, J. A.; Basto, P. A.; Escalante, N. K.; Pilarowski, G. O. W.; Bejnood, A.; Martins, M. M.; Kenkel, J. A.; Linde, I. L.; Bagchi, S.; Yuan, R.; Chang, S.; Spitzer, M. H.; Carmi, Y.; Cheng, J.; Tolentino, L. L.; Choi, O.; Wu, N.; Kong, C. S.; Gentles, A. J.; Sunwoo, J. B.; Satpathy, A. T.; Plevritis, S. K.; Engleman, E. G. Lymph Node Colonization Induces Tumor-Immune Tolerance to Promote Distant Metastasis. Cell 2022, 185, e23 DOI: 10.1016/j.cell.2022.04.019
- 36Liang, C.; Diao, S.; Wang, C.; Gong, H.; Liu, T.; Hong, G.; Shi, X.; Dai, H.; Liu, Z. Tumor Metastasis Inhibition by Imaging-Guided Photothermal Therapy with Single-Walled Carbon Nanotubes. Adv. Mater. 2014, 26, 5646– 5652, DOI: 10.1002/adma.201401825Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXps1yksr4%253D&md5=c243b95026adbbe71afe1380807870b0Tumor Metastasis Inhibition by Imaging-guided Photothermal Therapy with Single-walled Carbon NanotubesLiang, Chao; Diao, Shuo; Wang, Chao; Gong, Hua; Liu, Teng; Hong, Guosong; Shi, Xiaoze; Dai, Hongjie; Liu, ZhuangAdvanced Materials (Weinheim, Germany) (2014), 26 (32), 5646-5652CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)Cancer metastasis treatment with imaging-guided PDT with single-walled carbon nanotube is described.
- 37Watson, M. J.; Vignali, P. D. A.; Mullett, S. J.; Overacre-Delgoffe, A. E.; Peralta, R. M.; Grebinoski, S.; Menk, A. V.; Rittenhouse, N. L.; DePeaux, K.; Whetstone, R. D.; Vignali, D. A. A.; Hand, T. W.; Poholek, A. C.; Morrison, B. M.; Rothstein, J. D.; Wendell, S. G.; Delgoffe, G. M. Metabolic Support of Tumour-Infiltrating Regulatory T Cells by Lactic Acid. Nature 2021, 591, 645– 651, DOI: 10.1038/s41586-020-03045-2Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktF2iurg%253D&md5=bfe6b0c3f320fee905be55903bd9b65fMetabolic support of tumor-infiltrating regulatory T cells by lactic acidWatson, McLane J.; Vignali, Paolo D. A.; Mullett, Steven J.; Overacre-Delgoffe, Abigail E.; Peralta, Ronal M.; Grebinoski, Stephanie; Menk, Ashley V.; Rittenhouse, Natalie L.; DePeaux, Kristin; Whetstone, Ryan D.; Vignali, Dario A. A.; Hand, Timothy W.; Poholek, Amanda C.; Morrison, Brett M.; Rothstein, Jeffrey D.; Wendell, Stacy G.; Delgoffe, Greg M.Nature (London, United Kingdom) (2021), 591 (7851), 645-651CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumor microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumor cells show deregulated metab., leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumor for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metab. of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metab. of the glycolytic byproduct lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumor growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumors avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.
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Abstract
Figure 1
Figure 1. Enzyme-mimetic approach and characterization. (a) Natural LOX catalysis model and (b) proton abstraction from lactate toward natural LOX based on carbanion formation mechanism (PDB code: 5EBU). (c) N-centered nanozyme catalysis model. (d) Crystal structures of Co2N, Co3N, and Co4N. (e) N 1s XPS of the Co4N/C NEs. (f) Co 2p (sat. stands for satellite peak) XPS of Co3O4/C and Co4N/C NEs. (g) TEM images of Co4N/C NEs (inset: TEM image of Co4N/C NEs under higher magnification). (h) HRTEM image of Co4N/C NE. (i) HADDF image and element mapping of Co4N/C NEs (scale bar = 100 nm). (j) XRD patterns of Co2N/C, Co3N/C, and Co4N/C NEs (bottom: standard pattern for Co2N: 72-1368, Co3N: 06-0691, and Co4N: 41-0943).
Figure 2
Figure 2. LOX-mimicking activities of Co4N/C NEs. (a) Lactate consumption of Co3O4/C, N/C, Co2N/C, Co3N/C, and Co4N/C. (b) Influence of KSCN (a Co complexing agent) during the oxidation of lactate in the presence of Co2N/C, Co3N/C, and Co4N/C NEs. (c) Activation energy (Ea) for the catalytic reaction of the LOX-mimicking activity of Co2N/C, Co3N/C, and Co4N/C. (d) Relative activity of lactate oxidation catalyzed by Co4N/C NEs and natural LOX (100 ng mL–1) with various pH values. (e) Lactate consumption curves at different concentrations of Co4N/C NEs. (f) Michaelis–Menten kinetic curves and (g) Lineweaver–Burk plot for the Co4N/C NEs (LOX-mimicking activity). (h) Lactate consumption effect of Co4N/C NEs under air or N2 conditions. The stability of (i) lactate consumption and pyruvate generation and (j) H2O2 generation in four cycles. (k) Selectivity of Co4N/C NEs toward some cellular reductive biomolecules under US. (l) Schematic illustration of the LOX-mimic Ping-Pong mechanism of Co4N/C NEs.
Figure 3
Figure 3. DFT calculations and LOX-mimicking catalysis mechanism. (a) Calculated deformation charge density of Co2N, Co3N, and Co4N. (b) Calculated Co 3d density of states (DOS) of the Co2N, Co3N, and Co4N models. (c) Proposed reaction pathway of the Co4N model based on carbanion formation. (d) Transient state (TS) and corresponding barriers during lactate oxidation. (e) Free energy of the catalytic oxidation of lactate toward pyruvate on the Co2N, Co3N, and Co4N models. The yellow, blue, red, purple, and gray balls in (c) and (d) represent the N, Co, H, O, and C atoms, separately.
Figure 4
Figure 4. Enzyme-like activity of Co4N/C NEs in vitro. Cell experiments were divided into different groups: (1) Control, (2) US, (3) Co4N/C NEs, and (4) Co4N/C NEs + US. (a) Cellular uptake at different times was measured through Rhodamine B staining. (b) Lactate concentrations and (c) pH values of the supernatants of the culture media of 4T1 cells (n = 3). (d) Intracellular pH was detected through BCECF-AM staining. (e) The intracellular hypoxia level was measured using [Ru(dpp)3]2+Cl2 as a probe. (f) Intracellular ROS was detected through DCFH-DA staining. (g) Overall evaluation of the properties of US, Co4N/C NEs, and Co4N/C NEs + US during the anti-tumor process in vitro: (i) lactate consumption, (ii) intracellular pH elevation, (iii) hypoxia alleviation, (iv) ROS elevation, and (v) extracellular pH elevation. (h) Ratio (n = 4) and (i) populations of M1 (CD86high/CD206low) and M2 (CD86low/CD206high) macrophages after incubation with lactate and analysis by flow cytometry. Quantification of (j) TNF-α and (k) IL-10 (n = 3). (l) Cell viability of normal cells (NIH-3T3) and tumor cells (4T1) after different treatments. (m) Illustration of the immune modulation mechanism of Co4N/C NEs. The scale bars are 25 μm. Data are expressed as mean values ± standard deviations (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 5
Figure 5. In vivo immune activation and transcriptomic analysis. The mice were divided into different groups: (1) Saline, (2) US, (3) Co4N/C NEs, and (4) Co4N/C NEs + US. (a) Treatment protocol for antitumor studies. (b) Lactate content in various groups. (c) Ratio (n = 4) and (d) Population of M1 (CD86high/CD206low) and M2 (CD86low/CD206high) macrophages (gating on F4/80) after different treatments. (e) Population of maturing dendritic cells (CD86high/CD80high) analyzed by flow cytometry (gating on CD11c). (f) INF-γ level in serum after different treatments (n = 3). (g) Tumor volume variation within 14 days (n = 5). (h) Volcano plot of up-regulated genes (red) and down-regulated genes (blue) after treatments. (i) GO enrichment and (j) KEGG enrichment analysis of pathways associated with immune activation. (k) Enrichment of immunotherapy-related gene signatures. Data are expressed as mean values ± standard deviations (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 6
Figure 6. Lymph node immune responses and lymphatic tumor therapies. The mice were placed in different groups: (1) Saline, (2) US, (3) Co4N/C NEs, and (4) Co4N/C NEs + US. (a) Treatment protocol for anti-lymphatic tumor studies. (b) Immunofluorescence staining of T cells in tumor slices with anti-CD3 antibody (red). (c) Weight of spleen extracted from mice. (d) H&E staining of lymph node metastasis tumors (scale bar = 50 μm). (e) Body weight of mice within 14 days. (f) Survival curves of different groups (n = 6). (g) Representative digital photos and (h) number of metastatic nodules in lungs after the treatments. Data are expressed as mean values ± standard deviations (n = 5, *p < 0.05).
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- 1Huang, Y.; Ren, J.; Qu, X. Nanozymes: Classification, Catalytic Mechanisms, Activity Regulation, and Applications. Chem. Rev. 2019, 119, 4357– 4412, DOI: 10.1021/acs.chemrev.8b006721https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjsFSrsbo%253D&md5=bc90e3da0b0848b1abac07ca7916188fNanozymes: Classification, catalytic mechanisms, activity regulation, and applicationsHuang, Yanyan; Ren, Jinsong; Qu, XiaogangChemical Reviews (Washington, DC, United States) (2019), 119 (6), 4357-4412CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Because of the high catalytic activities and substrate specificity, natural enzymes have been widely used in industrial, medical, and biol. fields, etc. Although promising, they often suffer from intrinsic shortcomings such as high cost, low operational stability, and difficulties of recycling. To overcome these shortcomings, researchers have been devoted to the exploration of artificial enzyme mimics for a long time. Since the discovery of ferromagnetic nanoparticles with intrinsic horseradish peroxidase-like activity in 2007, a large amt. of studies on nanozymes have been constantly emerging in the next decade. Nanozymes are one kind of nanomaterials with enzymic catalytic properties. Compared with natural enzymes, nanozymes have the advantages such as low cost, high stability and durability, which have been widely used in industrial, medical, and biol. fields. A thorough understanding of the possible catalytic mechanisms will contribute to the development of novel and high-efficient nanozymes, and the rational regulations of the activities of nanozymes are of great significance. In this review, we systematically introduce the classification, catalytic mechanism, activity regulation as well as recent research progress of nanozymes in the field of biosensing, environmental protection, and disease treatments, etc. in the past years. We also propose the current challenges of nanozymes as well as their future research focus. We anticipate this review may be of significance for the field to understand the properties of nanozymes and the development of novel nanomaterials with enzyme mimicking activities.
- 2Ji, S.; Jiang, B.; Hao, H.; Chen, Y.; Dong, J.; Mao, Y.; Zhang, Z.; Gao, R.; Chen, W.; Zhang, R.; Liang, Q.; Li, H.; Liu, S.; Wang, Y.; Zhang, Q.; Gu, L.; Duan, D.; Liang, M.; Wang, D.; Yan, X.; Li, Y. Matching the Kinetics of Natural Enzymes with a Single-Atom Iron Nanozyme. Nat. Catal. 2021, 4, 407– 417, DOI: 10.1038/s41929-021-00609-x2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVOlt73K&md5=9eb2a22f5f1ce2ad43451fa042c878beMatching the kinetics of natural enzymes with a single-atom iron nanozymeJi, Shufang; Jiang, Bing; Hao, Haigang; Chen, Yuanjun; Dong, Juncai; Mao, Yu; Zhang, Zedong; Gao, Rui; Chen, Wenxing; Zhang, Ruofei; Liang, Qian; Li, Haijing; Liu, Shuhu; Wang, Yu; Zhang, Qinghua; Gu, Lin; Duan, Demin; Liang, Minmin; Wang, Dingsheng; Yan, Xiyun; Li, YadongNature Catalysis (2021), 4 (5), 407-417CODEN: NCAACP; ISSN:2520-1158. (Nature Portfolio)Abstr.: Developing artificial enzymes with the excellent catalytic performance of natural enzymes has been a long-standing goal for chemists. Single-atom catalysts with well-defined at. structure and electronic coordination environments can effectively mimic natural enzymes. Here, we report an engineered FeN3P-centered single-atom nanozyme (FeN3P-SAzyme) that exhibits comparable peroxidase-like catalytic activity and kinetics to natural enzymes, by controlling the electronic structure of the single-atom iron active center through the precise coordination of phosphorus and nitrogen. In particular, the engineered FeN3P-SAzyme, with well-defined geometric and electronic structures, displays catalytic performance that is consistent with Michaelis-Menten kinetics. We rationalize the origin of the high enzyme-like activity using d. functional theory calcns. Finally, we demonstrate that the developed FeN3P-SAzyme with superior peroxidase-like activity can be used as an effective therapeutic strategy for inhibiting tumor cell growth in vitro and in vivo. Therefore, SAzymes show promising potential for developing artificial enzymes that have the catalytic kinetics of natural enzymes. [graphic not available: see fulltext].
- 3Yu, B.; Wang, W.; Sun, W.; Jiang, C.; Lu, L. Defect Engineering Enables Synergistic Action of Enzyme-Mimicking Active Centers for High-Efficiency Tumor Therapy. J. Am. Chem. Soc. 2021, 143, 8855– 8865, DOI: 10.1021/jacs.1c035103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1aktb7F&md5=410b44c69b5f19461424b297ad0f4ab3Defect Engineering Enables Synergistic Action of Enzyme-Mimicking Active Centers for High-Efficiency Tumor TherapyYu, Bin; Wang, Wei; Sun, Wenbo; Jiang, Chunhuan; Lu, LehuiJournal of the American Chemical Society (2021), 143 (23), 8855-8865CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Perusing redox nanozymes capable of disrupting cellular homeostasis offers new opportunities to develop cancer-specific therapy, but remains challenging, because most artificial enzymes lack enzyme-like scale and configuration. Herein, for the first time, the authors leverage a defect engineering strategy to develop a simple yet efficient redox nanozyme by constructing enzyme-mimicking active centers and studied its formation and catalysis mechanism thoroughly. Specifically, the partial Fe doping in MoOx (donated as Fe-MoOv) activate structure reconstruction with abundant defect site generation, including Fe substitution and oxygen vacancy (OV) defects, which significantly enable the binding capacity and catalytic activity of Fe-MoOv nanozymes in a synergetic fashion. More intriguingly, plenty of delocalized electrons appear due to Fe-facilitated band structure reconstruction, directly contributing to the remarkable surface plasmon resonance effect in the near-IR (NIR) region. Under NIR-II laser irradn., the designed Fe-MoOv nanozymes are able to induce substantial disruption of redox and metab. homeostasis in the tumor region via enzyme-mimicking cascade reactions, thus significantly augmenting therapeutic effects. This study that takes advantage of defect engineering offers new insights into developing high-efficiency redox nanozymes.
- 4Colegio, O. R.; Chu, N. Q.; Szabo, A. L.; Chu, T.; Rhebergen, A. M.; Jairam, V.; Cyrus, N.; Brokowski, C. E.; Eisenbarth, S. C.; Phillips, G. M.; Cline, G. W.; Phillips, A. J.; Medzhitov, R. Functional Polarization of Tumour-Associated Macrophages by Tumour-Derived Lactic Acid. Nature 2014, 513, 559– 563, DOI: 10.1038/nature134904https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Wjs7nO&md5=19de6df0a2734ab8037ade191a02ed60Functional polarization of tumor-associated macrophages by tumor-derived lactic acidColegio, Oscar R.; Chu, Ngoc-Quynh; Szabo, Alison L.; Chu, Thach; Rhebergen, Anne Marie; Jairam, Vikram; Cyrus, Nika; Brokowski, Carolyn E.; Eisenbarth, Stephanie C.; Phillips, Gillian M.; Cline, Gary W.; Phillips, Andrew J.; Medzhitov, RuslanNature (London, United Kingdom) (2014), 513 (7519), 559-563CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Macrophages have an important role in the maintenance of tissue homeostasis. To perform this function, macrophages must have the capacity to monitor the functional states of their 'client cells': namely, the parenchymal cells in the various tissues in which macrophages reside. Tumors exhibit many features of abnormally developed organs, including tissue architecture and cellular compn. Similarly to macrophages in normal tissues and organs, macrophages in tumors (tumor-assocd. macrophages) perform some key homeostatic functions that allow tumor maintenance and growth. However, the signals involved in communication between tumors and macrophages are poorly defined. Here we show that lactic acid produced by tumor cells, as a byproduct of aerobic or anaerobic glycolysis, has a crit. function in signaling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumor-assocd. macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumor growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumor cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumors to promote their growth.
- 5Gatenby, R. A.; Gillies, R. J. Why Do Cancers Have High Aerobic Glycolysis?. Nat. Rev. Cancer 2004, 4, 891– 899, DOI: 10.1038/nrc14785https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXptFegt7w%253D&md5=0584954722468b0bfaa5821fa17f2db5Why do cancers have high aerobic glycolysis?Gatenby, Robert A.; Gillies, Robert J.Nature Reviews Cancer (2004), 4 (11), 891-899CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. If carcinogenesis occurs by somatic evolution, then common components of the cancer phenotype result from active selection and must, therefore, confer a significant growth advantage. A near-universal property of primary and metastatic cancers is upregulation of glycolysis, resulting in increased glucose consumption, which can be obsd. with clin. tumor imaging. We propose that persistent metab. of glucose to lactate even in aerobic conditions is an adaptation to intermittent hypoxia in pre-malignant lesions. However, upregulation of glycolysis leads to microenvironmental acidosis requiring evolution to phenotypes resistant to acid-induced cell toxicity. Subsequent cell populations with upregulated glycolysis and acid resistance have a powerful growth advantage, which promotes unconstrained proliferation and invasion.
- 6Chen, J.; Zhu, Y.; Wu, C.; Shi, J. Engineering Lactate-Modulating Nanomedicines for Cancer Therapy. Chem. Soc. Rev. 2023, 52, 973– 1000, DOI: 10.1039/D2CS00479H6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjtFGjtw%253D%253D&md5=88669225ca43466a3842c5cdafb424c6Engineering lactate-modulating nanomedicines for cancer therapyChen, Jiajie; Zhu, Yufang; Wu, Chengtie; Shi, JianlinChemical Society Reviews (2023), 52 (3), 973-1000CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Lactate in tumors has long been considered "metabolic junk" derived from the glycolysis of cancer cells and utilized only as a biomarker of malignancy, but is presently believed to be a pivotal regulator of tumor development, maintenance and metastasis. Indeed, tumor lactate can be a "fuel" for energy supply and functions as a signaling mol., which actively contributes to tumor progression, angiogenesis, immunosuppression, therapeutic resistance, etc., thus providing promising opportunities for cancer treatment. However, the current approaches for regulating lactate homeostasis with available agents are still challenging, which is mainly due to the short half-life, low bioavailability and poor specificity of these agents and their unsatisfactory therapeutic outcomes. In recent years, lactate modulation nanomedicines have emerged as a charming and efficient strategy for fighting cancer, which play important roles in optimizing the delivery of lactate-modulating agents for more precise and effective modulation and treatment. Integrating specific lactate-modulating functions in diverse therapeutic nanomedicines may overcome the intrinsic restrictions of different therapeutic modalities by remodeling the pathol. microenvironment for achieving enhanced cancer therapy. In this review, the most recent advances in the engineering of functional nanomedicines that can modulate tumor lactate for cancer therapy are summarized and discussed, and the fundamental mechanisms by which lactate modulation benefits various therapeutics are elucidated. Finally, the challenges and perspectives of this emerging strategy in the anti-tumor field are highlighted.
- 7Liu, X. H.; Yu, H. Y.; Huang, J. Y.; Su, J. H.; Xue, C.; Zhou, X. T.; He, Y. R.; He, Q.; Xu, D. J.; Xiong, C.; Ji, H. B. Biomimetic Catalytic Aerobic Oxidation of C-sp(3)-H Bonds under Mild Conditions Using Galactose Oxidase Model Compound Cu(II)L. Chem. Sci. 2022, 13, 9560– 9568, DOI: 10.1039/D2SC02606F7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitVChurnL&md5=21634a94efb07971a68a15fa2c1301deBiomimetic catalytic aerobic oxidation of C-sp(3)-H bonds under mild conditions using galactose oxidase model compound CuIILLiu, Xiao-Hui; Yu, Hai-Yang; Huang, Jia-Ying; Su, Ji-Hu; Xue, Can; Zhou, Xian-Tai; He, Yao-Rong; He, Qian; Xu, De-Jing; Xiong, Chao; Ji, Hong-BingChemical Science (2022), 13 (33), 9560-9568CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Developed highly efficient catalytic protocols for C-sp(3)-H bond aerobic oxidn. under mild conditions was a long-desired goal of chemists. Inspired by nature, a biomimetic approach for the aerobic oxidn. of C-sp(3)-H by galactose oxidase model compd. CuIIL and NHPI (N-hydroxyphthalimide) were developed. The CuIIL-NHPI system exhibited excellent performance in the oxidn. of C-sp(3)-H bonds to ketones, esp. for light alkanes. The biomimetic catalytic protocol had a broad substrate scope. Mechanistic studies revealed that the CuI-radical intermediate species generated from the intramol. redox process of CuIILH2 was crit. for O2 activation. Kinetic expts. showed that the activation of NHPI were the rate-detg. step. Furthermore, activation of NHPI in the CuIIL-NHPI system were demonstrated by time-resolved EPR results. The persistent PINO (phthalimide-N-oxyl) radical mechanism for the aerobic oxidn. of C-sp(3)-H bond were demonstrated.
- 8Sugiyama, S.; Kikumoto, T.; Tanaka, H.; Nakagawa, K.; Sotowa, K.-I.; Maehara, K.; Himeno, Y.; Ninomiya, W. Enhancement of Catalytic Activity on Pd/C and Te–Pd/C During the Oxidative Dehydrogenation of Sodium Lactate to Pyruvate in an Aqueous Phase Under Pressurized Oxygen. Catal. Lett. 2009, 131, 129– 134, DOI: 10.1007/s10562-009-9920-38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjs1Kktrk%253D&md5=7801102ff9a117a70638e3e783433a19Enhancement of Catalytic Activity on Pd/C and Te-Pd/C During the Oxidative Dehydrogenation of Sodium Lactate to Pyruvate in an Aqueous Phase Under Pressurized OxygenSugiyama, Shigeru; Kikumoto, Tetsuo; Tanaka, Haruki; Nakagawa, Keizo; Sotowa, Ken-Ichiro; Maehara, Keiko; Himeno, Yoshiyuki; Ninomiya, WataruCatalysis Letters (2009), 131 (1-2), 129-134CODEN: CALEER; ISSN:1011-372X. (Springer)The oxidative dehydrogenation of sodium lactate to sodium pyruvate in an aq. phase proceeded favorably using a Pd/C catalyst and Pd/C with Te promoter at 358 K with no adjustment in soln. pH under pressurized oxygen, although previous reports had stated that this reaction would not proceed using Pd/C while Pd/C doped with either Pb, Bi or Te showed activity at atm. pressure, 363 K, and pH 8.
- 9Yorita, K.; Matsuoka, T.; Misaki, H.; Massey, V. Interaction of Two Arginine Residues in Lactate Oxidase with the Enzyme Flavin: Conversion of FMN to 8-Formyl-FMN. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13039– 13044, DOI: 10.1073/pnas.2504722979https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXosVKqtL4%253D&md5=f5742e38bc30610f195f136a93cdac0aInteraction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMNYorita, Kazuko; Matsuoka, Takeshi; Misaki, Hideo; Massey, VincentProceedings of the National Academy of Sciences of the United States of America (2000), 97 (24), 13039-13044CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Two Arg residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-contg. enzymes that catalyze the oxidn. of α-hydroxy acids. In lactate oxidase from Aerococcus viridans, these residues were changed to Lys in 2 single mutations and in a double mutant form. In addn., Arg-181 was replaced by Met to det. the effect of removing the pos. charge on the residue. The effects of these replacements on the kinetic and thermodn. properties were detd. With all mutant forms, there were only small effects on the reactivity of the reduced flavin with O2. On the other hand, the efficiency of redn. of the oxidized flavin by L-lactate was greatly reduced, particularly with the R268K mutant forms. The results demonstrated the importance of the 2 Arg residues in the binding of the substrate and its interaction with the flavin, and were consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by Lys also resulted in a slow conversion of the 8-Me-substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different phys. and chem. properties from those of the FMN-enzyme.
- 10Xiao, Y. P.; Chen, P. H.; Lei, S.; Bai, F.; Fu, L. H.; Lin, J.; Huang, P. Biocatalytic Depletion of Tumorigenic Energy Sources Driven by Cascade Reactions for Efficient Antitumor Therapy. Angew. Chem., Int. Ed. 2022, 61, e202204584 DOI: 10.1002/anie.20220458410https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xitl2nsbnM&md5=1154551ae5fed8a9bf7c7a9284406854Biocatalytic Depletion of Tumorigenic Energy Sources Driven by Cascade Reactions for Efficient Antitumor TherapyXiao, Ya-Ping; Chen, Peng-Hang; Lei, Shan; Bai, Fang; Fu, Lian-Hua; Lin, Jing; Huang, PengAngewandte Chemie, International Edition (2022), 61 (42), e202204584CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Glucose and lactate play important roles for tumor growth. How to simultaneously deprive tumors of glucose and lactate is a big challenge. We have developed a cascade catalytic system (denoted as FPGLC) based on fluorinated polymer (FP) with co-loading of glucose oxidase (GOx), lactate oxidase (LOx), and catalase (CAT). GOx and LOx deprive glucose and lactate, resp., resulting in abundant hydrogen peroxide (H2O2) generation. Meanwhile, CAT catalyzes H2O2 into O2, which not only promotes catalytic reactions of GOx and LOx for consuming more glucose and lactate, but also alleviates tumor hypoxia. Benefiting from the excellent cross-membrane and transmucosal penetration capacities of FP, FPGLC rapidly accumulated in tumors and subsequently mediated enhanced cascade catalytic therapy under the guidance of photoacoustic imaging. These results demonstrate that the dual depletion of glucose and lactate with O2 supply is a promising strategy for efficient antitumor starvation therapy.
- 11Falivene, L.; Cao, Z.; Petta, A.; Serra, L.; Poater, A.; Oliva, R.; Scarano, V.; Cavallo, L. Towards the Online Computer-Aided Design of Catalytic Pockets. Nat. Chem. 2019, 11, 872– 879, DOI: 10.1038/s41557-019-0319-511https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWktLrE&md5=071d5b625c560172fc6daa496afbf3f8Towards the online computer-aided design of catalytic pocketsFalivene, Laura; Cao, Zhen; Petta, Andrea; Serra, Luigi; Poater, Albert; Oliva, Romina; Scarano, Vittorio; Cavallo, LuigiNature Chemistry (2019), 11 (10), 872-879CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)A review. The engineering of catalysts with desirable properties can be accelerated by computer-aided design. To achieve this aim, features of mol. catalysts can be condensed into numerical descriptors that can then be used to correlate reactivity and structure. Based on such descriptors, we have introduced topog. steric maps that provide a three-dimensional image of the catalytic pocket-the region of the catalyst where the substrate binds and reacts-enabling it to be visualized and also reshaped by changing various parameters. These topog. steric maps, esp. when used in conjunction with d. functional theory calcns., enable catalyst structural modifications to be explored quickly, making the online design of new catalysts accessible to the wide chem. community. In this Perspective, we discuss the application of topog. steric maps either to rationalize the behavior of known catalysts-from synthetic mol. species to metalloenzymes-or to design improved catalysts.
- 12Stoisser, T.; Brunsteiner, M.; Wilson, D. K.; Nidetzky, B. Conformational Flexibility Related to Enzyme Activity: Evidence for a Dynamic Active-Site Gatekeeper Function of Tyr(215) in Aerococcus Viridans Lactate Oxidase. Sci. Rep. 2016, 6, 27892, DOI: 10.1038/srep2789212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVWnsrrP&md5=b4bdc3706c8379a6ea7cc59344304a41Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidaseStoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, BerndScientific Reports (2016), 6 (), 27892CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidn. of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent Tyr residue in the substrate-binding pocket (Tyr-215 in A. viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr-215, the effects of substitutions of the Tyr residue (Y215F, Y215H) were analyzed kinetically, crystallog., and by mol. dynamics simulations. Enzyme variants showed slowed flavin redn. and oxidn. by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate showed that the H-bond between the phenolic OH group and the keto O atom in pyruvate was replaced with a potentially stronger hydrophobic interaction between the Phe residue and the Me group of pyruvate. Residues 200 through 215 or 216 appeared to be disordered in 2 of the 8 monomers in the asym. unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr-215 can thus lead to a kinetic bottleneck in product release.
- 13Noll, N.; Krause, A. M.; Beuerle, F.; Wurthner, F. Enzyme-Like Water Preorganization in a Synthetic Molecular Cleft for Homogeneous Water Oxidation Catalysis. Nat. Catal. 2022, 5, 867– 877, DOI: 10.1038/s41929-022-00843-x13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFCgtbnN&md5=ade688154ea42c10fda4b852484f1cf3Enzyme-like water preorganization in a synthetic molecular cleft for homogeneous water oxidation catalysisNoll, Niklas; Krause, Ana-Maria; Beuerle, Florian; Wuerthner, FrankNature Catalysis (2022), 5 (10), 867-877CODEN: NCAACP; ISSN:2520-1158. (Nature Portfolio)Abstr.: Inspired by the proficiency of natural enzymes, mimicking of nanoenvironments for precise substrate preorganization is a promising strategy in catalyst design. However, artificial examples of enzyme-like activation of H2O mols. for the challenging oxidative water splitting reaction are hardly explored. Here, we introduce a mononuclear Ru(bda) complex (M1, bda = 2,2'-bipyridine-6,6'-dicarboxylate) equipped with a bipyridine-functionalized ligand to preorganize H2O mols. in front of the metal center as in enzymic clefts. The confined pocket of M1 accelerates chem. driven water oxidn. at pH 1 by facilitating a water nucleophilic attack pathway with a remarkable turnover frequency of 140 s-1 that is comparable to the oxygen-evolving complex of photosystem II. Single crystal X-ray anal. of M1 under catalytic conditions allowed the observation of a seventh H2O ligand directly coordinated to a RuIII center. Another H2O substrate is preorganized via a well-defined hydrogen-bonding network for the crucial O-O bond formation by nucleophilic attack. [graphic not available: see fulltext].
- 14Yorita, K.; Janko, K.; Aki, K.; Ghisla, S.; Palfey, B. A.; Massey, V. On the Reaction Mechanism of L-lactate Oxidase: Quantitative Structure-Activity Analysis of the Reaction with Para-Substituted L-Mandelates. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 9590– 9595, DOI: 10.1073/pnas.94.18.959014https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXlvVSqtLk%253D&md5=e3b8ccd251336c7d24bf943861f7119bOn the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelatesYorita, Kazuko; Janko, Karl; Aki, Kenji; Ghisla, Sandro; Palfey, Bruce A.; Massey, VincentProceedings of the National Academy of Sciences of the United States of America (1997), 94 (18), 9590-9595CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The rate consts. for redn. of the flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H=3-7) on the rate consts. of flavin redn. were found, indicating that flavin redn. is a direct measure of α-C-H bond breakage. The rate consts. show only a small influence of the electronic characteristic of the substituents, but show a good correlation when combined with some substituent vol. parameters. A surprisingly good correlation is found with the mol. mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.
- 15Furuichi, M.; Suzuki, N.; Dhakshnamoorhty, B.; Minagawa, H.; Yamagishi, R.; Watanabe, Y.; Goto, Y.; Kaneko, H.; Yoshida, Y.; Yagi, H.; Waga, I.; Kumar, P. K.; Mizuno, H. X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with D-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation Mechanism. J. Mol. Biol. 2008, 378, 436– 446, DOI: 10.1016/j.jmb.2008.02.06215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlagtb4%253D&md5=7a686a4e8be9cce117861a5bebbde457X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with D-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation MechanismFuruichi, Makio; Suzuki, Nobuhiro; Dhakshnamoorhty, Balasundaresan; Minagawa, Hirotaka; Yamagishi, Ryosuke; Watanabe, Yuta; Goto, Yukari; Kaneko, Hiroki; Yoshida, Yoshihito; Yagi, Hirotaka; Waga, Iwao; Kumar, Penmetcha K. R.; Mizuno, HiroshiJournal of Molecular Biology (2008), 378 (2), 436-446CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Lactate oxidase (LOX) belongs to a family of FMN-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with D-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the D-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the active site, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystn. at pH 4.5, because the same flip is obsd. in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstrs. a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network; between His265-Asp174-Lys221 and, furthermore, brings mol. oxygen in between D-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O2 could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing mol. oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chem. steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.
- 16Tabacchi, G.; Zucchini, D.; Caprini, G.; Gamba, A.; Lederer, F.; Vanoni, M. A.; Fois, E. L-lactate Dehydrogenation in Flavocytochrome b2: a First Principles Molecular Dynamics Study. FEBS J. 2009, 276, 2368– 2380, DOI: 10.1111/j.1742-4658.2009.06969.x16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVKnt78%253D&md5=30385feb5fecfe1fae13f2bf50d30aeeL-Lactate dehydrogenation in flavocytochrome b2: A first principles molecular dynamics studyTabacchi, Gloria; Zucchini, Daniela; Caprini, Gianluca; Gamba, Aldo; Lederer, Florence; Vanoni, Maria A.; Fois, EttoreFEBS Journal (2009), 276 (8), 2368-2380CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)First principles mol. dynamics (MD) studies on active-site models of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidn. reaction, a still-debated issue. In the calcd. enzyme-substrate model complex, the L-lactate α-OH hydrogen is hydrogen bonded to the active-site base H373 Nε, whereas the Hα is directed towards flavin N5, suggesting that the reaction is initiated by α-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidn. led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to FMN, without intermediates, but with α-OH proton abstraction preceding Hα transfer and a calcd. free energy barrier (12.1 kcal/mol-1) consistent with that detd. exptl. (13.5 kcal/mol-1). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a no. of flavoenzymes. Namely, they highlighted the role of: (a) the FMN-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin redn.; (b) an active site water mol. belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction.
- 17Feng, X.; Song, Y.; Chen, J. S.; Xu, Z.; Dunn, S. J.; Lin, W. Rational Construction of an Artificial Binuclear Copper Monooxygenase in a Metal-Organic Framework. J. Am. Chem. Soc. 2021, 143, 1107– 1118, DOI: 10.1021/jacs.0c1192017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlt1CgsQ%253D%253D&md5=aaf8240e547a92c664b50c6a2b2cc8c4Rational Construction of an Artificial Binuclear Copper Monooxygenase in a Metal-Organic FrameworkFeng, Xuanyu; Song, Yang; Chen, Justin S.; Xu, Ziwan; Dunn, Soren J.; Lin, WenbinJournal of the American Chemical Society (2021), 143 (2), 1107-1118CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Artificial enzymic systems are extensively studied to mimic the structures and functions of their natural counterparts. However, there remains a significant gap between structural modeling and catalytic activity in these artificial systems. Herein we report a novel strategy for the construction of an artificial binuclear copper monooxygenase starting from a Ti metal-org. framework (MOF). The deprotonation of the hydroxide groups on the secondary building units (SBUs) of MIL-125(Ti) (MIL = Mat´eriaux de l'Institut Lavoisier) allows for the metalation of the SBUs with closely spaced CuI pairs, which are oxidized by mol. O2 to afford the CuII2(μ2-OH)2 cofactor in the MOF-based artificial binuclear monooxygenase Ti8-Cu2. An artificial mononuclear Cu monooxygenase Ti8-Cu1 was also prepd. for comparison. The MOF-based monooxygenases were characterized by a combination of thermogravimetric anal., inductively coupled plasma-mass spectrometry, X-ray absorption spectroscopy, Fourier-transform IR spectroscopy, and UV-vis spectroscopy. In the presence of coreductants, Ti8-Cu2 exhibited outstanding catalytic activity toward a wide range of monooxygenation processes, including epoxidn., hydroxylation, Baeyer-Villiger oxidn., and sulfoxidn., with turnover nos. of up to 3450. Ti8-Cu2 showed a turnover frequency at least 17 times higher than that of Ti8-Cu1. D. functional theory calcns. revealed O2 activation as the rate-limiting step in the monooxygenation processes. Computational studies further showed that the Cu2 sites in Ti8-Cu2 cooperatively stabilized the Cu-O2 adduct for O-O bond cleavage with 6.6 kcal/mol smaller free energy increase than that of the mononuclear Cu sites in Ti8-Cu1, accounting for the significantly higher catalytic activity of Ti8-Cu2 over Ti8-Cu1.
- 18Zhang, S.; Li, Y.; Sun, S.; Liu, L.; Mu, X.; Liu, S.; Jiao, M.; Chen, X.; Chen, K.; Ma, H.; Li, T.; Liu, X.; Wang, H.; Zhang, J.; Yang, J.; Zhang, X. D. Single-Atom Nanozymes Catalytically Surpassing Naturally Occurring Enzymes as Sustained Stitching for Brain Trauma. Nat. Commun. 2022, 13, 4744, DOI: 10.1038/s41467-022-32411-z18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitFarurrM&md5=1d16487dc70034d2091853ab06b6e67bSingle-atom nanozymes catalytically surpassing naturally occurring enzymes as sustained stitching for brain traumaZhang, Shaofang; Li, Yonghui; Sun, Si; Liu, Ling; Mu, Xiaoyu; Liu, Shuhu; Jiao, Menglu; Chen, Xinzhu; Chen, Ke; Ma, Huizhen; Li, Tuo; Liu, Xiaoyu; Wang, Hao; Zhang, Jianning; Yang, Jiang; Zhang, Xiao-DongNature Communications (2022), 13 (1), 4744CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Regenerable nanozymes with high catalytic stability and sustainability are promising substitutes for naturally-occurring enzymes but are limited by insufficient and non-selective catalytic activities. Herein, we developed single-atom nanozymes of RhN4, VN4, and Fe-Cu-N6 with catalytic activities surpassing natural enzymes. Notably, Rh/VN4 preferably forms an Rh/V-O-N4 active center to decrease reaction energy barriers and mediates a "two-sided oxygen-linked" reaction path, showing 4 and 5-fold higher affinities in peroxidase-like activity than the FeN4 and natural horseradish peroxidase. Furthermore, RhN4 presents a 20-fold improved affinity in the catalase-like activity compared to the natural catalase; Fe-Cu-N6 displays selectivity towards the superoxide dismutase-like activity; VN4 favors a 7-fold higher glutathione peroxidase-like activity than the natural glutathione peroxidase. Bioactive sutures with Rh/VN4 show recyclable catalytic features without apparent decay in 1 mo and accelerate the scalp healing from brain trauma by promoting the vascular endothelial growth factor, regulating the immune cells like macrophages, and diminishing inflammation.
- 19Fan, K.; Xi, J.; Fan, L.; Wang, P.; Zhu, C.; Tang, Y.; Xu, X.; Liang, M.; Jiang, B.; Yan, X.; Gao, L. In Vivo Guiding Nitrogen-Doped Carbon Nanozyme for Tumor Catalytic Therapy. Nat. Commun. 2018, 9, 1440, DOI: 10.1038/s41467-018-03903-819https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mjit1Khtg%253D%253D&md5=c50850cc01e120026c745b573ebe9915In vivo guiding nitrogen-doped carbon nanozyme for tumor catalytic therapyFan Kelong; Wang Peixia; Liang Minmin; Jiang Bing; Yan Xiyun; Xi Juqun; Zhu Chunhua; Tang Yan; Gao Lizeng; Fan Lei; Xu Xiangdong; Wang Peixia; Jiang Bing; Yan XiyunNature communications (2018), 9 (1), 1440 ISSN:.Nanomaterials with intrinsic enzyme-like activities (nanozymes), have been widely used as artificial enzymes in biomedicine. However, how to control their in vivo performance in a target cell is still challenging. Here we report a strategy to coordinate nanozymes to target tumor cells and selectively perform their activity to destruct tumors. We develop a nanozyme using nitrogen-doped porous carbon nanospheres which possess four enzyme-like activities (oxidase, peroxidase, catalase and superoxide dismutase) responsible for reactive oxygen species regulation. We then introduce ferritin to guide nitrogen-doped porous carbon nanospheres into lysosomes and boost reactive oxygen species generation in a tumor-specific manner, resulting in significant tumor regression in human tumor xenograft mice models. Together, our study provides evidence that nitrogen-doped porous carbon nanospheres are powerful nanozymes capable of regulating intracellular reactive oxygen species, and ferritinylation is a promising strategy to render nanozymes to target tumor cells for in vivo tumor catalytic therapy.
- 20Matthews, A.; Saleem-Batcha, R.; Sanders, J. N.; Stull, F.; Houk, K. N.; Teufel, R. Aminoperoxide Adducts Expand the Catalytic Repertoire of Flavin Monooxygenases. Nat. Chem. Biol. 2020, 16, 556– 563, DOI: 10.1038/s41589-020-0476-220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjtlanu7c%253D&md5=de80cfc4822f4e5ea788a87aa572e66cAminoperoxide adducts expand the catalytic repertoire of flavin monooxygenasesMatthews, Arne; Saleem-Batcha, Raspudin; Sanders, Jacob N.; Stull, Frederick; Houk, K. N.; Teufel, RobinNature Chemical Biology (2020), 16 (5), 556-563CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Abstr.: One of the hallmark reactions catalyzed by flavin-dependent enzymes is the incorporation of an oxygen atom derived from dioxygen into org. substrates. For many decades, these flavin monooxygenases were assumed to use exclusively the flavin-C4a-(hydro)peroxide as their oxygen-transferring intermediate. We demonstrate that flavoenzymes may instead employ a flavin-N5-peroxide as a soft α-nucleophile for catalysis, which enables chem. not accessible to canonical monooxygenases. This includes, for example, the redox-neutral cleavage of carbon-hetero bonds or the dehalogenation of inert environmental pollutants via atypical oxygenations. We furthermore identify a shared structural motif for dioxygen activation and N5-functionalization, suggesting a conserved pathway that may be operative in numerous characterized and uncharacterized flavoenzymes from diverse organisms. Our findings show that overlooked flavin-N5-oxygen adducts are more widespread and may facilitate versatile chem., thus upending the notion that flavin monooxygenases exclusively function as nature's equiv. to org. peroxides in synthetic chem.
- 21Sheng, J.; Wang, L.; Deng, L.; Zhang, M.; He, H.; Zeng, K.; Tang, F.; Liu, Y.-N. MOF-Templated Fabrication of Hollow Co4N@N-Doped Carbon Porous Nanocages with Superior Catalytic Activity. ACS Appl. Mater. Interfaces 2018, 10, 7191– 7200, DOI: 10.1021/acsami.8b0057321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVKku74%253D&md5=05bd112c36f451a274b597795f22c8f4MOF-Templated Fabrication of Hollow Co4N@N-Doped Carbon Porous Nanocages with Superior Catalytic ActivitySheng, Jianping; Wang, Liqiang; Deng, Liu; Zhang, Min; He, Haichuan; Zeng, Ke; Tang, Feiying; Liu, You-NianACS Applied Materials & Interfaces (2018), 10 (8), 7191-7200CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Metallic Co4N catalysts have been considered as one of the most promising non-noble materials for heterogeneous catalysis because of their high elec. cond., great magnetic property, and high intrinsic activity. However, the metastable properties seriously limit their applications for heterogeneous water phase catalysis. In this work, a novel Co-metal-org. framework (MOF)-derived hollow porous nanocages (PNCs) composed of metallic Co4N and N-doped carbon (NC) were synthesized for the first time. This hollow three-dimensional (3D) PNC catalyst was synthesized by taking advantage of Co-MOF as a precursor for fabricating 3D hollow Co3O4@C PNCs, along with the NH3 treatment of Co-oxide frames to promote the in situ conversion of Co-MOF to Co4N@NC PNCs, benefiting from the high intrinsic activity and electron cond. of the metallic Co4N phase and the good permeability of the hollow porous nanostructure as well as the efficient doping of N into the carbon layer. Besides, the covalent bridge between the active Co4N surface and PNC shells also provides facile pathways for electron and mass transport. The obtained Co4N@NC PNCs exhibit excellent catalytic activity and stability for 4-nitrophenol redn. in terms of low activation energy (Ea = 23.53 kJ mol-1), high turnover frequency (52.01 × 1020 mol. g-1 min-1), and high apparent rate const. (kapp = 2.106 min-1). Furthermore, its magnetic property and stable configuration account for the excellent recyclability of the catalyst. It is hoped that our finding could pave the way for the construction of other hollow transition metal-based nitride@NC PNC catalysts for wide applications.
- 22Yang, Y.; Zeng, R.; Xiong, Y.; DiSalvo, F. J.; Abruna, H. D. Cobalt-Based Nitride-Core Oxide-Shell Oxygen Reduction Electrocatalysts. J. Am. Chem. Soc. 2019, 141, 19241– 19245, DOI: 10.1021/jacs.9b1080922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitF2nurvI&md5=83e9f48a566bee508eecc87e7eae0f2eCobalt-Based Nitride-Core Oxide-Shell Oxygen Reduction ElectrocatalystsYang, Yao; Zeng, Rui; Xiong, Yin; DiSalvo, Francis J.; Abruna, Hector D.Journal of the American Chemical Society (2019), 141 (49), 19241-19245CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Developing high-performance, low-cost, and conductive nonprecious electrocatalysts for the O redn. reaction (ORR) was a key challenge for advancing fuel cell technologies. Here, the authors report on a novel family of Co nitrides (CoxN/C, x = 2, 3, 4) as ORR electrocatalysts in alk. fuel cells. Co4N/C exhibited the highest ORR activity among the three types of Co nitrides studied, with a half-wave potential (E1/2) of 0.875 V vs. RHE in 1 M KOH, rivaling that of com. Pt/C (0.89 V). Also, Co4N/C showed an 8-fold improvement in mass activity at 0.85 V, when compared to Co oxide, Co3O4/C, and a negligible degrdn. (ΔE1/2 = 14 mV) after 10,000 potential cycles. The superior performance was ascribed to the formation of a conductive nitride core surrounded by a naturally formed thin oxide shell (∼2 nm). The conductive nitride core effectively mitigated the low cond. of the metal oxide, and the thin oxide shell on the surface provided the active sites for the ORR. Strategies developed herein represent a promising approach for the design of other novel metal nitrides as electrocatalysts for fuel cells.
- 23Wang, L.; Zhang, W.; Zheng, X.; Chen, Y.; Wu, W.; Qiu, J.; Zhao, X.; Zhao, X.; Dai, Y.; Zeng, J. Incorporating Nitrogen Atoms into Cobalt Nanosheets as a Strategy to Boost Catalytic Activity toward CO2 Hydrogenation. Nat. Energy 2017, 2, 869– 876, DOI: 10.1038/s41560-017-0015-x23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVehu74%253D&md5=ca887835cd0d140245e8726037aae2aeIncorporating nitrogen atoms into cobalt nanosheets as a strategy to boost catalytic activity toward CO2 hydrogenationWang, Liangbing; Zhang, Wenbo; Zheng, Xusheng; Chen, Yizhen; Wu, Wenlong; Qiu, Jianxiang; Zhao, Xiangchen; Zhao, Xiao; Dai, Yizhou; Zeng, JieNature Energy (2017), 2 (11), 869-876CODEN: NEANFD; ISSN:2058-7546. (Nature Research)Hydrogenation of CO2 into fuels and useful chems. could help to reduce reliance on fossil fuels. Although great progress has been made over the past decades to improve the activity of catalysts for CO2 hydrogenation, more efficient catalysts, esp. those based on non-noble metals, are desired. Here we incorporate N atoms into Co nanosheets to boost the catalytic activity toward CO2 hydrogenation. For the hydrogenation of CO2, Co4N nanosheets exhibited a turnover frequency of 25.6 h-1 in a slurry reactor under 32 bar pressure at 150 °C, which was 64 times that of Co nanosheets. The activation energy for Co4N nanosheets was 43.3 kJ mol-1, less than half of that for Co nanosheets. Mechanistic studies revealed that Co4N nanosheets were reconstructed into Co4NHx, wherein the amido-hydrogen atoms directly interacted with the CO2 to form HCOO* intermediates. In addn., the adsorbed H2O* activated amido-hydrogen atoms via the interaction of hydrogen bonds.
- 24Zhang, Y.; Ouyang, B.; Xu, J.; Jia, G.; Chen, S.; Rawat, R. S.; Fan, H. J. Rapid Synthesis of Cobalt Nitride Nanowires: Highly Efficient and Low-Cost Catalysts for Oxygen Evolution. Angew. Chem., Int. Ed. 2016, 55, 8670– 8674, DOI: 10.1002/anie.20160437224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptVyrtrs%253D&md5=0ed485fdb2c5a8a291fc2aef670672e7Rapid Synthesis of Cobalt Nitride Nanowires: Highly Efficient and Low-Cost Catalysts for Oxygen EvolutionZhang, Yongqi; Ouyang, Bo; Xu, Jing; Jia, Guichong; Chen, Shi; Rawat, Rajdeep Singh; Fan, Hong JinAngewandte Chemie, International Edition (2016), 55 (30), 8670-8674CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Electrochem. splitting of water to produce hydrogen and oxygen is an important process for many energy storage and conversion devices. Developing efficient, durable, low-cost, and earth-abundant electrocatalysts for the oxygen evolution reaction (OER) is of great urgency. To achieve the rapid synthesis of transition-metal nitride nanostructures and improve their electrocatalytic performance, a new strategy has been developed to convert cobalt oxide precursors into cobalt nitride nanowires through N2 radio frequency plasma treatment. This method requires significantly shorter reaction times (about 1 min) at room temp. compared to conventional high-temp. NH3 annealing which requires a few hours. The plasma treatment significantly enhances the OER activity, as evidenced by a low overpotential of 290 mV to reach a c.d. of 10 mA cm-2, a small Tafel slope, and long-term durability in an alk. electrolyte.
- 25Chen, J.; Ma, Q.; Zheng, X.; Fang, Y.; Wang, J.; Dong, S. Kinetically Restrained Oxygen Reduction to Hydrogen Peroxide with Nearly 100% Selectivity. Nat. Commun. 2022, 13, 2808, DOI: 10.1038/s41467-022-30411-725https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVaqurnE&md5=7a4ed9c319e9ef813fb27a13419d0950Kinetically restrained oxygen reduction to hydrogen peroxide with nearly 100% selectivityChen, Jinxing; Ma, Qian; Zheng, Xiliang; Fang, Youxing; Wang, Jin; Dong, ShaojunNature Communications (2022), 13 (1), 2808CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Hydrogen peroxide has been synthesized mainly through the electrocatalytic and photocatalytic oxygen redn. reaction in recent years. Herein, we synthesize a single-atom rhodium catalyst (Rh1/NC) to mimic the properties of flavoenzymes for the synthesis of hydrogen peroxide under mild conditions. Rh1/NC dehydrogenates various substrates and catalyzes the redn. of oxygen to hydrogen peroxide. The max. hydrogen peroxide prodn. rate is 0.48 mol gcatalyst-1 h-1 in the phosphorus acid aerobic oxidn. reaction. We find that the selectivity of oxygen redn. to hydrogen peroxide can reach 100%. This is because a single catalytic site of Rh1/NC can only catalyze the removal of two electrons per substrate mol.; thus, the subsequent oxygen can only obtain two electrons to reduce to hydrogen peroxide through the typical two-electron pathway. Similarly, due to the restriction of substrate dehydrogenation, the hydrogen peroxide selectivity in com. Pt/C-catalyzed enzymic reactions can be found to reach 75%, which is 30 times higher than that in electrocatalytic oxygen redn. reactions.
- 26Hui, S.; Ghergurovich, J. M.; Morscher, R. J.; Jang, C.; Teng, X.; Lu, W.; Esparza, L. A.; Reya, T.; Le, Z.; Yanxiang Guo, J.; White, E.; Rabinowitz, J. D. Glucose Feeds the TCA Cycle via Circulating Lactate. Nature 2017, 551, 115– 118, DOI: 10.1038/nature2405726https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M7itVeitA%253D%253D&md5=52d11bb82116a571b4bf551ecd2c4690Glucose feeds the TCA cycle via circulating lactateHui Sheng; Ghergurovich Jonathan M; Morscher Raphael J; Jang Cholsoon; Teng Xin; Lu Wenyun; Rabinowitz Joshua D; Hui Sheng; Morscher Raphael J; Jang Cholsoon; Teng Xin; Lu Wenyun; Rabinowitz Joshua D; Ghergurovich Jonathan M; Esparza Lourdes A; Reya Tannishtha; Le Zhan; Yanxiang Guo Jessie; White Eileen; Rabinowitz Joshua D; Le Zhan; White Eileen; Yanxiang Guo Jessie; Yanxiang Guo JessieNature (2017), 551 (7678), 115-118 ISSN:.Mammalian tissues are fuelled by circulating nutrients, including glucose, amino acids, and various intermediary metabolites. Under aerobic conditions, glucose is generally assumed to be burned fully by tissues via the tricarboxylic acid cycle (TCA cycle) to carbon dioxide. Alternatively, glucose can be catabolized anaerobically via glycolysis to lactate, which is itself also a potential nutrient for tissues and tumours. The quantitative relevance of circulating lactate or other metabolic intermediates as fuels remains unclear. Here we systematically examine the fluxes of circulating metabolites in mice, and find that lactate can be a primary source of carbon for the TCA cycle and thus of energy. Intravenous infusions of (13)C-labelled nutrients reveal that, on a molar basis, the circulatory turnover flux of lactate is the highest of all metabolites and exceeds that of glucose by 1.1-fold in fed mice and 2.5-fold in fasting mice; lactate is made primarily from glucose but also from other sources. In both fed and fasted mice, (13)C-lactate extensively labels TCA cycle intermediates in all tissues. Quantitative analysis reveals that during the fasted state, the contribution of glucose to tissue TCA metabolism is primarily indirect (via circulating lactate) in all tissues except the brain. In genetically engineered lung and pancreatic cancer tumours in fasted mice, the contribution of circulating lactate to TCA cycle intermediates exceeds that of glucose, with glutamine making a larger contribution than lactate in pancreatic cancer. Thus, glycolysis and the TCA cycle are uncoupled at the level of lactate, which is a primary circulating TCA substrate in most tissues and tumours.
- 27Sekine, H.; Yamamoto, M.; Motohashi, H. Tumors Sweeten Macrophages with Acids. Nat. Immunol. 2018, 19, 1281– 1283, DOI: 10.1038/s41590-018-0258-027https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2qtbfE&md5=6ad30efef43b5e3963a737bbbb0839c8Tumors sweeten macrophages with acidsSekine, Hiroki; Yamamoto, Masayuki; Motohashi, HozumiNature Immunology (2018), 19 (12), 1281-1283CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)Acidic microenvironments induced by highly glycolytic tumor cells promote the noninflammatory polarization of tumor-assocd. macrophages, which leads to immunoevasion.
- 28Bohn, T.; Rapp, S.; Luther, N.; Klein, M.; Bruehl, T. J.; Kojima, N.; Aranda Lopez, P.; Hahlbrock, J.; Muth, S.; Endo, S.; Pektor, S.; Brand, A.; Renner, K.; Popp, V.; Gerlach, K.; Vogel, D.; Lueckel, C.; Arnold-Schild, D.; Pouyssegur, J.; Kreutz, M.; Huber, M.; Koenig, J.; Weigmann, B.; Probst, H. C.; von Stebut, E.; Becker, C.; Schild, H.; Schmitt, E.; Bopp, T. Tumor Immunoevasion via Acidosis-Dependent Induction of Regulatory Tumor-Associated Macrophages. Nat. Immunol. 2018, 19, 1319– 1329, DOI: 10.1038/s41590-018-0226-828https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2qur7M&md5=753dc4e3bb15b5d6f38c2825f4d78cd1Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophagesBohn, Toszka; Rapp, Steffen; Luther, Natascha; Klein, Matthias; Bruehl, Till-Julius; Kojima, Nobuhiko; Aranda Lopez, Pamela; Hahlbrock, Jennifer; Muth, Sabine; Endo, Shogo; Pektor, Stefanie; Brand, Almut; Renner, Kathrin; Popp, Vanessa; Gerlach, Katharina; Vogel, Dennis; Lueckel, Christina; Arnold-Schild, Danielle; Pouyssegur, Jacques; Kreutz, Marina; Huber, Magdalena; Koenig, Jochem; Weigmann, Benno; Probst, Hans-Christian; von Stebut, Esther; Becker, Christian; Schild, Hansjoerg; Schmitt, Edgar; Bopp, TobiasNature Immunology (2018), 19 (12), 1319-1329CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a mol. mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-assocd. macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a mol. mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.
- 29Certo, M.; Tsai, C. H.; Pucino, V.; Ho, P. C.; Mauro, C. Lactate Modulation of Immune Responses in Inflammatory Versus Tumour Microenvironments. Nat. Rev. Immunol. 2021, 21, 151– 161, DOI: 10.1038/s41577-020-0406-229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1KhtL3P&md5=33df655fbc49326d6d7a4a03008f5f32Lactate modulation of immune responses in inflammatory versus tumour microenvironmentsCerto, Michelangelo; Tsai, Chin-Hsien; Pucino, Valentina; Ho, Ping-Chih; Mauro, ClaudioNature Reviews Immunology (2021), 21 (3), 151-161CODEN: NRIABX; ISSN:1474-1733. (Nature Research)A review. Abstr.: The microenvironment in cancerous tissues is immunosuppressive and pro-tumorigenic, whereas the microenvironment of tissues affected by chronic inflammatory disease is pro-inflammatory and anti-resoln. Despite these opposing immunol. states, the metabolic states in the tissue microenvironments of cancer and inflammatory diseases are similar: both are hypoxic, show elevated levels of lactate and other metabolic byproducts and have low levels of nutrients. In this Review, we describe how the bioavailability of lactate differs in the microenvironments of tumors and inflammatory diseases compared with normal tissues, thus contributing to the establishment of specific immunol. states in disease. A clear understanding of the metabolic signature of tumors and inflammatory diseases will enable therapeutic intervention aimed at resetting the bioavailability of metabolites and correcting the dysregulated immunol. state, triggering beneficial cytotoxic, inflammatory responses in tumors and immunosuppressive responses in chronic inflammation.
- 30Macintyre, A. N.; Gerriets, V. A.; Nichols, A. G.; Michalek, R. D.; Rudolph, M. C.; Deoliveira, D.; Anderson, S. M.; Abel, E. D.; Chen, B. J.; Hale, L. P.; Rathmell, J. C. The Glucose Transporter Glut1 is Selectively Essential for CD4 T Cell Activation and Effector Function. Cell Metab. 2014, 20, 61– 72, DOI: 10.1016/j.cmet.2014.05.00430https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpvFWmsbw%253D&md5=5be0f77c6e21a18df8d94d80bafa8fe6The Glucose Transporter Glut1 Is Selectively Essential for CD4 T Cell Activation and Effector FunctionMacintyre, Andrew N.; Gerriets, Valerie A.; Nichols, Amanda G.; Michalek, Ryan D.; Rudolph, Michael C.; Deoliveira, Divino; Anderson, Steven M.; Abel, E. Dale; Chen, Benny J.; Hale, Laura P.; Rathmell, Jeffrey C.Cell Metabolism (2014), 20 (1), 61-72CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)CD4 T cell activation leads to proliferation and differentiation into effector (Teff) or regulatory (Treg) cells that mediate or control immunity. While each subset prefers distinct glycolytic or oxidative metabolic programs in vitro, requirements and mechanisms that control T cell glucose uptake and metab. in vivo are uncertain. Despite expression of multiple glucose transporters, Glut1 deficiency selectively impaired metab. and function of thymocytes and Teff. Resting T cells were normal until activated, when Glut1 deficiency prevented increased glucose uptake and glycolysis, growth, proliferation, and decreased Teff survival and differentiation. Importantly, Glut1 deficiency decreased Teff expansion and the ability to induce inflammatory disease in vivo. Treg cells, in contrast, were enriched in vivo and appeared functionally unaffected and able to suppress Teff, irresp. of Glut1 expression. These data show a selective in vivo requirement for Glut1 in metabolic reprogramming of CD4 T cell activation and Teff expansion and survival.
- 31Balin, S. J.; Pellegrini, M.; Klechevsky, E.; Won, S. T.; Weiss, D. I.; Choi, A. W.; Hakimian, J.; Lu, J.; Ochoa, M. T.; Bloom, B. R.; Lanier, L. L.; Stenger, S.; Modlin, R. L. Human Antimicrobial Cytotoxic T Lymphocytes, Defined by NK Receptors and Antimicrobial Proteins, Kill Intracellular Bacteria. Sci. Immunol. 2018, 3, eaat7668 DOI: 10.1126/sciimmunol.aat7668There is no corresponding record for this reference.
- 32Liu, Y.; Liang, G.; Xu, H.; Dong, W.; Dong, Z.; Qiu, Z.; Zhang, Z.; Li, F.; Huang, Y.; Li, Y.; Wu, J.; Yin, S.; Zhang, Y.; Guo, P.; Liu, J.; Xi, J. J.; Jiang, P.; Han, D.; Yang, C. G.; Xu, M. M. Tumors Exploit FTO-Mediated Regulation of Glycolytic Metabolism to Evade Immune Surveillance. Cell Metab. 2021, 33, 1221– 1233.e11, DOI: 10.1016/j.cmet.2021.04.00132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVShs77E&md5=74422d1b32aa1faa1a04f0fd3a25eaceTumors exploit FTO-mediated regulation of glycolytic metabolism to evade immune surveillanceLiu, Yi; Liang, Guanghao; Xu, Hongjiao; Dong, Wenxin; Dong, Ze; Qiu, Zhiwei; Zhang, Zihao; Li, Fangle; Huang, Yue; Li, Yilin; Wu, Jun; Yin, Shenyi; Zhang, Yawei; Guo, Peijin; Liu, Jun; Xi, Jianzhong Jeff; Jiang, Peng; Han, Dali; Yang, Cai-Guang; Xu, Meng MichelleCell Metabolism (2021), 33 (6), 1221-1233.e11CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)The ever-increasing understanding of the complexity of factors and regulatory layers that contribute to immune evasion facilitates the development of immunotherapies. However, the diversity of malignant tumors limits many known mechanisms in specific genetic and epigenetic contexts, manifesting the need to discover general driver genes. Here, we have identified the m6A demethylase FTO as an essential epitranscriptomic regulator utilized by tumors to escape immune surveillance through regulation of glycolytic metab. We show that FTO-mediated m6A demethylation in tumor cells elevates the transcription factors c-Jun, JunB, and C/EBPβ, which allows the rewiring of glycolytic metab. Fto knockdown impairs the glycolytic activity of tumor cells, which restores the function of CD8+ T cells, thereby inhibiting tumor growth. Furthermore, we developed a small-mol. compd., Dac51, that can inhibit the activity of FTO, block FTO-mediated immune evasion, and synergize with checkpoint blockade for better tumor control, suggesting reprogramming RNA epitranscriptome as a potential strategy for immunotherapy.
- 33Adema, G. J.; Hartgers, F.; Verstraten, R.; de Vries, E.; Marland, G.; Menon, S.; Foster, J.; Xu, Y.; Nooyen, P.; McClanahan, T.; Bacon, K. B.; Figdor, C. G. A Dendritic-Cell-Derived C-C Chemokine That Preferentially Attracts Naive T Cells. Nature 1997, 387, 713– 717, DOI: 10.1038/4271633https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktVWms7g%253D&md5=8be13f000a50bf8f569af22bb0a5e47eA dendritic-cell-derived C-C chemokine that preferentially attracts naive T cellsAdema, Gosse J.; Hartgers, Franca; Verstraten, Riet; de Vries, Edwin; Marland, Gill; Menon, Satish; Foster, Jessica; Xu, Yuming; Nooyan, Pete; McClanahan, Terrill; Bacon, Kevin B.; Figdor, Carl G.Nature (London) (1997), 387 (6634), 713-717CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)Dendritic cells form a system of highly efficient antigen-presenting cells. After capturing antigen in the periphery, they migrate to lymphoid organs where they present the antigen to T cells. Their seemingly unique ability to interact with the sensitized native T cells gives dendritic cells a central role in the initiation of immune responses and allows them to be used in therapeutic strategics against cancer, viral infection and other diseases. How they interact preferentially with naive rather than activated T lymphocytes is still poorly understood. Chemokines direct the transport of white blood cells in immune surveillance. Here the authors report the identification and characterization of a C-C chemokine (DC-CK1) that is specifically expressed by human dendritic cells at high levels. Tissue distribution anal. demonstrates that dendritic cells present in germinal centers and T-cell areas of secondary lymphoid organs express this chemokine. The authors show that DC-CK1, in contrast to RANTES, MIP-1α and interleukin-8, preferentially attracts naive T cells (CD45RA+). The specific expression of DC-CK1 by dendritic cells at the site of initiation of an immune response, combined with its chemotactic activity for native T cells, suggests that CD-CK1 has an important rule in the induction of immune responses.
- 34Chaffer, C. L.; Weinberg, R. A. A Perspective on Cancer Cell Metastasis. Science 2011, 331, 1559– 1564, DOI: 10.1126/science.120354334https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjs1Cgtbo%253D&md5=a361a1380ba9a72d974189059ebd9210A Perspective on Cancer Cell MetastasisChaffer, Christine L.; Weinberg, Robert A.Science (Washington, DC, United States) (2011), 331 (6024), 1559-1564CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Metastasis causes most cancer deaths, yet this process remains one of the most enigmatic aspects of the disease. Building on new mechanistic insights emerging from recent research, we offer our perspective on the metastatic process and reflect on possible paths of future exploration. We suggest that metastasis can be portrayed as a 2-phase process: The first phase involves the phys. translocation of a cancer cell to a distant organ, whereas the second encompasses the ability of the cancer cell to develop into a metastatic lesion at that distant site. Although much remains to be learned about the second phase, we feel that an understanding of the first phase is now within sight, due in part to a better understanding of how cancer cell behavior can be modified by a cell-biol. program called the epithelial-to-mesenchymal transition.
- 35Reticker-Flynn, N. E.; Zhang, W.; Belk, J. A.; Basto, P. A.; Escalante, N. K.; Pilarowski, G. O. W.; Bejnood, A.; Martins, M. M.; Kenkel, J. A.; Linde, I. L.; Bagchi, S.; Yuan, R.; Chang, S.; Spitzer, M. H.; Carmi, Y.; Cheng, J.; Tolentino, L. L.; Choi, O.; Wu, N.; Kong, C. S.; Gentles, A. J.; Sunwoo, J. B.; Satpathy, A. T.; Plevritis, S. K.; Engleman, E. G. Lymph Node Colonization Induces Tumor-Immune Tolerance to Promote Distant Metastasis. Cell 2022, 185, e23 DOI: 10.1016/j.cell.2022.04.019There is no corresponding record for this reference.
- 36Liang, C.; Diao, S.; Wang, C.; Gong, H.; Liu, T.; Hong, G.; Shi, X.; Dai, H.; Liu, Z. Tumor Metastasis Inhibition by Imaging-Guided Photothermal Therapy with Single-Walled Carbon Nanotubes. Adv. Mater. 2014, 26, 5646– 5652, DOI: 10.1002/adma.20140182536https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXps1yksr4%253D&md5=c243b95026adbbe71afe1380807870b0Tumor Metastasis Inhibition by Imaging-guided Photothermal Therapy with Single-walled Carbon NanotubesLiang, Chao; Diao, Shuo; Wang, Chao; Gong, Hua; Liu, Teng; Hong, Guosong; Shi, Xiaoze; Dai, Hongjie; Liu, ZhuangAdvanced Materials (Weinheim, Germany) (2014), 26 (32), 5646-5652CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)Cancer metastasis treatment with imaging-guided PDT with single-walled carbon nanotube is described.
- 37Watson, M. J.; Vignali, P. D. A.; Mullett, S. J.; Overacre-Delgoffe, A. E.; Peralta, R. M.; Grebinoski, S.; Menk, A. V.; Rittenhouse, N. L.; DePeaux, K.; Whetstone, R. D.; Vignali, D. A. A.; Hand, T. W.; Poholek, A. C.; Morrison, B. M.; Rothstein, J. D.; Wendell, S. G.; Delgoffe, G. M. Metabolic Support of Tumour-Infiltrating Regulatory T Cells by Lactic Acid. Nature 2021, 591, 645– 651, DOI: 10.1038/s41586-020-03045-237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktF2iurg%253D&md5=bfe6b0c3f320fee905be55903bd9b65fMetabolic support of tumor-infiltrating regulatory T cells by lactic acidWatson, McLane J.; Vignali, Paolo D. A.; Mullett, Steven J.; Overacre-Delgoffe, Abigail E.; Peralta, Ronal M.; Grebinoski, Stephanie; Menk, Ashley V.; Rittenhouse, Natalie L.; DePeaux, Kristin; Whetstone, Ryan D.; Vignali, Dario A. A.; Hand, Timothy W.; Poholek, Amanda C.; Morrison, Brett M.; Rothstein, Jeffrey D.; Wendell, Stacy G.; Delgoffe, Greg M.Nature (London, United Kingdom) (2021), 591 (7851), 645-651CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumor microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumor cells show deregulated metab., leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumor for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metab. of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metab. of the glycolytic byproduct lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumor growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumors avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.
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Additional experimental details including experimental methods, materials characterization, DFT results, multienzyme cascade data, and other in vitro and in vivo data (PDF)
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