物理化学学报 >> 2017, Vol. 33 >> Issue (3): 620-626.doi: 10.3866/PKU.WHXB201612011

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漆酶催化邻苯二酚开环的自由基反应机制

陈明1,王林2,谭天1,罗学才2,郑在2,尹若春2,苏吉虎1,*(),杜江峰1   

  1. 1 中国科学技术大学近代物理系,中国科学院微观磁共振重点实验室,合肥230026
    2 安徽大学生命科学学院生物制造创意中心,合肥230601
  • 收稿日期:2016-09-29 发布日期:2017-03-07
  • 通讯作者: 苏吉虎 E-mail:sujihu@ustc.edu.cn
  • 基金资助:
    国家重点基础研究发展规划项目(973)(2013CB921802);亚热带农业生物资源保护与利用国家重点实验室开放课题基金(KSL-CUSAb-2012-03)

Radical Mechanism of Laccase-Catalyzed Catechol Ring-Opening

Ming CHEN1,Lin WANG2,Tian TAN1,Xue-Cai LUO2,Zai ZHENG2,Ruo-Chun YIN2,Ji-Hu SU1,*(),Jiang-Feng DU1   

  1. 1 CAS Key Laboratory of Microscale Magnetic Resonance, Department of Modern Physics, University of Science and Technology of China, Hefei 230026, P. R. China
    2 Inspiration Center for Bio-manufacture, School of Life Sciences, Anhui University, Hefei 230601, P. R. China
  • Received:2016-09-29 Published:2017-03-07
  • Contact: Ji-Hu SU E-mail:sujihu@ustc.edu.cn
  • Supported by:
    the National Key Basic Research Program of China (973)(2013CB921802);Fundamental Research Funds for the Central Universities, China, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, China(KSL-CUSAb-2012-03)

摘要:

酶催化反应是绿色化学领域中的研究热点之一.在常用的酶中,漆酶是一种多铜氧化酶.在此,漆酶被用来研究邻苯二酚的氧化和芳香环开环的自由基过程.实验表明,邻苯二酚的初始氧化产物是一个半醌自由基,再发生分子间加成反应而生成二聚体和三聚体(通过质谱鉴定),和分子内加成而生成可被5, 5-二甲基-1-氧化吡咯啉(DMPO)所捕获的含有支链烷基自由基的呋喃衍生物.后者经核磁共振氢谱(1H-NMR)加以分析.同时,通过17O同位素标记追踪方法,我们发现水直接参与该分子内加成反应,并释放出17O标记的羟基自由基(·17OH).此外,除了3-甲基邻苯二酚和4-甲基邻苯二酚两种类似物,此自由基过程在间苯二酚、对苯二酚、萘二酚、3-硝基邻苯二酚和4-硝基邻苯二酚等底物中均未发生.据此,我们对C4-C5位点的选择性活化与开环的机理展开分析和讨论,并将该机理与邻苯二酚双加氧酶导致的邻苯二酚双氧中间位置和相邻位置的开环过程相比较.这些结果将有益于漆酶的改造和仿生.

关键词: 漆酶, 自由基氧化, 芳香环开环, 电子顺磁共振波谱, 自旋捕获技术

Abstract:

Enzyme-catalyzed reactions are a prominent field of research in green chemistry. Laccase is a multicopper oxidase, which we used to study the oxidation of catechol. A mechanism for this ring-opening reaction is also proposed. A o-benzosemiquinone radical was the initial nascent product of catechol oxidation during the catalytic reaction. This radical underwent two reaction pathways:(1) formation of an intramolecular adduct, which gave a carbon-centered furan-derived radical trapped by 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO); (2) formation of an intermolecular adduct producing dimeric and trimeric oligomers, as resolved by mass spectrometry. Products of the furan-like intermediate were also characterized by 1H-NMR. Simultaneously, a hydroxyl radical (·OH) originating from the water solvent was identified by 17O-isotope tracing. The kinetics of this radical were also evident with substrates including 3-and 4-methyl catechol, but not with resorcinol and hydroquinone isomers, 3-and 4-nitro catechol, and 2, 3-dihydroxynaphthalene. The mechanism of selective activation and ring-opening at the C4-C5 site is discussed. This reaction is distinct from intra-and extra-diol ringcleavages catalyzed by catechol dioxygenase. These results are meaningful for mimicking laccase catalysis to further protein design.

Key words: Laccase, Radical oxidation, Aromatic ring cleavage, Electron paramagnetic resonance spectroscopy, Spin trapping

MSC2000: 

  • O643