关键词: 没药醇合酶, 倍半萜, 青蒿, 立体异构体, α-没药醇

Abstract: The gas and liquid chromatography method were established to identify the absolute configuration of α-bisabolol and to determine the absolute configuration of Artemisia annua α-bisabolol synthase products. Separation and preparation of four stereoisomers of α-bisabolol were performed by chiral liquid phase. The absolute configurations of four kinds of stereoisomers were determined by gas chromatography and standard, combined with optical rotation determination. Identification of four stereoisomers of α-bisabolol by gas and liquid chromatography, chemical synthesis of substrate farnesyl pyrophosphate(FPP). By prokaryotic expression, protein purification and in vitro catalysis, the absolute configuration of two bisabolol synthases in vitro was identified. Consistent with the literature report, Lippia dulcis α-bisabolol- DOI:10.3969/j.issn.1000-1565.2020.04.011青蒿α-bisabolol合酶产物绝对构型的确定穆星¹,杨倩¹,刘会云¹,向英¹,曹飞²,李振秋¹(1.河北大学 生命科学学院,河北 保定 071002;2. 河北大学 药学院,河北 保定 071002)摘 要:建立气相和液相色谱方法用于鉴定α-没药醇(α-bisabolol)绝对构型,确定青蒿α-bisabolol合酶产物的绝对构型. 手性液相方法分离并制备α-bisabolol的4种立体异构体,结合旋光度测定、气相色谱和标准品确定4种立体异构体的绝对构型. 分别建立气相和液相色谱2种方法来鉴定α-bisabolol的4种立体异构体混合物. 化学合成酶促反应的底物法尼基焦磷酸(farnesyl pyrophosphate,FPP),原核表达、蛋白纯化后进行体外催化,鉴定2个α-bisabolol合酶体外催化产物α-bisabolol的绝对构型. 甜舌草α-bisabolol合酶(Lippia dulcis bisabolol synthase, LdBOS)体外催化FPP,产物构型为(+)-epi-α-bisabolol,与文献报道一致. 青蒿α-bisabolol合酶(Artemisia annua bisabolol synthase, AaBOS)体外催化FPP生成的产物为(+)-α-bisabolol. Agilent CP-Chirasil-Dex-CB气相色谱柱和Chiralpak IA液相色谱柱可分别用以分离、鉴定α-bisabolol的4种手性异构体. AaBOS和LdBOS的催化产物6位碳构型相同,暗示这2种酶与青蒿紫穗槐二烯合酶(Artemisia annua amorpha-4,11-diene synthase, AaADS)的催化机理类似. 利用气相和液相色谱的方法分析手性化合物,具有灵敏度高、稳定性强、简便易行等优点. 手性化合物α-bisabolol绝对构型的分离、鉴定,在医药、食品和化工领域具有重要意义.关键词:没药醇合酶;倍半萜;青蒿;立体异构体;α-没药醇中图分类号:Q785 文献标志码:A 文章编号:1000-1565(2020)04-0409-06Determination of absolute configuration of Artemisia annua α-bisabolol synthase productsMU Xing¹, YANG Qian¹, LIU Huiyun¹, XIANG Ying¹, CAO Fei², LI Zhenqiu¹(1. College of Life Sciences, Hebei University, Baoding 071002, China; 2. College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China )Abstract: The gas and liquid chromatography method were established to identify the absolute configuration of α-bisabolol and to determine the absolute configuration of Artemisia annua α-bisabolol synthase products. Separation and preparation of four stereoisomers of α-bisabolol were performed by chiral liquid phase. The absolute configurations of four kinds of stereoisomers were determined by gas chromatography and standard, combined with optical rotation determination. Identification of four stereoisomers of α-bisabolol by gas and liquid chromatography, chemical synthesis of substrate farnesyl pyrophosphate(FPP). By prokaryotic expression, protein purification and in vitro catalysis, the absolute configuration of two bisabolol synthases in vitro was identified. Consistent with the literature report, Lippia dulcis α-bisabolol- 收稿日期:2019-04-16 基金项目:国家自然科学基金资助项目(31570305; 30900111) 第一作者:穆星(1994—),女,河北保定人,河北大学在读硕士研究生,主要从事分子细胞生物学研究. E-mail:mxmuxing@126.com 通信作者:李振秋(1974—),男,河北石家庄人,河北大学教授,主要从事分子细胞生物学研究. E-mail:cell@hbu.edu.cn第4期穆星等:青蒿α-bisabolol合酶产物绝对构型的确定synthase(LdBOS)catalyzes FPP in vitro, and the product configuration was(+)-epi-α-bisabolol; Artemisia annua α-bisabolol synthase(AaBOS)catalysed FPP in vitro, and the product was(+)-α-bisabolol. The catalytic product of AaBOS and LdBOS have the same carbon configuration at position 6, suggesting that the catalytic mechanism of these two enzymes is similar to that of the Artemisia annua amorpha-4,11-diene synthase(AaADS). The analysis of chiral compounds by gas and liquid chromatography has many advantages, such as high sensitivity, strong stability, easy to operate and so on. The separation and identification of the absolute configuration of chiral compounds α-bisabolol are of great significance in the fields of medicine, food and chemical industry.

Key words: bisabolol synthase, sesquiterpene, Artemisia annua, stereoisomers, α-bisabolol