分段式微流控非水相酶促合成二咖啡酰奎尼酸的催化机理

The chemo-enzymatic de novo synthesis strategy has become a fundamental approach to produce natural drugs with high-bioactivity, low-content and complex structures. In our preliminary study, we first discovered a chemo-enzymatic synthesis of anti-viral 3, 5-dicaffeoylquinic acid with high reaction rate and stereo-selectivity using microfluidic biocatalysis technology. Compared with the traditional chemical total synthesis consisting of 9 steps with 20.5% yield, our process only consisted of 4 steps and the yield was significantly increased to 49.5%. Due to the positive preliminary results, a novel method of enzymatic synthesis in a non-aqueous phase utilizing a segmented-flow microfluidics technique will be further established. The aim of this project is to comprehensively answer the fundamental scientific questions of microscale enzyme-catalyzed esterification synthesis in a non-aqueous phase by studying the enzymatic synthesis of 3, 5-dicaffeoylquinic acid. These questions include: 1) explore the mechanism of substrate specificity and stereo-selectivity in the enzymatic catalysis via investigating enzymatic kinetics and affinity differences between substrates and enzymes; 2) explain the control mechanism of the micro-fluidic mass transfer on the enzymatic kinetics by elucidating the parameters of reaction, flow and system of micro-channel; 3) reveal the novel technological principle of the microfluidic coupling reaction and separation in non-aqueous phase via establishingting the segmented-flow of ionic liquid-solvent. Based on this proposed study, the catalytic mechanism of microfluidic enzymatic synthesis in a non-aqueous phase will be clarified, which will be favorable in developing the fundamental theory of industrial biocatalysis. Also it will provide new methodologies and approaches for microfluidic pharmaceutical biosynthesis and application in the research & development of anti-viral drugs.

化学酶法从头合成已成为制备高活性、低含量和复杂结构天然药物的根本途径。我们以化学酶法首次合成抗病毒3,5-二咖啡酰奎尼酸，应用"微流控生物催化"提高反应速率和立体选择性，仅4步反应得率可达49.5%，而化学全合成9步仅为20.5%。由此，我们基于"Miniaturizing Biocatalysis"新概念建立"分段式微流控非水相酶促合成"新方法，以酶促合成二咖啡酰奎尼酸为研究对象，全面剖析微尺度下非水相酶促酯类合成的共性基础问题：①研究酶促动力学和酶与底物的亲和力差异，探索酶的底物特异性和立体选择性机理；②考察微通道内的反应、流体及系统参数，分析微流传质对非水相酶促合成的调控机制；③构建"离子液体-溶剂"分段流，揭示微流控非水相反应-分离耦合的新工艺原理。从而阐明分段式微流控非水相酶促合成的催化机理，丰富工业生物催化的基础理论，为微流控药物生物合成及其在抗病毒药物研发中的应用提供新思路。

化学酶法从头合成已成为制备高活性、低含量和复杂结构天然药物的根本途径。我们以化学酶法首次合成抗病毒3,5-二咖啡酰奎尼酸，应用“微流控生物催化”提高反应速率和立体选择性，仅4 步反应得率可达49.5%，而化学全合成9 步仅为20.5%。由此，我们基于“Miniaturizing Biocatalysis”新概念建立“分段式微流控非水相酶促合成”新方法，以酶促合成二咖啡酰奎尼酸过程中的一系列咖啡酸酯类中间体为研究对象，全面剖析微尺度下非水相酶促酯类合成的共性基础问题，首次系统地研究了微尺度下生物催化的过程机制，对于分段式微流控非水相酶促合成的催化机理及其反应分离耦合作用规律获得了更加深入的认识和理解。主要研究成果总结为三个方面内容：（1）基于酯交换策略，以含烷基碳数1~8的饱和脂肪醇的咖啡酸酯为底物，分析了脂肪酶催化酯化和酯交换反应的底物特异性和催化的立体选择性；（2）自主设计了连续流的固定床式微通道反应器，通过探讨微流控连续流体系中反应、流体及系统参数对达姆科勒数、有效因子及产量三者的作用关系，揭示了微环境中达姆科勒数调控催化常数与传质系数的作用机制；（3）基于络合剂三辛基氧化膦（TOPO）优选“离子液体-溶剂”两相体系，建立了一套分段式微流控酶促反应-分离耦合催化新技术，揭示了微尺度下反应-分离耦合对集成生物催化的调控规律，既缓解了产物抑制现象，又实现了产物的高效分离。相关研究结果已申请国家发明专利6项（含授权4项），发表学术论文12篇（其中SCI收录11篇，EI收录9篇），参加学术会议3次，培养研究生3名。研究成果对阐明微流控药物生物合成中酶与底物、微环境与传质、反应与分离的相互作用及其对生物催化过程的调控机理具有重要的指导意义，为进一步丰富工业生物催化理论提供了新颖的学术观点，并为微流控药物生物合成新理论和新技术的形成与发展提供了思路。