含离子液体体系中生物转化法制备药物中间体雄烯二酮应用基础研究

Androst-4-ene-3,17-dione (AD) is an important precursor of steroid drugs, and microbial transformation to produce AD from phytosterols sees a promising application. However, the poor water solubility of substrate and the cell toxicity of product are major problems causing low bioconversion efficiency. The utilization of biphasic systems involving green solvents such as ionic liquids (ILs) is one of the effective strategies, which can induce in situ product removal and has great practical significance in the innovation of green process engineering for sustainable environment. In this work, representative ILs are selected to study the relationships of ILs structures with their physicochemical properties, including hydrophobicity, polarity, H-bond basicity, the liquid-liquid interfacial tension and phase composition of IL/water system. The effects of cationic and anionic structures on ILs solvating power and extraction capability, mass transfer behavior of substrate and product, and biocompatibility of ILs with cells will be investigated. Then novel ILs, consisting of biomolecule lactic acid and long-chain fatty acid as anion donors and quaternary phosphonium and quaternary ammonium as cation donors, will be designed and regulated rationally to attain efficient bioconversion at high substrate feeding concentration. The mechanism of bioconversion process in IL/water two phase system will also be discussed, and the focus will be laid on the interactions between IL functional group and key enzyme in metabolic pathway. Furthermore, ILs recovery for environmental concerns, ecotoxicity and biodegradability will also be assessed. A general evaluation procedure of ILs application in a given whole-cell bioconversion can be expected to construct in this research, and the achievement will benefit for the development of green catalytic process involving ILs.

雄烯二酮是重要的甾体药物中间体，以自然界中丰富的植物甾醇为底物通过生物转化法生产雄烯二酮具有良好的应用前景。但是甾醇底物的低水溶性和产物的细胞毒性导致生物法效率偏低，制约了该技术的发展。本课题拟以绿色溶剂离子液体(IL)构建IL/水两相体系，结合原位萃取以解除产物对细胞活性的抑制，促进底物溶解，提高过程转化效率。首先测定不同类别IL的理化性质包括疏水性、极性、氢键碱性和IL/水液-液界面张力及相组成。研究阴阳离子结构与IL溶解和萃取性能、底物/产物传质行为及甾醇转化过程中IL生物相容性的关系。在此基础上，以生物分子乳酸和长链脂肪酸为阴离子供体，季铵盐和季鏻盐为阳离子供体设计合成新型IL用于甾醇转化过程，通过改变IL结构理性调节其性能以实现高底物投料浓度下的生物转化。探讨含IL体系生物转化机理，重点剖析IL基团对甾醇转化关键酶活性和代谢途径的作用机制。评估IL回收、生态毒性和生物可降解性，研究成果有助于推动IL绿色催化工艺的发展。

在对商业化离子液体进行系统评估的基础上，本文设计合成了生物相容性胆碱-氨基酸离子液体和强溶解性季铵/季鏻盐-乳酸/长链脂肪酸离子液体，建立了离子液体结构、性质与生物转化之间的关系，为新型离子液体在全细胞生物转化中的应用提供了参考。针对疏水性分枝杆菌细胞易团聚、甾醇底物低水溶性、产物抑制和甾核降解等技术难题，构建了含离子液体的共溶剂体系和两相生物转化体系应用于分枝杆菌降解植物甾醇侧链制备药物中间体雄烯二酮，并结合细胞固定化技术和原位产物分离技术等过程强化策略以提高其产量。对分枝杆菌进行全基因组测序，得到了多个甾醇侧链降解关键酶编码基因，利用分子动力学模拟手段研究了胆碱-氨基酸离子液体对关键酶结构和活性的影响，探讨了含离子液体体系中全细胞转化过程的作用机制，为多功能性离子液体的开发提供了理论指导。此外，对含深共熔溶剂体系中植物甾醇生物转化过程进行了有益的探索，研究结果有助于推动甾体药物中间体绿色合成工艺的可持续性发展。