Upgrade and repair of high performance liquid chromatography

高效液相色谱升级改造

• 批准号: RTI-2018-00367
• 负责人: Ro, DaeKyun
• 金额: $ 1.45万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=642174
• 关键词: Upgrade; repair; performance; liquid; chromatography

Isoprenoids are the largest class of natural products with more than 70,000 known structures. Despite their structural complexities, all isoprenoids are biosynthesized by a simple building block, five-carbon isopentenyl diphosphate (IPP). As many isoprenoids have direct implications in human society as pharmaceuticals, nutraceuticals, flavors, and polymers, how this simple IPP can be transformed to a diverse array of isoprenoids has been intensively studied. My research program focuses on elucidating biochemical mechanisms for the formations of diverse isoprenoids with an aim at providing knowledge for biotechnological productions of naturally scarce isoprenoids. Two major isoprenoids of interest in my laboratory are the biopolymer of natural rubber and pharmaceutically important sesquiterpene lactones. Natural rubber is a poly-cis-isoprene biopolymer, comprised of more than a thousand IPP monomers. Despite progresses in polymer chemistry, synthetic rubber derived from petroleum cannot fully replace natural rubber in manufacturing industries because the physical properties of synthetic rubber cannot surpass those of natural rubber (e.g., elasticity, heat dissipation, and abrasion resistance). Natural rubber is, therefore, an irreplaceable biomaterial synthesized from plant. On the other hand, sesquiterpene lactones form an important group of small isoprenoids, members of which have been used as anti-malarial (e.g., artemisinin) and anti-prostate cancer (e.g., thapsigargin) drug. However, most sesquiterpene lactones are present at low quantities in nature and their chemical synthesis is economically not viable due to their stereochemical complexities. The enzymatic synthesis of sesquiterpene lactones in surrogate hosts has emerged as an alternative biological production. However, the biochemical mechanism of how these isoprenoids are synthesized remains elusive, limiting biotechnological productions of these compounds. My research has identified a novel protein complex necessary for natural rubber biosynthesis, using lettuce as a model system. We have also elucidated a complete biosynthetic pathway of the simplest sesquiterpene lactone, costunolide. Advancing the biochemical knowledge for natural rubber and sesquiterpene lactone has been dependant on the routine use of an analytical instrument, high performance liquid chromatography (HPLC) coupled with a photodiode array detector (for sesquiterpene lactone analysis) and an evaporative light scattering detector (for natural rubber analysis). Reliable detection, quantitation, and purification of small or polymeric isoprenoids using HPLC are essential analytical practices. Timely upgrade and repair of HPLC are critically required to continue the metabolic studies of the isoprenoids.

类异戊二烯是最大的一类天然产物，拥有 70,000 多种已知结构。尽管结构复杂，但所有类异戊二烯都是通过简单的结构单元五碳异戊烯基二磷酸 (IPP) 生物合成的。由于许多类异戊二烯对人类社会有直接影响，如药物、营养品、香料和聚合物，因此如何将这种简单的 IPP 转化为多种类异戊二烯已得到深入研究。我的研究项目侧重于阐明形成不同类异戊二烯的生化机制，旨在为天然稀有类异戊二烯的生物技术生产提供知识。我的实验室感兴趣的两种主要类异戊二烯是天然橡胶的生物聚合物和药学上重要的倍半萜内酯。天然橡胶是一种聚顺式异戊二烯生物聚合物，由一千多种IPP单体组成。尽管高分子化学取得了进步，但源自石油的合成橡胶在制造业中仍无法完全取代天然橡胶，因为合成橡胶的物理性能无法超越天然橡胶（例如弹性、散热性和耐磨性）。因此，天然橡胶是一种不可替代的植物合成生物材料。另一方面，倍半萜内酯形成了一组重要的小类异戊二烯，其中的成员已被用作抗疟疾（例如青蒿素）和抗前列腺癌（例如毒胡萝卜素）药物。然而，大多数倍半萜内酯在自然界中的含量很低，并且由于其立体化学复杂性，化学合成在经济上不可行。在替代宿主中酶法合成倍半萜内酯已成为一种替代生物生产方法。然而，这些类异戊二烯合成的生化机制仍然难以捉摸，限制了这些化合物的生物技术生产。我的研究以生菜为模型系统，发现了天然橡胶生物合成所需的新型蛋白质复合物。我们还阐明了最简单的倍半萜内酯木香内酯的完整生物合成途径。天然橡胶和倍半萜内酯生化知识的推进依赖于分析仪器、高效液相色谱 (HPLC) 以及光电二极管阵列检测器（用于倍半萜内酯分析）和蒸发光散射检测器（用于天然橡胶）的常规使用。橡胶分析）。使用 HPLC 可靠地检测、定量和纯化小或聚合类异戊二烯是重要的分析实践。为了继续类异戊二烯的代谢研究，迫切需要及时升级和修复 HPLC。