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，已知结构超过70，000种。尽管它们的结构复杂，但所有类异戊二烯都是由一个简单的结构单元，五碳异戊烯基二磷酸（IPP）生物合成的。由于许多类异戊二烯在人类社会中作为药物、营养品、香料和聚合物具有直接的意义，因此已经深入研究了如何将这种简单的IPP转化为各种各样的类异戊二烯。我的研究计划的重点是阐明不同的类异戊二烯形成的生物化学机制，旨在为自然稀缺的类异戊二烯的生物技术生产提供知识。我实验室感兴趣的两个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。尽管在聚合物化学方面取得了进展，但在制造工业中，源自石油的合成橡胶不能完全取代天然橡胶，因为合成橡胶的物理性质不能超过天然橡胶的物理性质（例如，弹性、散热和耐磨性）。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，倍半萜内酯形成一组重要的小类异戊二烯，其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以低量存在，并且由于它们的立体化学复杂性，它们的化学合成在经济上不可行。在替代宿主中酶促合成倍半萜内酯已成为一种替代的生物生产。然而，这些类异戊二烯如何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。我的研究已经确定了天然橡胶生物合成所必需的一种新的蛋白质复合物，使用生菜作为模型系统。我们还阐明了一个完整的生物合成途径的最简单的倍半萜内酯，木香。推进天然橡胶和倍半萜内酯的生物化学知识一直依赖于分析仪器的常规使用，高效液相色谱法（HPLC）与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（用于天然橡胶分析）。可靠的检测，定量和纯化的小或聚合的类异戊二烯使用高效液相色谱法是必不可少的分析实践。及时升级和修复HPLC是继续类异戊二烯代谢研究的关键。