Microscale LC-MS and NMR Methodology for Rapid Natural *

Rapid Natural 的微型 LC-MS 和 NMR 方法 *

• 批准号: 7125188
• 负责人: JIMMY ORJALA
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125188
• 关键词: Cyanophyta; NIH Roadmap Initiative tag; analytical method; bioassay; biological products; biotherapeutic agent; drug discovery /isolation; high throughput technology; liquid chromatography mass spectrometry; method development; microorganism culture; microorganism growth; nuclear magnetic resonance spectroscopy; protease inhibitor

DESCRIPTION (provided by applicant): Drug discovery from natural products can be prohibitively expensive and inefficient due to the labor and materials required for preparative-scale isolation of an active component, often before its novelty and potency can be determined. We propose to apply 10-fold more sensitive methods of mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis early in the isolation of a bioactive natural product. The novel MS interface provides a high sensitivity and resolution previously attainable only with nanobore LC-MS, with the use of larger LC columns, which allow enough material to be collected for NMR analysis. The micro-NMR method can acquire NMR spectra of all collected fractions under full automation, with a limit of quantitation below 1 mu/g, and recover the samples for bioassay. Together, these methods enable identification and quantitation of an active species concurrent with its initial isolation, eliminating the lengthy and expensive large-scale purification needed by traditional methods. The proposed methodology will be demonstrated and developed in the isolation of 3 recently-discovered protease inhibitor activities in extracts of cyanobacteria, a slow-growing, low-yield organism that has not been practical to study by traditional methods. The higher sensitivity and non-destructive nature of the new analytical methods enables enough material for analysis and bioassay to be obtained from 100-fold smaller growths, or for 100 times more strains to be grown with the same resources. The ability to perform extraction and purification on an analytical scale rather than preparative scale reduces the time required from weeks to days. These dramatic improvements have the potential to reinvigorate the field of natural products discovery. The specific aims are: 1) To implement more-sensitive, higher-recovery LC-MS methods using the recently-developed nano-splitter device. 2) To implement high-throughput NMR of fractions collected from LC-MS analysis, combining use of microcoil probes with a method for lossless loading of small volumes (2 uL) from 96-well plates. 3) To provide bioguided fractionation of cyanobacterial extracts, which are optimized for high-throughput micro-NMR and LC-MS analysis. The objective of this proposal is to improve on natural product discovery. This has the potential to directly impact on public health, by overcoming current limitations of traditional methods and reinvigorate the important field of drug discovery from natural sources.

描述（由申请人提供）：从天然产物中发现药物可能非常昂贵且效率低下，因为通常在确定活性成分的新颖性和效力之前，需要大量的劳动力和材料来分离活性成分。我们建议在生物活性天然产物的分离早期应用灵敏度高 10 倍的质谱 (MS) 和核磁共振 (NMR) 分析方法。新颖的 MS 接口提供了以前只能通过纳米孔 LC-MS 才能实现的高灵敏度和分辨率，并使用较大的 LC 柱，从而可以收集足够的材料用于 NMR 分析。微核磁共振方法可以全自动获取所有收集组分的核磁共振谱，定量限低于1μ/g，并回收样品进行生物测定。总之，这些方法能够在初始分离的同时对活性物质进行鉴定和定量，从而消除了传统方法所需的漫长且昂贵的大规模纯化。所提出的方法将在蓝藻提取物中分离出 3 种最近发现的蛋白酶抑制剂活性进行论证和开发，蓝藻是一种生长缓慢、产量低的生物体，通过传统方法进行研究并不实用。新分析方法具有更高的灵敏度和非破坏性，能够从较小的 100 倍生长物中获得足够的材料进行分析和生物测定，或者使用相同的资源生长 100 倍以上的菌株。在分析规模而不是制备规模上进行提取和纯化的能力将所需的时间从几周缩短到几天。这些巨大的进步有可能重振天然产物发现领域。具体目标是：1）使用最近开发的纳米分流器装置实施更灵敏、更高回收率的LC-MS方法。 2) 为了对从 LC-MS 分析中收集的组分进行高通量 NMR，将微线圈探针的使用与从 96 孔板无损加载小体积 (2 uL) 的方法相结合。 3) 提供蓝藻提取物的生物引导分级分离，并针对高通量微核磁共振和液质联用分析进行了优化。该提案的目的是改进天然产物的发现。通过克服传统方法目前的局限性，并重振天然药物发现的重要领域，这有可能直接影响公共健康。