Microscale LC-MS and NMR Methodology:Rapid Natural (RMI)

描述(由申请人提供)：从天然产品中发现药物的成本可能高得令人望而却步，而且效率低下，这是因为制备规模分离活性成分所需的劳动力和材料，通常是在确定其新颖性和效力之前。我们建议在分离生物活性天然产物的早期应用10倍更灵敏的质谱学(MS)和核磁共振(NMR)分析方法。新的MS界面提供了以前只有纳米孔LC-MS才能获得的高灵敏度和分辨率，使用了更大的LC柱，允许收集足够的材料用于核磁共振分析。该方法可以在全自动条件下获得所有样品的核磁共振波谱，定量限在1mU/g以下，并可回收样品进行生物测定。总而言之，这些方法可以在初步分离活性物种的同时对其进行鉴定和定量，从而消除了传统方法所需的冗长和昂贵的大规模纯化。所提出的方法将在分离蓝藻提取物中最近发现的3种蛋白酶抑制活性方面进行演示和开发，蓝藻是一种生长缓慢、产量低的生物，用传统方法研究它并不实用。新的分析方法具有更高的灵敏度和非破坏性，使得能够从100倍的较小生长中获得足够的分析和生物测定材料，或者使用相同的资源培养100倍以上的菌株。能够在分析规模而不是制备规模上进行提取和纯化，将所需的时间从几周减少到几天。这些戏剧性的改进有可能重振天然产品发现领域。具体目标是：1)利用最近开发的纳米分离器装置实现更灵敏、更高回收率的LC-MS方法。2)对从LC-MS分析中收集的馏分进行高通量核磁共振分析，结合使用微线圈探针和从96孔板上无损加载小体积(2 Ul)的方法。3)提供蓝藻提取物的生物导向分级，优化了高通量的显微核磁共振和LC-MS分析。这项提议的目标是改进天然产品的发现。这有可能直接影响公众健康，因为它克服了目前传统方法的局限性，重振了从自然来源发现药物这一重要领域。

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.