Microscale LC-MS and NMR Methodology for Rapid Natural *

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，因为通常在确定其新奇和效力之前，活性成分的重复性规模分离所需的劳动力和材料。我们建议在生物活性天然产物的分离早期应用10倍更灵敏的质谱（MS）和核磁共振（NMR）分析方法。新的MS界面提供了以前仅用纳米孔LC-MS可获得的高灵敏度和分辨率，使用更大的LC柱，这允许收集足够的材料用于NMR分析。微核磁共振方法可以在全自动化下获得所有收集的馏分的核磁共振谱，定量限低于1 μ/g，并回收样品用于生物测定。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冗长且昂贵的大规模纯化。所提出的方法将被证明和开发中的3个最近发现的蛋白酶抑制剂活性的蓝藻，生长缓慢，低产量的有机体，并没有实际的研究传统的方法提取物的分离。新的分析方法具有更高的灵敏度和非破坏性，能够从100倍的小生长中获得足够的分析和生物测定材料，或者用相同的资源培养100倍的菌株。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数周减少到数天。这些巨大的改进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离器装置实现更灵敏、更高回收率的LC-MS方法。2)为了对从LC-MS分析中收集的馏分进行高通量NMR，将微线圈探针的使用与从96孔板中无损加载小体积（2 uL）的方法相结合。3)提供蓝藻提取物的超临界分级分离，该分级分离针对高通量微核磁共振和液相色谱-质谱分析进行了优化。该提案的目的是改善天然产品的发现。通过克服传统方法目前的局限性，并重振从自然来源发现药物的重要领域，这有可能对公共卫生产生直接影响。

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.