Applications of New Mass Spectral Techniques

该项目的目标是开发新的质谱学技术，以便为涉及(1)化学结构确定、(2)复杂混合物分析和(3)生物系统中痕量组分测量的问题提供创新和/或更快速的解决方案。基质辅助激光解吸电离(MALDI)质谱仪、电喷雾电离质谱仪(ESI/MS)、串联质谱仪(MS/MS)、液质联用(LC/MS)、毛细管电泳质谱(CE/MS)联用和精确质量测定是目前研究的热点。采用后续峰采集的在线CE样品浓缩技术正在研究中，作为CE与高分辨率MALDI/MS离线结合和源后衰变碎片分析的一种制备规模方法。一项协同的质谱学和分子模拟研究正在进行中，以研究在从液体中电离(ESI/MS和FAB/MS)过程中控制碱金属离子阳离子的因素。在正常情况下，碱金属离子阳离子化是不可取的，因为它会降低灵敏度和/或使光谱解释复杂化。然而，对于某些类型的化合物，如低聚糖和倍半萜类化合物，选择性和可控的阳离子作用可以带来分析优势。抗疟疾天然产物青蒿素(青蒿素)的衍生物，包括一系列具有抗肿瘤活性的三噁烷二聚体，正被用作模型化合物，因为它们需要碱金属离子阳离子来获得有用的质谱图。对双氢青蒿素的初步研究表明，加合物的形成是阳离子和电离技术的选择性。为了使分子模拟计算具有预测性，需要考虑液体基质中的阳离子溶剂化作用，而不仅仅是气相阳离子结合能。因此，在电离过程中从液体中形成的阳离子加合物至少部分地代表了分析物在基质中的状态。在小分子的MALDI/MS过程中也观察到类似的阳离子现象，其中从固相发生解吸和电离。 通过对新化合物和合成中间体的结构表征，快原子轰击质谱仪(FAB/MS)被用于支持LMC的合成工作。一些建立在受限制的甘油支架(二取代DAG-内酯)上的合成化合物已被鉴定为蛋白激酶C(PK-C)的有效激动剂。根据包括R1和R2的取代基的结构，这些DAG-内酯似乎具有一定程度的PK-C同工酶专一性。一种固相组合方法正被应用于LMC中，以研究R1和R2的化学多样性，以产生更具体的C1域配体。重要的是，在生物评价之前，必须对这些合成产品进行快速表征并确定其结构。利用FAB/MS开发、评估和实施了这些组合文库的快速质谱学表征策略。典型的DAG-内酯衍生物的正离子FAB质谱图由完整的分子离子(M)或质子化分子(MH)以及来自酰基和亚烷基部分的几个碎片离子组成。这一光谱是可预测的，可用于鉴定简单混合物中存在的DAG-内酯衍生物。最初，对选定的库列或行的成员进行单独检查，以确认质谱图是可预测的，并评估化学的效率。然后，这些信息被用来组装涵盖整个化学空间的简单的4至6组分的混合物；然后对这些混合物进行分析和解释。随后对混合物分析过程中未检测到的库组件进行单独分析。混合分析与单独分析直接相关，大大减少了要检查的样品数量，从而增强了分析的周转性。一套完整的初步评估、样品制备、质谱分析、数据解释和报告生成过程可在2天内完成。每个文库成分的等量也被存档，以供将来可能的分析，如果以后的生物学评估需要额外的特征。流动注射电喷雾电喷雾质谱(ESI/MS)不适用于这些DAG-内酯类化合物的分析，因为它们的亲油性较高，且碱性有限。然而，流动注射-APCI/MS产生的光谱由MH以及各种碎片和溶剂加成离子组成，这种方法在光谱信息方面似乎是对FAB/MS的补充。MALDI/MS也被评估为快速表征这些小分子文库的工具。我们的最终目标是在一天内完成所有96个库组件的结构表征。通过三维数据库搜索确认被确认为HIV-1整合酶抑制剂的化合物的结构同一性的研究仍在继续。从海洋苔藓动物Caulibugula intermis中分离得到一系列新的具有细胞毒性的异喹啉类化合物和亚氨基类化合物，作为其结构解析的一部分，对其进行了精确的质量分析。

The objective of this project is development of new mass spectral techniques in order to provide innovative and/or more rapid solutions to problems involving (1) chemical structure determination, (2) complex mixture analysis and (3) measurement of trace components in biological systems. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry, electrospray ionization mass spectrometry (ESI/MS), tandem mass spectrometry (MS/MS), combined liquid chromatography-mass spectrometry (LC/MS), combined capillary electrophoresis-mass spectrometry (CE/MS) and accurate mass measurement are the techniques of current interest. On-line CE sample concentration techniques with subsequent peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS and post-source decay fragmentation analysis. A collaborative mass spectrometric and molecular modeling study to investigate the factors controlling alkali metal ion cationization during ionization from the liquid phase (ESI/MS and FAB/MS) is ongoing. Under normal circumstances, alkali metal ion cationization is not desirable because it can decrease sensitivity and/or complicate spectral interpretation. However, for certain classes of compounds, such as oligosaccharides and sesquiterpenoids, selective and controlled cationization can confer an analytical advantage. Derivatives of the antimarial natural product artemesinin (qinghaosu), including a series of trioxane dimers with antitumor activity, are being used as model compounds since they require alkali metal ion cationization to obtain useful mass spectra. An initial investigation focusing on dihyroartemisinin shows that adduct formation is cation and ionization technique selective. For molecular modeling calculations to be predictive, a consideration of cation solvation in the liquid matrix is required rather than just gas phase cation binding energies. Thus it appears that cation adducts formed during ionization from a liquid phase represent at least partially the state of the analyte in the matrix. A similar cationization phenomenon is observed during MALDI/MS of small molecules where desorption and ionization occurs from the solid phase. Fast atom bombardment mass spectrometry (FAB/MS) is employed to support the LMC synthetic effort through structural characterization of new compounds and synthetic intermediates. A number of synthetic compounds built on a constrained glycerol scaffold (disubstituted DAG-lactone) have been identified as potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents comprising R1 and R2, these DAG-lactones appear to have some degree of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic products be rapidly characterized and their structures established before biological evaluation. Strategies for the rapid mass spectral characterization of these combinatorial libraries have been developed, evaluated and implemented using FAB/MS. The positive ion FAB mass spectrum of a typical DAG-lactone derivative consists of the intact molecular ion (M+) or protonated molecule (MH+) as well as several fragment ions derived from both the acyl and alkylidene portions. This spectrum is predictable and can be used to identify the DAG-lactone derivatives present in simple mixtures. Initially, the members of one selected library column or row are examined individually to confirm that mass spectra are predictable and to evaluate the efficiency of the chemistry. This information is then used to assemble simple 4- to 6-component mixtures encompassing the entire chemical space; these mixtures are then analyzed and interpreted. Library components not detected during mixture analysis are subsequently analyzed individually. Mixture analysis correlates directly with individual analysis and substantially reduces the number of samples to be examined, resulting in enhanced analytical turn-around. A complete sequence of initial evaluation, sample preparation, mass spectral analysis, data interpretation and report generation can be completed in 2 days. Aliquots of each library component are also archived for possible future analysis should later biological evaluation warrant additional characterization. Flow-injection ESI/MS is not suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. However, flow injection-APCI/MS produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions, and this approach appears to be complementary to FAB/MS in terms of spectral information. MALDI/MS is also being evaluated as a tool for the rapid characterization of these small molecule libraries. Our ultimate goal is the structural characterization of all 96 library components in one day. Studies to confirm the structural identity of compounds identified as HIV-1 integrase inhibitors through 3-dimensional database searching continue. A series of novel cytotoxic isoquinoline quinones and iminoquinones isolated from the marine bryozoan Caulibugula intermis have been characterized by accurate mass analysis as part of their structural elucidation.