Applications of New Mass Spectral Techniques

该项目的目标是开发新的质谱技术，以提供创新和/或更快速的解决方案，解决涉及（1）化学结构测定，（2）复杂混合物分析和（3）生物系统中痕量成分的测量。基质辅助激光解吸电离（MALDI）质谱、电喷雾电离质谱（ESI/MS）、串联质谱（MS/MS）、液相色谱-质谱联用（LC/MS）、毛细管电泳-质谱联用（CE/MS）和精确质量测量是当前感兴趣的技术。在线CE样品浓缩技术与随后的峰收集正在研究作为一种用于离线组合CE与高分辨率MALDI/MS和源后衰变裂解分析的分析尺度方法。正在进行一项质谱和分子建模协作研究，以调查液相电离（ESI/MS和FAB/MS）过程中控制碱金属离子阳离子化的因素。在正常情况下，碱金属离子阳离子化是不可取的，因为它可以降低灵敏度和/或复杂的光谱解释。然而，对于某些类别的化合物，如寡糖和倍半萜类化合物，选择性和受控的阳离子化可以赋予分析优势。抗疟天然产物青蒿素的衍生物，包括一系列具有抗肿瘤活性的三恶烷二聚体，由于它们需要碱金属离子阳离子化才能获得有用的质谱，因此被用作模型化合物。对二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选择性的。对于要预测的分子模拟计算，需要考虑液体基质中的阳离子溶剂化，而不仅仅是气相阳离子结合能。因此，似乎在从液相电离期间形成的阳离子加合物至少部分地代表基质中分析物的状态。在小分子的MALDI/MS期间观察到类似的阳离子化现象，其中从固相发生解吸和电离。 采用快速原子轰击质谱法（FAB/MS）通过新化合物和合成中间体的结构表征来支持LMC合成工作。许多建立在受限甘油支架上的合成化合物（二取代的DAG-内酯）已被鉴定为蛋白激酶C（PK-C）的有效激动剂。根据包含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同工酶特异性。一个固相组合的方法被应用在LMC研究化学多样性在R1和R2，以产生更具体的C1结构域配体。重要的是，这些合成产品的快速表征和生物学评价前建立其结构。这些组合库的快速质谱表征的策略已被开发，评估和实施使用FAB/MS。典型的DAG-内酯衍生物的正离子FAB质谱由完整的分子离子（M+）或质子化分子（MH+），以及几个碎片离子来自酰基和亚烷基部分。该光谱是可预测的，可用于鉴别简单混合物中存在的DAG-内酯衍生物。最初，单独检查一个选定库列或行的成员，以确认质谱是可预测的，并评估化学效率。然后，这些信息被用来组装简单的4至6组分混合物，涵盖整个化学空间;然后对这些混合物进行分析和解释。随后单独分析在混合物分析期间未检测到的文库组分。混合物分析与单个分析直接相关，大大减少了待检测样品的数量，从而提高了分析周转率。从初始评估、样品制备、质谱分析、数据解释到生成报告的完整流程可在2天内完成。还将每个文库组分的等分试样存档，以备将来可能进行的分析（如果后续生物学评价需要进行额外表征）。流动注射ESI/MS不适合分析这些DAG-内酯，因为它们的高亲油性和有限的碱性。然而，流动注射-APCI/MS产生的光谱，包括MH+以及各种碎片和溶剂加合物离子，这种方法似乎是互补的FAB/MS的光谱信息。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对所有96个文库组分进行结构表征。通过三维数据库检索确认被鉴定为HIV-1整合酶抑制剂的化合物的结构特性的研究仍在继续。一系列新的细胞毒性异喹啉醌和亚氨基醌分离的海洋苔藓虫Caulibugula intermis已通过准确的质量分析，其结构解析的一部分。

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.