Applications of New Mass Spectral Techniques

电喷雾电离质谱法（ESI/MS）、大气压化学电离质谱法（APCI-MS）、串联质谱法（MS/MS）、基质辅助激光解吸电离（MALDI）质谱法、用于元素组成测定的精确质量测量以及毛细管电泳（CE）和质谱法的结合仍然是当前关注的技术。MALDI/MS使用特殊的能量传递表面和直接激光解吸电离（LDI）而不存在矩阵，显示出对小分子（分子量范围= 200-1000）的快速分析的一些希望。用于传统MALDI/MS的低分子量基质中的离子通常模糊了质谱的低质量范围，因为它们比样品多出100到1000倍。LDI质谱可以从各种各样的分子结构中获得，包括那些不容易吸收紫外线激光能量的化合物。目前正在进行比较LDI和传统MALDI/MS的研究，以确定各自在小分子分析中的优势和局限性。我们的目标是选择MALDI或LDI方法，以提供最佳机会提供明确的分子量信息。正常和非常规（如石墨）MALDI基质都在本研究中进行了研究。LDI/MS光谱通常表现出广泛的碱金属离子阳离子化，这可能使分子量的测定出现问题，特别是当样品不纯或混合物时。使用铅笔芯中的石墨获得的MALDI光谱也显示出相同的行为，除了根据铅笔芯的来源有很大的变化。以3,4-二羟基苯甲酸（DHB）为基体，获得了最佳的MALDI整体小分子质谱。MALDI/MS与高效液相色谱（HPLC）的离线结合也在研究中，作为复杂生物和合成混合物靶向分析的替代方法。该方法包括在MALDI样品板上自动收集和定位HPLC洗脱液以及适当的基质，用于后续的质谱分析，包括精确的质量测量和源后衰减碎片分析或质谱/质谱分析。与在线LC/MS相比，这种方法的一个优点是生成原始分离样品的存档以供重新分析。合成衍生物建立在约束二酰基化甘油支架（dag -内酯）是有效的激动剂的蛋白激酶C （PK-C）。根据酰基R1和R2对应的取代基的结构，这些dag -内酯表现出不同程度的PK-C同工酶特异性。一种固相组合方法被应用于LMC中，以研究R1和R2的化学多样性，以产生更特异性的C1结构域配体。重要的是，这些合成文库的快速表征和它们的结构建立在生物学评价之前。用于初步表征这些合成dag -内酯文库的一种方法采用结合分析策略，通过快速原子轰击（FAB）质谱法对合成的简单混合物进行快速分析。这种方法大大减少了要检查的样品数量，结果在提供广泛的结构和样品信息的同时，提高了分析周转率。它也适用于快速准确的质量测量，以确定元素组成，并提供额外的结构信息。由于这些化合物的高亲脂性和有限的碱度导致电离非常差和可变，因此ESI/MS不适合分析这些dag -内酯。单个dag -内酯的流动注射APCI/MS产生由MH+以及各种片段和溶剂加合离子组成的光谱。后一种方法，特别是如果使用MS/MS技术，在光谱信息方面似乎是FAB/MS的补充，尽管它不适合混合分析。然而，流动注射APCI/MS可实现自动化，并允许在无人值守的情况下对同一样品进行多次分析。MALDI/MS作为快速表征这些小分子文库的工具也很有用，尽管可变的碱金属离子阳离子化使光谱解释变得复杂。基质优化（见上文）和样品沉积过程的自动化正在研究中，作为解决这一问题的方法，并提供更快速的分析方法。我们的目标是在一天内对所有96个库组件进行全面的结构表征。口服活性DNA甲基转移酶（DNMT）抑制剂2(1H)-嘧啶核苷（zebularine）的核苷和核苷酸前药仍在研究中。在体内和体外均表现出较高的抑制DNA甲基化的活性，但与其他已知的DNMT抑制剂相比效力较低。据推测，在药物与DNMT酶之间形成紧密复合物，从而发挥DNMT抑制剂的作用之前，需要将zebularine掺入DNA中。我们之前已经表明，斑马草碱的细胞内代谢激活既复杂又低效，只导致非常有限的DNA掺入。由于DNA整合需要激活的2'-脱氧核苷酸，因此DNA整合的代谢瓶颈似乎是核糖核苷酸还原酶将zebularine-5'-二磷酸转化为2'-脱氧zebularine-5'-二磷酸。2'-脱氧西蓝碱本身对DNA甲基化没有抑制活性。我们发现2'-脱氧西蓝碱缺乏活性是由于它是哺乳动物细胞激酶的一种极差的底物，负责初始磷酸化步骤。斑马碱的第二个问题是，在母体药物进入细胞并被磷酸化之前，它在体内会被醛氧化酶迅速代谢。核苷前药是一种抗醛氧化酶的药物，可以被血浆酯酶激活，在LMC中被合成，试图增加细胞内的斑马碱浓度，从而激活整体代谢。色谱和质谱方法被用于辅助这些前药的合成，并评价它们在体外的化学和酶稳定性。作为原理证明，将使用CE和MS对处理细胞的提取物进行分析，以评估是否有任何磷酸化代谢物的产生增强。

Electrospray ionization mass spectrometry (ESI/MS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), tandem mass spectrometry (MS/MS), matrix-assisted laser desorption ionization (MALDI) mass spectrometry, accurate mass measurement for elemental composition determination, and combined capillary electrophoresis (CE) and mass spectrometry remain the techniques of current interest. MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix shows some promise for the rapid analysis of small molecules (molecular weight range = 200-1000). The ions from the low-molecular-weight matrices used for conventional MALDI/MS often obscure the low mass range of the mass spectrum because they are in 100- to 1000-fold excess to the sample. LDI mass spectra could be obtained from a wide variety of molecular structures including those compounds that would not be expected to readily absorb energy from a UV laser. Studies are ongoing to compare LDI with conventional MALDI/MS in order to define the advantages and limits of each for small molecule analysis. The goal is to choose the MALDI or LDI method that gives the best chance of providing unambiguous molecular weight information. Both normal and unconventional (e.g. graphite) MALDI matrices are being investigated in this study. LDI/MS spectra usually exhibit extensive alkali metal ion cationization and this can make determination of the molecular weight problematic, especially if the sample is impure or is a mixture. MALDI spectra obtained using graphite from pencil lead also show the same behavior in addition to substantial variation depending on the source of the pencil lead. The best overall small molecule MALDI mass spectra are obtained using 3,4-dihydroxybenzoic acid (DHB) as a matrix. The off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) is also under investigation as an alternate approach for the targeted analysis of complex biological and synthetic mixtures. This approach involves automatically collecting and spotting the HPLC eluent along with an appropriate matrix on a MALDI sample plate for subsequent MS analysis, which can include accurate mass measurement and post-source decay fragmentation analysis or MS/MS. An advantage of this approach over on-line LC/MS is the generation of an archive of the original separated sample for re-analysis. Synthetic derivatives built on a constrained diacylated glycerol scaffold (DAG-lactones) are potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents corresponding to the acyl groups R1 and R2, these DAG-lactones exhibit varying degrees 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 libraries be rapidly characterized and their structures established before biological evaluation. One method used for the initial characterization of these synthetic DAG-lactone libraries employs a combine-and-analyze strategy with rapid analysis of the resultant simple mixtures by fast atom bombardment (FAB) mass spectrometry. This approach substantially reduces the number of samples to be examined, results in enhanced analytical turn-around while providing extensive structural and sample information. It is also amenable to rapid accurate mass measurement to determine elemental composition and provide an additional dimension of structure information. ESI/MS is not suitable for analysis of these DAG-lactones because the high lipophilicity and limited basicity of these compounds results in very poor and variable ionization. Flow-injection APCI/MS of individual DAG-lactones produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach, especially if MS/MS techniques are used, appears to be complementary to FAB/MS in terms of spectral information, although it is not suitable for mixture analysis. However, flow-injection APCI/MS is amenable to automation and allows multiple analyses of the same sample to be carried out unattended. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries although variable alkali metal ion cationization complicates spectral interpretation. Optimization of matrix (see above) and automation of the sample deposition process are under investigation as an approach to resolve this problem and provide an even more rapid analysis approach. Our goal is the comprehensive structural characterization of all 96 library components in one day. Nucleoside and nucleotide prodrugs of the orally active DNA methyltransferase (DNMT) inhibitor 2(1H)-pyrimidinone riboside (zebularine) continue under investigation. Zebularine exhibits high activity in inhibiting DNA methylation both in vitro and in vivo but low potency relative to other known inhibitors of DNMT. It is postulated that incorporation of zebularine into DNA is required before the drug can function as an inhibitor of DNMT by formation of a tight complex between it and the enzyme. We have previously shown that the intracellular metabolic activation of zebularine is both complex and inefficient, resulting in only very limited DNA incorporation. Since DNA incorporation requires the activated 2'-deoxynucleotide, the metabolic bottleneck for DNA incorporation appears to be the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by ribonucleotide reductase. 2'-Deoxyzebularine itself shows no inhibitory activity for DNA methylation. We have found that this lack of activity for 2'-deoxyzebularine is due to it being an extremely poor substrate for the mammalian cellular kinases responsible for the initial phosphorylation step. A second problem with zebularine has been the rapid in vivo metabolism of the parent drug by aldehyde oxidase before it can enter cells and be phosphorylated. Nucleoside prodrugs, which are resistant to aldehyde oxidase and which can be activated by plasma esterases, are being synthesized in the LMC in an attempt to increase intracellular concentrations of zebularine and hence overall metabolic activation. Chromatographic and mass spectral methods are being used to assist the synthesis of these prodrugs and to evaluate their chemical and enzymatic stability in vitro. As a proof of principle, analysis of extracts of treated cells by CE and MS will be used to evaluate whether there is any enhancement of the production of phosphorylated metabolites.