Applications of New Mass Spectral Techniques

电喷雾电离质谱（ESI/MS）、大气压化学电离质谱（APCI-MS）、串联质谱（MS/MS）、基质辅助激光解吸电离（MALDI）质谱、毛细管电泳（CE）和质谱联用以及精确的质量测量是当前关注的技术。MALDI/MS的初步研究使用特殊的能量传递表面和直接激光脱附电离（LDI），而不存在矩阵，显示出快速分析小分子（分子量范围= 200-1000）的希望。该方法正在与使用常规和非常规（如石墨）基质的传统MALDI/MS方法进行比较，以确定每种方法在小分子分析中的优势和局限性。MALDI/MS与高效液相色谱（HPLC）和CE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高效液相色谱洗脱液，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进行再分析。类似的方法正在应用于CE，其中在线样品浓缩技术包括在峰收集之前对大容量样品注射进行电泳聚焦，作为CE与高分辨率MALDI/MS离线组合的制备规模方法正在研究中。我们的目标是自动化收集过程，因为涉及的体积非常小（纳升）。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）。根据由R1和R2组成的取代基的结构，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，通过快速原子轰击质谱法对合成混合物进行快速分析。这种方法与个体分析直接相关，大大减少了待检查样品的数量，结果提高了分析周转率，并适用于准确的质量测量。由于这些dag -内酯具有高亲脂性和有限的碱度，因此LC/MS和流动注射ESI/MS都不适合分析。然而，单个dag -内酯的流动注射APCI/MS产生的光谱由MH+以及各种片段和溶剂加合物离子组成。后一种方法在光谱信息方面似乎是对FAB/MS的补充，尽管它不适合用于混合物分析。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。目前正在研究无矩阵MALDI（见上文的LDI）作为解决此问题的方法，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）的核苷酸前药仍在研究中。斑马碱在抑制DNA甲基化方面表现出高活性，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。然而，在小鼠结肠癌细胞中，用黑胃果蝇脱氧核糖核苷激酶（Dm-dNK）转染后，2'-脱氧zebularine可以被磷酸化。我们观察到2'-脱氧西蓝碱对这些表达Dm-dNK的细胞具有显著的抗增殖作用，而对野生型肿瘤细胞无细胞毒作用。因此，我们正在与FDA合作伙伴一起制作和评估2'-deoxzebularine-5'-monophosphate （2'-dZeb-MP）的热酶激活前药，以规避这种代谢效率低下的激活并提高药物效力。我们通过在生理条件下具有热不稳定保护基团的环状（2'-dZeb-MP）二聚体可以生成2'-dZeb-MP，证明了这种前药概念在原则上是可行的。CE和MS分析表明，磷酸保护基团的去除顺序相似，并且得到的二核苷酸二聚体可以被磷酸二酯酶- i转化为2'-dZeb-MP。我们的直接目标是将各种环（2'-dZeb-MP）二聚体衍生物的激活动力学与其体外活性联系起来

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, combined capillary electrophoresis (CE) and mass spectrometry, and accurate mass measurement are the techniques of current interest. Preliminary studies of MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix show promise for the rapid analysis of small molecules (molecular weight range = 200-1000). This approach is being compared with conventional MALDI/MS using both normal and unconventional (e.g. graphite) matrices in order to define the advantages and limits of each for small molecule analysis. The off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) and with CE 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 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 reanalysis. A similar approach is being applied with CE where on-line sample concentration techniques involving electrophoretic focusing of large-volume sample injections prior to peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS. Our goal is to automate the collection process because of the very small (nanoliter) volumes involved. 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 comprising 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 mixtures by fast atom bombardment mass spectrometry. This approach correlates directly with individual analysis, substantially reduces the number of samples to be examined, results in enhanced analytical turn-around and is amenable to accurate mass measurement. Neither LC/MS nor flow-injection ESI/MS are suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. However, 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 appears to be complementary to FAB/MS in terms of spectral information, although it is not suitable for mixture analysis. 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. Matrix-less MALDI (see LDI above) is currently 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. 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 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. 2'-Deoxyzebularine could be phosphorylated, however, in murine colon cancer cells that had been transfected with a Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK). We observed that 2'-deoxyzebularine possessed a significant antiproliferative effect in these Dm-dNK expressing cells, while it had no cytotoxic effect against wild type tumor cells. Thus, in conjunction with our FDA collaborators, we are making and evaluating thermally and enzymatically activated prodrugs of 2'-deoxzebularine-5'-monophosphate (2'-dZeb-MP) in order to circumvent this metabolically inefficient activation and increase drug potency. We have demonstrated that this prodrug concept is feasible in principle by showing that 2'-dZeb-MP can be generated from a cyclic(2'-dZeb-MP) dimer with thermally labile protecting groups under physiological conditions. Analysis by CE and MS showed that removal of the phosphate protecting groups occurs sequentially with similar rate constants and that the resulting dinucleotide dimer can be converted to 2'-dZeb-MP by phosphodiesterase-I. Our immediate goal is to correlate the kinetics of activation of various cyclic(2'-dZeb-MP) dimer derivatives with their in vitro activity