【作者】 陈晓岚；

【导师】 赵玉芬；

【作者基本信息】 郑州大学 ， 有机化学， 2004， 博士

【摘要】 作为近年来涌现的新学科，化学生物学（Chemical Biology）融合了化学、生物学、物理学、信息科学等多个相关学科的理论、技术和研究方法，跳出了传统的思路和方法，从更深的层面去研究生命过程。虽然目前还没有一个公认的化学生物学的定义和研究范围，但从分子的基础去研究和了解大分子之间、化学小分子与生物大分子之间的相互作用，以及这些作用对生命体系的调节、控制都是很多研究的共同点。磷酰胺及其酯类衍生物大多具有广泛的生物活性，它们参与生命有机体内的物质转化并在其生命过程中发挥着重要作用。本文中，首先选择结构相对较简单苯二酚和氨基酚类化合物作为起始改造模型，研究Atherton-Todd反应应用于这类化合物的较佳反应条件，探讨产物的ESI-MS／MS裂解规律；研究了含有-COOH的芳环类化合物参与Atherton-Todd反应的特点及作用机理；首次将经简化的Atherton-Todd反应应用于4-氨基安替比林磷酰化结构改造；最终将较成熟的简化的Atherton-Todd反应扩大到黄酮和异黄酮的结构改造上，合成一批新型的磷酰化黄酮和异黄酮；电喷雾质谱（Electrospray ionization mass spectrometry,ESI-MS）是近年来发展起来的一类新的软电离质谱仪，已广泛应用于弱相互作用、非共价复合物的检测和研究，本文以现代生物质谱ESI为主，其它手段为辅，开展磷酰化黄酮和黄酮的与蛋白发生弱相互作用的研究，得出磷酰化黄酮更易与蛋白发生弱相互作用的结论，确立磷酰基在分子与蛋白发生相互作用时的关键性的作用。 一、以亚磷酸二乙酯为磷酰化试剂，通过Atherton-Todd反应分别对邻、间、对苯二酚和氨基苯酚六种化合物进行了磷酰化改造；首次通过此方法得到了相应的二磷酰酯衍生物并给出了其波谱数据；对该类磷酸酯类化合物的电喷雾质谱裂解规律研究结果表明：只有当两个磷酰化基团处于相邻位置时，其五元环磷化合物碎片离子的形成才成为可能，较稳定的五元环离子可以作为判断此类磷酸酯取代基在苯环上相对位置的方法。 二、采用Atherton-Todd反应对水杨酸进行磷酰化结构改造，来合成水杨酸羟基磷酸酯，产率偏低。通过ESI—MS／MS和³¹P NMR跟踪反应，探明机理，发现水杨酸的-COOH和-OH均参与了磷酰化反应，与-OH不同的是，-COOH郑州大学博创七学位论文更具反应活性，一COOH在形成混醉高能键后，能迅速接受另一水杨酸分子一COOH的进攻，使反应复杂化。混配高能键的形成在整个反应进程中起着非常重要的作用，导致了大部分的磷酚化试剂最终被转化成四异丙基焦磷酸酷。 三、将Atherton一Todd反应运用到4一氨基安替比林官能团的结构改造上。采用二异丙基亚磷酸醋（DIPPH）对4一氨基安替比林进行磷酞化，生成无色菱形的新的磷酞产物N一DIPP一4一Antiprine，其结构被x一ray晶体衍射、以及详细的NMR波谱数据所证实确认。 四、将经改造的并较简化的Atheron一Todd反应扩大到黄酮和异黄酮的磷酞化结构改造上，选择了7一经基黄酮，白杨素，7一轻基4’一氯异黄酮以及7一轻基4’-氯异黄酮为反应物，成功合成了一系列新的磷酞化黄酮及磷酞化异黄酮，所有的产物结构均由ESI一MS/MS，NMR和IR证实，其中三个目标产物培养成了单晶，单晶结构经X一ray晶体衍射数据证实。 五、大量ESI图谱显示，经改造的系列磷酞化黄酮易和溶菌酶、肌红蛋白、细胞色素C和胰岛素这一系列蛋白发生弱相互作用，但ESI则未检测到黄酮一蛋白质非共价复合物的存在。磷酞化黄酮对蛋白质更具亲和力。ESI证实了磷酞化黄酮的磷酞化部分以及碱性氨基酸残基作为作用位点在此非共价复合物形成中的不容忽视的作用，也进一步展示了蛋白的构象对弱相互作用形成的复合物化学计量的影响;以荧光法为方法的必要补充，研究比较了气相和液相中弱相互作用的情况，根据荧光碎灭双倒数图计算了磷酞化黄酮与测试蛋白之间的结合常数，证明随着温度的升高，磷酸化黄酮与测试蛋白的结合常数逐渐降低，说明了两者之间形成了复合物，从荧光光谱可以看出磷酞化黄酮对测试蛋白产生荧光碎灭，且碎灭过程是由于形成了复合物而引起的静态碎灭，根据F6ster能量传递原理计算出磷酞化黄酮在测试蛋白分子上的结合距离，并根据热力学参数确定了磷酞化黄酮与测试蛋白之间的作用力类型为电荷作用力。结果显示Esl提供了关于小分子与蛋白作用的较真实的情况，说明液态中形成的复合物能够在离子化气化过程中被保存并最终被检测到。另外，本实验室又从细胞水平开展了磷酞化黄酮和黄酮对HELA肿瘤细胞抑制活性的初步研究，表明磷酞化黄酮比黄酮具有更强的抑制活性。本实验方法为进一步研究小分子与蛋白质的弱相互作用，筛选更具活 摘要性的化合物提供快速、直观、可信的方法。更多还原

【Abstract】 It is an established fact that esters of phosphoric acid have wide bio-activities and play a vital role in many biological processes. Cellular functions are often triggered by weak non-covalent enzyme-substrate, protein-ligand, protein-protein or antibody-antigen interactions. Since its introduction, ESI-MS has served a powerful tool in providing evidence in support of the existence of non-covalently associated marcromolecular complexes in the gas phase. In the study described in this thesis, aminophenols and dihydroxybenzenes were first phosphorylated by modification of the classical Atheron-Todd procedure in our primary stage; Then the mechanism of the reaction between salicylic acid and dialkyl phosphite was studied by electrospray ionozation mass spectrometry and 3lP NMR; 4-aminoantipine was further phosphorylated by modification of the classical Atheron-Todd procedure; finally, we have been able to enlarge the scope of the Atheron-Todd reaction to flavone and isoflavone phosphorylation. Electrospray ionization results showed that the phosphorylated flavonoids could form non-covalent complexes with many proteins such as lysozyme, myoglobin, bovine insulin and cytochrome c, while non-covalent complexes were not detected with the mixed solution of the chrysin and proteins. The phosphorylated flavonoids possess relatively stronger affinities and form non-covalent complexes with the proteins more easily than the unphosphorylated compounds.1).Aminophenols and dihydroxybenzenes were phosphorylated by modification of the classical Atheron-Todd procedure. Their structures were elucidated by NMR and ESI-MS/MS. Results showed that the cleavage pathways of these phosphorylated compounds were closely related to the relative positions of the phorsporylating groups. Comparatively stable five-membered ring ions were produced when the two phosphorylated functional groups were in the ortho position. These stable five-membered ring ions can be considered as indicators for ortho functional groups of phenols.2).When the Atheron-Todd reaction was employed to phosphorylate salicylicacid, the yield was quite low. But further experiments show that the Atheron-Todd reaction could successfully be applied to phenol and ethyl-salicylate phosphorylation. The reaction between salicylic acid and dialkyl phosphite was then traced by electrospray ionozation mass spectrometry and 31P NMR. The mechanism proposed based on ESI-MS results and 3IP NMR profiles. The results show that both the -COOH and -OH of salicylic acid were phosphorylated, but the -COOH possessed stronger reaction ability from the onset. Finally, most of the DIPPH was translated into tetraisopropyl pyrophosphate through reacting with -COOH. The energy-rich carboxylic-phosphoric mixed anhydrides played important roles in the whole process.3).4-aminoantipine was phosphorylated by our modified classical Atheron-Todd procedure. Their structures were elucidated by NMR, ESI-MS and x-ray data.4).We have been able to enlarge the scope of the Atheron-Todd reaction to flavone phosphorylation. The procedures are practical. 7-hydroxyflvone, chrysin, 4’-chloro-7-hydroxyisoflavone and 4’-fluo-7-hydroxy-isoflavone were phosphorylated by the Atheron-Todd reaction. The expected phosphates were obtained in good yields. All the structures of target products were determined by ESI-MS/MS, NMR and IR for the first time. The structures of three phosphated fiavones were further confirmed by X-ray diffraction.5).The mixed solutions of the five phosphorylated flavonoids and different proteins, such as insulin, lysozyme and cytochrome c were detected by ESI-MS. The results show that all the phosphorylated flavonoids could form non-covalent complexes with proteins mentioned above, while noncovalent complexes were not detected from the mixed solution of the chrysin or 7-hydroxyflavone with proteins. The phosphorylated flavonoids possess relatively stronger affinities and form non-covalent complexes with the proteins more easily than the unphosphorylated compounds. The protonated basic amino更多还原