【作者】 周宁；

【导师】 赵玉芬；

【作者基本信息】 厦门大学 ， 有机化学， 2003， 博士

【摘要】 利用组合化学技术建立的肽库已被广泛用于寻找与受体和酶结合的生物活性配体、研究抗体—抗原的相互作用以及探索蛋白—蛋白相互作用的详细信息等。最近，肽和细胞特异性相互作用的研究也引起人们的关注。此外，寻找药物研究领域中具有生物活性的肽也是肽库应用的一个广阔领域。因此，肽库巨大的应用价值吸引了许多科学家致力于肽库的构建、筛选和应用等领域的跨学科研究。 目前，肽库的化学合成方法主要有以下五种：1．多中心合成法：2．茶叶袋法；3．裂分合成法；4．光控合成法；5．纤维素和交联聚苯乙烯—聚乙烯为载体的组合合成。这些方法除了茶叶袋法外大多采用固相组合合成技术，而液相组合合成技术与固相方法相比，则发展比较缓慢。有机磷试剂作为一类肽缩合试剂已被认识并在多肽合成中得到应用，但其使用对象是已保护的氨基酸，且很少见用于肽库构建的报导。赵玉芬教授领导的生命有机磷研究小组多年的研究结果发现，N，O-二(三甲基硅基)氨基酸(BTMS—AA)与O，O-二芳基磷酰氯(如O，O-亚苯基磷酰氯)反应生成N-磷酰-α-氨基酸后，可以发生自组装反应，得到七肽或八肽自组装产物。 本文利用核磁共振，电喷雾质谱等现代分析手段，对氨基酸在简单磷试剂三氯氧磷辅助下的成肽反应作了详细的研究，构建了非极性氨基酸的均肽库、杂肽库以及缀合物库。详细的研究结果如下： 研究了二十种天然氨基酸在三氯氧磷辅助下的成肽情况，发现非极性氨基酸能在有机溶剂中形成2～14的均肽，且所成肽的长度随着时间的延长而增长，但并不是无限增加，而是到了一定时间以后有一个接近平衡的状态，这个时间一般在96小时左右。在反应时间恒定的情况下，所成肽的长度随着反应所用溶剂极性的增加而增加，随着所用氨基酸极性的增加而减短。一些极性氨基酸能在三氯氧磷辅助下形成短肽。利用电喷雾质谱研究了氨基酸均肽在多级质谱中的裂解规律，发现氨基酸的均肽或均肽酯在多级电喷雾质谱中易发生b型断裂，形中文摘要成相应的碎片离子，这一结果对于不同长度多肤的序列分析具有重要的借鉴意义。 研究了磷试剂辅助下氨基酸自组装成肤反应的机理，发现在三氯氧磷辅助下，硅烷基化氨基酸的自组装成肤反应经历了一个五配位磷的中间体，反应过程类似N一磷酞一a一氨基酸的自组装成肤反应，经历了氨基酸的活化、肤链的延长以及肤链的终止三个阶段。在三氛氧磷辅助下，氨基酸自身的成肤反应机理一般认为是经过梭端活化成混配后另一氨基组分进攻混配的拨基形成了酞氨键。我们根据自己的实验以及量子化学的理论计算结果，提出了一种可能的五配位磷中间体的反应机理。 研究了两种氨基酸同时在三氯氧磷辅助下的成肤情况，发现非极性氨基酸亮氨酸和撷氨酸与三氯氧磷的反应形成了种类最丰富的杂肤库，肤库中各种长度肤的含量在质谱图上呈波兹曼分布，Bi。一To。厂M软件分析的结果表明非极性氨基酸形成的肤库具有序列多样性，这样的杂肤库对于生物活性物质的筛选具有十分重要的意义。 以L一苯丙氨酸、L一亮氨酸及L一撷氨酸和三氯氧磷的反应产物为底物，通过选择不同的含轻基或氨基的生物活性分子如薄荷醇 (L一menthol)、金鸡纳陡(L一einehonidine)、节胺(benZylamine)以及核昔类药物山T等来终止反应，得到各种可能含生物活性配体的小型缀合物肤库(月太的长度2~n)，并用磷酞化试剂对亮氨酸的均肤库进行了N一端磷酞化，利用电喷雾质谱技术鉴定了缀合物肤库的组成。这样的缀合物肤库和磷酞化肤库对于研究生物活性小分子对蛋白、核酸以及细胞的识别作用将提供新的方法并获得新的认识，对于组合化学的发展以及研究肤与生命体系的识别作用都具有重要的理论意义和潜在应用价值。 研究了磷酞化肤在电喷雾多级质谱中的裂解规律。研究结果发现，磷酞化多肤的负离子多级质谱和正离子多级质谱都表现出和磷酞化氨基酸不一样的裂解规律，多级质谱图中均有从准分子离子峰首先脱去分子量为164的中性分子(即磷酞基，磷酞化氨基酸是失去异丙烯)的碎裂反应，我们推测其裂解途径可能是经过了两个五元环质子传递中文摘要过程。进一步的研究发现，随着肤链的延长，在多级质谱图上失去磷酞化基团(一164)碎片峰的强度也随之增加，而失去异丙烯的碎片峰强度随之减弱，且失去磷酞化基团所需的能量会随之降低。我们认为多肤骨架上多个极性酞胺基团吸电子诱导效应的叠加使得P一N键上的电子云更多的偏向于N原子，N端负电性增加，更易结合质子，其磷氮键就较磷酞化氨基酸上的磷氮键容易断裂，因此在多级质谱上首先是磷酞基脱去而不是异丙基失去。 利用商品化的竞争ELISA试剂盒，对合成的三个均肤库进行了初步的生物活性筛选工作。研究结果发现，在加入亮氨酸，苯丙氨酸，撷氨酸均肤库的情况下，酶标抗体与抗原的结合更加紧密，不易被洗脱，导致在相同洗板情况下，有更多的抗体结合在抗原上。在相同时间内，对底物的利用增加，显色效果增强。这一结果提示我们，液相合成的亮、苯丙、撷氨酸?更多还原

【Abstract】 Peptide libraries have been widely used to identify and examine the specificities of immune system, for screening bioactive ligands that bind to DNA and enzymes, and recognized as powerful tools for searching detailed information about protein-protein interactions and so on. Also, it has been widely used by the drug industry to discover biological active compounds. There are some methods for constructing peptide libraries such as: multipin synthesis, tea-bag method, split synthesis and combinatorial library by light directed. Organophosphorus compounds have been used as coupling regents to synthesize bioactive peptides, but rarely reported for construction of peptide libraries. In previous work of Prof. Zhao Yu-Fen, it was discovered that polypeptides were formed by self-assembly of N, O-bis(trimethylsilyl) amino acids with the assistance of O, O-phenylene phosphoryl chloride.In this paper, the reactions of amino acids and phosphorus oxychloride were studied. The homo-peptide libraries, hetro-peptide libraries and the peptide conjugate libraries were constructed by this method. 31P NMR and ESI-MS/MS were used to study the mechanism and products of the reaction and confirm the structures of the peptides. These results were illustrated as following:The reactions of twenty natural amino acid and phosphorus oxychloride were studied. It was found that nonpolar a-amino acids could be assembled into 2-14 homo-peptides by phosphorus oxychloride activation, after quenching with water or various alcohols, the reaction mixtures yielded the corresponding peptides or peptide esters, respectively, which were analyzed by ESI-MS and ESI-MS/MS. As the reaction time was prolonged, the length of peptides increased, but after 96 hours later, the maximum length of the peptides appeared to reach equilibrium. It was found that withthe same reaction time, the lengths of peptides increased as the polarity of the solvent increased and decreased as the polarity of amino acids increased. Some polar amino acids could be assembled into 2-4 short peptides. Multistage electrospray mass spectrometry of homo-peptides and peptide esters were studied, and the results showed that the fragmentation pathway for these protonated homo-peptides yielded mainly "b" type ions, consistent with the conclusion that the products were the oligo-peptides and peptide esters.The reaction mechanisms of peptide formation by phosphorus oxychloride activation were investigated. It was found that with phosphorus oxychloride assistance, trimethylsilyl amino acids could form cyclic or spiro penta-coordinate phosphorus compounds firstly, then assembled into polypeptide. There were three steps involved in the self organization reactions of trimethylsilyl amino acids, namely, the activation of amino acid, the elongation of peptide chain and the termination of the chain reaction. The result of theoretical calculations showed that the reactions of amino acids and phosphorus oxychloride might go through penta-coordinate phosphorus compounds intermediate.The reactions of two non-polar amino acids with phosphorus oxychloride were investigated. It was found that in addition to the homo-peptides, the hetro-peptides were also formed. Several hetro-peptide libraries were synthesized and analyzed by Bio-Tools?software with ESI-MS/MS technique. The products of the libraries were studied and the diversity of the peptide libraries was discussed. It was discovered that the reaction of phosphorus oxychloride with L-Val/L-Leu produced the most abundant hetro-peptide libraries with sixty and eight kinds of peptides sub-libraries based on molecular weight difference.Glycosylation and phosphorylation of peptides drug play an important role in the pharmacokinetic phase. The synthesis of modified glycopeptide and phosphoryl peptide may therefore represent a mean to improve thevabsorption and distribution of bio-available therapeutic peptides. It was proposed that glycopeptide and phosphoryl peptide not only could increase their resistance to enzymatic degradation, but a更多还原