分子间磷酰基转移反应的研究

【作者】 苏玉倩；

【导师】 赵玉芬；

【作者基本信息】 郑州大学 ， 药物化学， 2005， 硕士

【摘要】 十多年来,赵玉芬院士研究小组已系统研究了二十种天然α-氨基酸磷酰化产物的化学活性,得到了丰硕且有重大意义的成果。N-磷酰-α-基酸在水-醇体系中能够发生成酯反应、成肽反应、磷原子上的酯交换反应和N→O迁移反应等四种类型的反应,这一点已作为N-磷酰-α-氨基酸的基本性质得到了大家的共识。 本论文在大量实验事实的基础上,首次发现N-磷酰-α-氨基酸除了上述的四类反应外,还可以发生分子间的磷酰基转移反应,即磷酰基可以从一个氨基酸残基转移到另一个氨基酸残基上,同时,这种转移反应也可以进一步推广到非氨基酸体系。本论文从以下三个部分对这种新的磷酰基转移反应进行说明: 第一部分:磷酰基转移反应的初步发现。我们利用O,O-亚苯基磷酰氯合成丝组二肽时,在用高效液相色谱-电喷雾-离子阱多级质谱(HPLC-ESI-MS/MS)联用技术对成肽产物进行了分离和鉴定的过程中,发现在丝组二肽的合成产物里含有几乎等量的副产物组组二肽(His-His)。用常规的理论难以解释组组二肽的生成,因此我们对产物的形成进行了深入的探讨,推测在反应体系中,硅醚保护的组氨酸对氨基酸五配位磷化合物中磷原子存在着亲核进攻竞争反应,初步提出了磷酰基可在不同氨基酸分子之间发生转移反应,反应结果是生成了不同的二肽产物。 第二部分:为了进一步验证分子间的磷酰基转移反应,我们将上部分讨论的O,O-亚苯基磷酰氯改换成为另外一种磷酰化试剂(二异丙氧基磷酸酯,DIPPH)。进行同样的反应后,用ESI-MS进行检测,发现反应产物的混合物中,不仅有His-His二肽,还同时检测出了His-His-His和His-His-His-His的存在,这些都进一步佐证磷酰基确实发生了转移反应。与此同时,我们设计了另外一种反应,将组氨酸的羧基进行酯化保护,然后再与二异丙氧基磷酰化的丝氨酸反应。实验结果显示,因为羧基已被甲酯化,可以阻止肽的生成,在发生分子间磷酰基转移反应后,不会进一步生成His-His等组氨酸寡肽。 第三部分:磷酰基转移反应的推广。前两部分仅涉及到了丝氨酸、组氨酸两更多还原

【Abstract】 About more than ten years, Professor Yu-fen Zhao, the member of CAS studied systemically the chemical activity of twenty natural N-diisopropyloxyphosphoryl amino acids (DIPP-aa), and got plentiful and substantial achievements. DIPP-aa that was presented in water-alcohol tended to self-activate to give peptides, esters, phosphoryl esterification and N→O migration of the phosphoryl group, and it was known by us as the basic property of DIPP-aa. Also, it’s interesting to study reactions of DIPP-aa further.In this paper, based on a lot of experiments, we found phosphoryl group could transfer from aat to aa2 when N-diisopropyloxyphosphoryl amino acid (DIPP-aai) reacted with another amino acids(aa2), that is to say, phosphoryl group could give intermolecular phosphoryl transfer reaction between two amino acids. The study could be divided three parts.The first part: the primary finding of phosphoryl transfer reaction. In this part, When we used the reaction of 0,0-phenylene phosphoryl O- trimethylsilyl serine trimethylsilyl ester and N,N-bis(trimethylsilyl) histidine trimethylsilyl ester and prepared seryl-histidine dipeptide, with the assistance, of HPLC-ESI-MS/MS, we found for the first time, there was almost equimolar amount of histidyl-histidine dipeptide in product as well as seryl-histidine dipeptide. The mechanism was speculated that there was phosphoryl transfer reaction between penta-coordinated phosphoryl serine and histidine in the reaction process. As a result, two types of dipeptide were produced.The second part: Based on a series of experiments, we found the extent of phosphoryl transfer reaction was much higher when DIPP-Ser protected by HMDS reacted with histidine protected by NMDS. Detected by ESI-MS, it was found that after phosphoryl group transfer from serine to histidine, histidine had self-assembled into His-His, His-His-His, His-His-His-His, respectively. Structures of these peptides were further confirmed by ESI-MS/MS.The third part: the generalizing of phosphoryl transfer reaction. According to the former study, we enlarged the range of research from serine and histidine to another amino acids. The result indicated basicity of amino acids was a vital factor for this kind of phosphoryl transfer reactions. With regard to the reaction of DIPP-aa1 and aa2, when basicity of aa2 was stronger than that of aa1, phosphoryl transfer could comeinto being; when basicity of aa2 was lower than that of aa1, phosphoryl transfer couldn’t occur. In order to confirm this rule further, as a non-amino acid compound, hexamethylene diamine which has stronger basicity than any amino acids was used to react with DIPP-His in the same reaction condition as before. As we expected, because of the strong basicity of hexamethylene diamine, phosphoryl group could transfer quickly from histidine to it.更多还原