【作者】 李欣；

【导师】 刘艳； 赵玉芬；

【作者基本信息】 厦门大学 ， 化学生物学， 2020， 硕士

【摘要】 生命起源的研究是要解释生命从何而来,然而其意义远不止追根溯源,还在于了解生命与环境、整体与部分、结构与功能的辩证关系,从而认识和阐明生命的本质。磷元素在生命起源与进化过程中起到了重要的催化与调控作用。丝组二肽是现代蛋白水解酶的前生源潜在模型,然而,早期地球环境下有效活性磷源是否可以促进丝组二肽的前生源合成?原始细胞膜在生命起源之初与蛋白、核酸之间有着重要的协同作用。那么,前生源环境下细胞膜前体是否对磷活化下的成肽、成核苷反应产生协同促进作用?上述两个基本科学问题,目前尚不清楚,缺乏系统的研究。因此,本论文主要针对以上两个问题展开研究,并主要取得了如下研究成果:一、基于HPLC-MS技术,在模拟的Lost City热液区的碱性水溶液以及海洋潮汐的干-湿循环条件下,以无机磷P3m及DAP活化的Phe、A/U及油酸(OA)反应体系为化学反应模型,研究脂肪酸膜前体对无机磷活化的成肽、成核苷酸反应的影响,发现:1)通过优化HPLC-MS分离、分析方法,鉴定出在模拟的Lost City热液区的碱性水溶液中,无机磷P3m活化Phe及核苷A反应,可以得到5’-aa-AMP的类似物酰胺-核苷酸aa-N-AMP,结合多维核磁谱图确定主产物峰为2’-Phe-N-AMP;2)在上述反应体系中引入油酸,没有检测到aa-N-AMP的生成,相对于没有油酸囊泡的参与的体系,油酸的引入对原料的转化反而不太有利;可能的原因是膜前体形成囊泡的pH条件(脂肪酸的pKa)与磷试剂活化氨基酸的条件(pH 11)不兼容;3)模拟海洋潮汐的干-湿循环条件下,研究膜前体油酸/辛酸对磷试剂P3m/DAP活化Phe、A生成寡聚核苷酸及寡肽的影响。通过SIPAL衍生化策略结合HPLC-MS技术,检测到反应体系中AMP和Phe2的生成,比对EIC峰面积发现脂肪酸的加入对成肽和核苷酸不利,并且饱和脂肪酸辛酸相比于长链不饱和脂肪酸油酸更为不利;4)建立1,2-环甘油磷酸酯和1,3-环甘油磷酸酯混合物的分离方法,鉴定出DIPP-Ser与甘油反应所得的主要膜前体产物是1,2-环甘油磷酸酯。总的来说,上述实验结果表明膜前体脂肪酸的pKa与P3m活化的最佳反应pH条件之间的兼容性对原始细胞膜协同作用的发挥至关重要。二、基于SIPAL衍生策略结合HPLC-MS技术,研究前生源冰冻环境下,无机磷试剂P3m“一锅法”活化丝氨酸和组氨酸及其他氨基酸的竞争成肽反应,并利用微波合成仪器加速反应加以佐证。研究发现:1)pH=11,-20℃冰冻的前生源模拟环境下,P3m“一锅法”活化Ser、His的自组装成肽反应体系以及80℃,2h微波辅助加速反应,都可以实现Ser-His/His-Ser等相关功能二肽的有效获得;2)在冰冻环境下,P3m“一锅法”活化Ser、His以及其他古老氨基酸(Ala/Asp/Pro)的竞争成肽反应体系中,有多种二肽产物生成,特别是Ser-His或者His-Ser、His-His在以上三组反应均可生成,并且His与其他氨基酸的成肽反应更为容易,Ser不易成肽;3)Ser/His反应体系中,第三个氨基酸的引入会对Ser-His/His-Ser的生成产生影响,Ala/Asp/Pro中,Asp的引入对于Ser-His和His-Ser的成肽效果优于Ala和Pro,说明Asp在Ser/His自组装成肽过程可能存在促进作用;4)在上述成肽反应体系中,针对Ser-His/His-Ser二肽的生成,除了 Ser-His/His-Ser混合物的生成外,还有少量Ser-His的差向异构体的检出,说明冰冻的前生源环境也存在氨基酸的消旋现象。本论文研究意义在于为无机磷活化下“核酸、蛋白、膜”三合一共起源模型提供有效的实验支持,证实无机磷磷活化下丝组二肽前生源合成路径,为功能小肽——丝组二肽作为蛋白水解酶的潜在进化雏形提供实验支持,从而帮助更好地理解磷在生命起源过程中的催化及调控作用。更多还原

【Abstract】 The study on the origin of life is not only explain where life comes from,its significance goes far beyond tracing the source,but also to understand the dialectical relationship between life and environment,whole and part,structure and function,so as to understand and clarify the essence of life.Phosphorus plays important catalysis and regulation roles in the origin and evolution of life.The Ser-His dipeptide is a potential probiotic model of modern proteolytic enzymes.However,could the available phosphorus source promote the synthesis of Ser-His in the early Earth environment?The original cell membrane has an important synergy relationship between proteins and nucleic acids at the beginning of life.Then,does the precursor of the cell membrane under the probiotic environment synergistically promote the peptide-and nucleotideformation with phosphorus activation?The two basic scientific issues are currently unclear and lack of systematic research.Therefore,our research focuses on the above-mentained two issues,and the following experimental results have achieved:Firstly,based on HPLC-MS technology,the reaction system of Phe,A or U and oleic acid(OA)with inorganic phosphorus P3m and DAP activation was investigated in detailed under the simulated alkaline aqueous environment of the Lost City hydrothermal zone and the dry-wet cycle conditions of ocean tides.The effect of fatty acid membrane precursors on the peptide and nucleotide formation in this reaction system was emphatically discussed.1)By optimizing the HPLC-MS separation and analysis method,it was found that aa-N-AMP,which is analogs of 5’-aa-AMP was produced in this reaction system of P3m activating Phe and A under the alkaline aqueous solution of the simulated Lost City hydrothermal zone.Combined with multi-dimensional NMR,the structure of the main product peak of aa-N-AMP is determined as 2’-Phe-N-AMP;2)To introduce oleic acid in the above reaction system could not detect the formation of aa-N-AMP.Compared with the reaction system without fatty acid OA vesicles,introducing oleic acid is not beneficial to the conversion of raw materials;the possible reason is the pH conditions of precursor vesicle formation are not compatible with the conditions for phosphorus reagents to activate amino acids(pH 11);3)We studied on the synthesis of oligonucleotides,oligopeptides and membranes by different phosphorus reagents P3m and DAP activating Phe,A and oleic acid or caprylic acid under the dry-wet cycle of simulated ocean tides.The AMP and Phe2 were detected in this reaction system through the SIPAL derivatization strategy combined with LC-MS technology.Compared with the EIC peak area of AMP and Phe2 respectively,it was found that the addition of fatty acids was unfavorable for peptide and nucleotides formation.Saturated fatty acid oleic acid has more negative role for peptide and nucleotides formation than the unsaturated fatty acid;4)The separation method of 1,2-cycloglycerol phosphate and 1,3-cycloglycerol phosphate mixture was established,and the main membrane precursor product obtained by treating DIPP-Ser with glycerol was identified as 1,2-cycloglycerol phosphate.Overall,the above experimental results indicate that the compatibility between the pKa of membrane precursor and the optimal reaction pH conditions for P3m activation is critical to the synergy of the original cell membrane.Secondly,based on SIPAL derivation strategy combined with LC-MS technology,the competitive peptide-forming reactions of Ser,His and other amino acids with P3m activation under the prebiotic freezing environment were explored systematically and double checked by microwave acceleration..1)"one-pot method" of Ser and His self-assembly peptide formation with P3m activation was investigated under the simulated probiotic frozen environment and 80℃ 2 h microwave-assisted condition.It was found that under above two conditions,Ser-His/His-Ser and other related functional dipeptides could be obtained effectively acquisition of;2)Under the frozen environment,P3m activates Ser,His,and other ancient amino acids(Ala/Asp/Pro)in the competitive peptide-forming reaction system,a variety of dipeptide products are produced,especially,Ser-His or His-Ser and His-His can be generated in the above three groups of reactions,and the peptide forming reaction of His and other amino acids is easier than Ser;3)In the Ser/His reaction system,to introduce the third amino acid will affect the formation of Ser-His/His-Ser.Among the three amino acids,Ala/Asp/Pro,Asp has better performance on the peptide formation of Ser-His than Ala and Pro.It indicates that Asp may promote the process of Ser/His self-assembly into peptides;4)In the above peptide-forming reaction system,in addition to the generation of Ser-His/His-Ser dipeptide,a small amount of Ser-His epimer was detected.It indicates that amino acids undergo racemization under the prebiotic frozen environment.In short,the above research results provide effective experimental support for the co-origin model of "nucleic acid,protein and membrane" activated by inorganic phosphorus,and prove that the prebiotic synthetic pathway of Ser-His dipeptide under the activation of inorganic phosphorus.It will provide experimental support for Ser-His as a potential evolutionary prototype of proteolytic enzymes,thereby helping to better understand the catalytic and regulatory role of phosphorus in the origin of life.更多还原