N-磷酸化氨基酸的合成与反应性质 ——生命化学中P-N键模型化合物的研究

论文摘要

为了理解P-N键在生命起源化学和生物化学中的可能作用，本论文系统地研究了模型化合物N-磷酸化氨基酸（N-phosphono-amino acids，简称NPAAs）的化学性质。氨基酸-磷酰胺内酸酐（Cyclic acylphosphoramidates，简称CAPAs）是一种集磷酸-羧酸混酐和磷酰胺两个官能团于一身的磷酸活化的氨基酸。自CAPA的结构被提出以来，CAPAs的化学反应几乎没有得到进一步的研究，所以本论文的第二章研究了以甘氨酸-CAPA和丙氨酸-CAPA为代表α-CAPAs分子，通过同位素标记实验(18O，15N)证明了CAPA分子具有两个亲电中心。同时，CAPA和亲核试剂如水，氨基酸，磷酸和甲醇分子反应可以生成各种磷酰化产物，如NPAAs和N-磷上单取代的磷酰化氨基酸。为了制备足量的NPAAs以及它们的类似物样品来研究NPAAs的性质和反应活性，本论文的第三章发展了两个合成方法。首先，利用第二章发现的CAPA反应性质优化了偏三聚磷酸钠（Trimetaphosphate简称P3m）和α-氨基酸的反应并通过此反应在水溶液中合成大量的α-NPAAs（产率60～91％）。这一反应的副产物无机磷酸盐可以回收并进一步转化为P3m，因此它是一个绿色环保的合成方法。第二，本论文发展了P3m／CaO方法用于制备非α-氨基酸的单磷酰化和焦磷酸化产物，这一方法利用钙离子切割并沉淀N-三聚磷酰胺衍生物的磷酸链。本论文的第四章对合成的NPAAs以及它们的类似物进行了核磁-pH滴定实验，准确地得到了一系列无邻近羧基的磷酰胺类似物的磷酰基氧和磷酰胺氮原子的酸解离结合常数（pKas）。然而，α-NPAAs的磷31核磁-pH滴定曲线在pH=0～5的区域出现了反常的变化趋势，使得对它们的磷酰基氧和羧基氧原子的pKas测定变得困难。进一步实验结果和数据分析表明α-NPAAs的磷酰基氧和羧基氧原子的pKas数值匹配，此结果为α-NPAAs的分子内氢键假说提供了支持。本论文第五章研究了NPAAs在水溶液中的各种亲核反应，包括水解，N→N磷酰基转移和N→O磷酰基转移反应。实验结果表明NPAAs的邻近官能团羧基和羟基可以催化NPAAs的水解；此外，本章还通过ESI串联质谱（正离子和负离子）研究了各种离子化状态的NPAAs在气相中的裂解途径。本论文的第六章主要研究NPAAs的生命起源化学反应，结果表明NPAAs具有成肽和磷酰基转移的能力。这一过程需要高浓度的NPAAs溶液（pH=7～8，常温）来诱导二级反应，即NPAA通过两个分子之间的缩合生成N-焦磷酸化氨基酸，N-焦磷酸化氨基酸进一步生成具有成肽活性的CAPA中间体。二价镁离子可以催化NPAA到单磷酸核苷的磷酰基转移，生成焦磷酸核苷。我们认为上述非酶促反应可能曾经存在于原始地球上。基于上述各章发现的NPAA的性质和反应活性，本论文的第七章提出了一全新的磷酰基转移机理，这一机理可以为阐明N-磷酸化赖氨酸中间体参与的生物磷酰基转移的机制提供线索。

论文目录

• Dedication
• Acknowledgement
• 摘要
• Abstract
• Chapter1:Introduction
• 1.1 Goal
• 1.2 Text framework and guide
• 1.3 Nomenclature
• 1.4 Reference
• Chapter2:Prebiotic formation of N-phosphoryl amino acids via cyclic acylphosphoramidate （CAPA）
• 2.1 Introduction
• 2.1.1 Prebiotic formation of amino acids and polyphosphates
• 2.1.2 Reaction of amino acids with trimetaphosphate （P3m）
• 2.1.3 Significance of cyclic acylphosphoramidate （CAPA）
• 2.2 Results and Discussion
• 2¹⁸O—pathway to prebiotic formation of NPAAs'>2.2.1 Bi-Electrophilicity of CAPAs towards H₂¹⁸O—pathway to prebiotic formation of NPAAs
• 2.2.2 Bi-Electrophilicity of CAPAs towards amino group
• 2.2.3 Electrophilicity of CAPAs towards phosphate and methanol—pathway to prebiotic formation of N-（mono-substituted）phosphoryl amino acids
• 2.2.4 CAPAs vs. NCAs
• 2.2.4.1 Bi-electrophilicity
• 2.2.4.2 Peptide formation, phosphate activation and phosphorylation
• 2.3 Summary and perspective
• 2.4 Experimental section
• 2.5 Reference
• Chapter 3: Preparation of N-phosphono-amino acids （NPAAs） and their analogues
• 3.1 Introduction
• 3.2 Results and Discussion
• 3.2.1 N-phosphorylation of alpha-amino acids by trimetaphosphate （P3m）
• 3.2.1.1 General procedure
• 3.2.1.2 Recycling of polyphosphates
• 3.2.1.3 Purification and Purity determination
• 3.2.1.4 pH dependence of synthesis of NPAA
• 3m/CaO'>3.2.2 N-phosphorylation of amines without containing a alpha carboxyl group by P₃m/CaO
• 3, isopropyl amine and pyrrolidine'>3.2.2.1 Phosphorylation of NH₃, isopropyl amine and pyrrolidine
• 3.2.2.2 N-Phosphorylation of β-alanine, γ-amino-butyric acids and lysine
• 3.2.2.3 N-Phosphorylation of ethanol amine and propanol amine
• 3.3 Summary and perspective
• 3.4 Experimental section
• 3.5 Reference
• Chapter 4: NMR Titration of NPAAs
• 4.1 Introduction
• 4.2 Results
• 31P NMR-pH titration of simple phosphoramidates'>4.2.1³¹P NMR-pH titration of simple phosphoramidates
• 31P NMR-pH titration ofphosphoramidic acid, N-isopropylphosphoramidic acid,2-hydroxyethylphosphoramidic acid and 3-hydroxypropyl phosphoramidic acid'>4.2.1.1³¹P NMR-pH titration ofphosphoramidic acid, N-isopropylphosphoramidic acid,2-hydroxyethylphosphoramidic acid and 3-hydroxypropyl phosphoramidic acid
• 31P NMR-pH titration of N^ε-phosphono-lysine'>4.2.1.2³¹P NMR-pH titration of N^ε-phosphono-lysine
• 31P NMR-pH titration of NPAAs'>4.2.2³¹P NMR-pH titration of NPAAs
• 31P NMR titration of N-phosphono-proline'>4.2.2.1 Full-pH ³¹P NMR titration of N-phosphono-proline
• 31P NMR-pH titration of N-phospho-amino acids with primary amino group'>4.2.2.2³¹P NMR-pH titration of N-phospho-amino acids with primary amino group
• 4.3 Discussion
• a of phosphoramidate nitrogen'>4.3.1 pK_a of phosphoramidate nitrogen
• a of phosphoryl oxygen in phosphoramidates'>4.3.2 pK_a of phosphoryl oxygen in phosphoramidates
• as of α-NPAAs'>4.3.3 pK_as of α-NPAAs
• a match and intramolecular strong H-bond between phosphate and carboxylate in α-NPAAs'>4.3.4 Implications to pK_a match and intramolecular strong H-bond between phosphate and carboxylate in α-NPAAs
• a match in α-NPAAs'>4.3.4.1 pK_a match in α-NPAAs
• 4.3.4.2 Intramolecular H-bond in α-NPAAs
• 4.3.4.3 Conformational analysis of α-NPAAs
• 4.4 Summary and perspective
• 4.5 Experimental section
• 4.6 Reference
• Chapter 5: Reactivity of NPAAs
• 5.1 Introduction
• 5.2 Results and Discussion
• 5.2.1 Hydrolysis of NPAAs and related analogues
• 2L in N^ε-P-Lys due to successive protonations'>5.2.1.1 Comparison of hydrolysis rates of species HL and H₂L in N^ε-P-Lys due to successive protonations
• 5.2.1.2 Rate enhancement contributed from vicinal carboxyl group in NPAAs
• 5.2.1.3 Rate enhancement contributed from vicinal hydroxyl group in phosphoramidates
• 5.2.2 Intermolecular N→O phosphate transfer mediated by N-P-Pro
• 5.2.3 N→Nphosphate transfer mediated by N-P-Pro
• 5.2.4 Reactivity of NPAAs in gas phase
• - and [NaHL]^- in negative ESI MS'>5.2.4.1 Fragmentation pathways of NPAA monoainon [H2L]^- and [NaHL]^- in negative ESI MS
• 3HL]⁺and [Na₂H₂L]⁺ in positive ESI MS'>5.2.4.2 Fragmentation pathways of NPAA monocation [Na₃HL]⁺and [Na₂H₂L]⁺ in positive ESI MS
• 5.3 Summary and perspective
• 5.4 Experimental section
• 5.5 Reference
• Chapter 6: Peptide Formation and Phosphate Transfer Mediated by NPAAs---Prebiotic Roles of NPAAs
• 6.1 Introduction
• 6.2 Results and Disscution
• 6.3 Summary and perspective
• 6.4 Experimental section
• 6.5 Reference
• Chapter 7: Implications to biological phosphate transfer
• 7.1 Introduction
• 7.2 Reactivity of NPAAs provides implications to the biological phosphate transfer mechanism
• 7.2.1 Knowledge on model compounds NPAAs
• 7.2.2 Novel enzymatic phosphate transfer mechanisms with N-phospholysine as intermediate
• 7.3 Reference
• Supporting information
• NMR spectra
• Academic communications during Ph.D study