METASTABLE PHASES DIRECT POLYMER EMERGENCE AND EVOLUTION IN DYNAMIC CHEMICAL NETWORKS Open Access

Chen, Chenrui (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/st74cq56t?locale=en
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Abstract

The de novo construction of peptide surrogate polymers linked with reversible acetals is demonstratedd in novel dynamic chemical networks. By changing the oxidation state of the amino acid, we demonstrate environmentally responsive dynamic peptide network that allows for specific oligomer synthesis and prion-like selection. Moreover, these dynamic networks are susceptible to infection by protein templates that template morphologically distinct supramolecular materials. This process crosses the critical chemical threshold necessary for self-organizing alternative polypeptide scaffolds and abiotic biopolymer evolution and provides the first empirical model for this polymer threshold of life.

Table of Contents

Chapter 1: Potential of Dynamic Chemical Network for Chemical Evolution ................... 1

1.1 Chemical evolution - fundamental to Biological evolution ............................................. 1 1.2 Two types of information transfer in evolution: digital and analog (genetic and epigenetic) .................................................................................................................................. 3 1.3 Emergence of the ribosome through mutualism between nucleic acid and amino acid polymers ............................................................................................................................. 5 1.4 Dynamic Combinatorial Chemistry and Dynamic Chemical Network ............................ 7 1.4.1 Reversible Reactions in DCN ................................................................................... 8 1.4.2 External Templating Shifts Equilibrium in DCNs .................................................. 10 1.4.3 Internal Templating Shifts Equilibrium and Drives Self-assembly in DCNs ......... 11 1.4.4 Self-replicating DCN may serve as the chemical threshold for biopolymer emergence and evolution ....................................................................................................... 14 1.5 Construction of Dynamic Chemical Network in terms of three dimensions ................. 15 Chapter 2: Exploring Reversible Acetal Linkages for Constructing Dynamic Chemical Networks ................................................................................................. 17 2.1 Introduction .................................................................................................................... 17 2.1.1 Reversible Imine and Acetal linkages in Nucleic Acid Dynamic Chemical Networks ............................................................................................................................... 17 2.1.2 Extending Reversible Acetal linkages in Peptide Dynamic Chemical Networks ... 20 2.1.3 Understanding Stereochemistry in Acetal Chiral Centers ...................................... 22 2.2 Materials and Methods ................................................................................................... 23 2.2.1 Reagents .................................................................................................................. 23 2.2.2 Preparation of Free Amine Substrates by Neutralizing Ser, Cys and Asn Ester Hydrochloride Salts ............................................................................................................... 24 2.2.3 Mixing of Ser/Cys/Asn with the aldehyde for N, O-/ N, S-/ N, N-acetal product, respectively ............................................................................................................. 25 2.3 Results and Discussions ................................................................................................. 25 2.3.1 N, O-acetal Linkage ................................................................................................ 25 2.3.2 Anomeric effect observed by experiments in polar solvents .................................. 30 2.3.3 Kinetics and thermodynamics of N,O-acetal in different solvents ......................... 31 2.3.4 N,N-acetal linkage .................................................................................................. 33 2.3.5 Diastereoselectivity of N,N-acetal in MeCN .......................................................... 39 2.3.6 Kinetics and Thermodynamic Comparison of N,O-; N,S-; N,N-acetal Condensations in MeCN ........................................................................................................ 41 2.3.7 Temperature effect on diastereoselectivity of N,O-; N,S-; N,N-acetals ................. 42 2.4 Discussion ...................................................................................................................... 45 Chapter 3: NF-CHO Dynamic Chemical Network ....................................................... 47 3.1 Introduction .................................................................................................................... 47 3.2 Materials and Methods ................................................................................................... 48 3.2.1 Materials ................................................................................................................. 48 3.2.2 NMR Analysis ........................................................................................................ 53 3.2.3 Dynamic Peptide Network Preparation .................................................................. 53 3.2.4 HPLC and LC-MS Analyses ................................................................................... 54 3.2.5 IMS-MS .................................................................................................................. 54 3.2.6 Transmission Electron Microscopy and Electron Diffraction ................................ 55 3.2.7 Microwave Assisted Solid-Phase Peptide Synthesis .............................................. 55 3.3 Results and Discussions ................................................................................................. 57 3.3.1 Construction of NF-CHO Dynamic Chemical Networks (NF-DCN) ..................... 57 3.3.2 pH Dependence of NF-DCN ................................................................................... 58 3.3.3 Temperature Dependence of NF-DCN ................................................................... 59 3.3.4 NF-DCN Aging and Kinetic Modeling................................................................... 61 3.3.5 Identification of Assemblies in the NF-DCN ......................................................... 69 3.3.6 Probing the Critical Concentrations for Network members to Self-assemble ........ 70 3.3.7 Rapid Analysis of DCN .......................................................................................... 72 3.4 Discussions ..................................................................................................................... 73 Chapter 4: NFF-CHO Dynamic Chemical Network ..................................................... 79 4.1 Introduction .................................................................................................................... 79 4.2 Materials and Methods ................................................................................................... 80 4.2.1 Materials ................................................................................................................. 80 4.2.2 Synthesis of Building Blocks .................................................................................. 80 4.2.3 NMR Analysis ........................................................................................................ 86 4.2.4 Dynamic Peptide Network Preparation .................................................................. 87 4.2.5 HPLC and LC-MS Analyses ................................................................................... 87 4.2.6 IMS-MS .................................................................................................................. 88 4.2.7 Transmission Electron Microscopy and Electron Diffraction ................................ 88 4.2.8 Thioflavine T (ThT) Fluorescence .......................................................................... 89 4.2.9 X-ray diffraction (XRD) Analyses.......................................................................... 89 4.3 Results and Discussions ................................................................................................. 89 4.3.1 Construction of NFF-CHO Dynamic Chemical Network (NFF-DCN) .................. 89 4.3.2 Direct Analysis of the Network Composition by HPLC ........................................ 90 4.3.3 pH Profile of NFF-DCN ......................................................................................... 93 4.3.4 Kinetics Study of NFF-DCN Reveals an Emergent Phase That Is Far From Equilibrium ................................................................................................. 96 4.3.5 Identification of Assemblies in the NF-DCN ....................................................... 104 4.3.6 Characterization of Fiber Assemblies in the NFF-DCN ....................................... 105 4.3.7 Supramoleclar Assemblies' Responsiveness to pH Change ................................. 109 4.4 Discussions ................................................................................................................... 110 4.4.1 Comparison of the Kinetics of NFF-DCN to NF-DCN ........................................ 111 Chapter 5: Understanding the pathway of assemblies in Chemical Networks and their responsiveness to templates ..................................................................... 116 5.1 Introduction .................................................................................................................. 116 5.2 Two Steps of Amyloid Peptide Self-assembly ............................................................. 116 5.2.1 Are Oligomers in the Pathway to Mature Fibers? ................................................ 118 5.2.2 IMS-MS Technique for Probing the Oligomeric Intermediates ........................... 119 5.2.3 Are Protofibrils in the Pathway to Mature Fibers? ............................................... 124 5.3 Materials and Methods ................................................................................................. 129 5.3.1 Materials ............................................................................................................... 129 5.3.2 Dynamic Peptide Network Preparation ................................................................ 129 5.3.3 HPLC and LC-MS Analyses ................................................................................. 130 5.3.4 IMS-MS ................................................................................................................ 131 5.3.5 Transmission Electron Microscopy and Electron Diffraction .............................. 131 5.3.6 Microwave Assisted Solid-Phase Peptide Synthesis ............................................ 132 5.3.7 Alexa Binding of Seeded Assemblies in NFF-DCN............................................. 133 5.3.8 Fluorescent Peptide Seeds Preparation ................................................................. 133 5.3.9 Dual Color Fluorescence Imaging of Alexa 633 and Rhodamine Labeled Peptide 133 5.4 Results .......................................................................................................................... 134 5.4.1 Using IMS-MS to Probe the Oligomeric Intermediates on Pathway of Network Assemblies ........................................................................................................................... 134 5.4.2 Are Particles and Molten Globule on Pathway for Network Assemblies to Mature Fibers? 138 5.4.3 Are Protofibrils (Twisted Fibers) on Pathway for Network Assemblies to Mature Fibers? 142 5.4.4 Using Seeding experiment to Probe Protofibrils on Pathway of Network Assemblies ........................................................................................................................... 144 5.4.5 Exogenous Seeding of NFF-DCN by H-NFNFNF-NH2 ...................................... 146 5.4.6 Exogenous Seeding of NFF-DCN by Ac-KLVFFAL-NH2 Peptide Nanotubes ... 149 5.4.7 Alexa 633 Binds Co-assembly of NFF-DCN with E22L ..................................... 154 5.4.8 Dual Color Experiment for Visualizing Growth of New Assemblies .................. 156 5.5 Discussions ................................................................................................................... 160 Chapter 6: Conclusion and Perspectives .................................................................... 166

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