Bioconjugate materials for the study of pigment mobility in light-harvesting systems, protein-based formulations for hydrophobic actives, and conformational changes in conjugate vaccines by Jake Jaffe A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Matthew B. Francis, Chair Professor Felix R. Fischer Professor Douglas S. Clark Spring 2016 Bioconjugate materials for the study of pigment mobility in light-harvesting systems, protein-based formulations for hydrophobic actives, and conformational changes in conjugate vaccines Copyright © 2016 By: Jake Jaffe Abstract Bioconjugate materials for the study of pigment mobility in light-harvesting systems, protein-based formulations for hydrophobic actives, and conformational changes in conjugate vaccines by Jake Jaffe Doctor of Philosophy in Chemistry University of California, Berkeley Professor Matthew B. Francis, Chair Techniques for the preparation, purification, and characterization of protein-based materials have allowed for advances in fields ranging from medicine to materials science. While great attention has been paid to the chemistries used in the attachment of molecules of interest to proteins, the importance of the linking group is often overlooked. Each chapter of this dissertation describes the use of linkers as functional design elements in three distinct projects. In Chapter One, the development and characterization of a minimal model for investigating the role of pigment mobility in photosynthetic light-harvesting antenna systems will be discussed. In this model system, pigment- protein and pigment-pigment interactions were altered through the use of pigment-protein linkers of various lengths and rigidities. Chapter Two explores new biocompatible bond cleavage reactions for the preparation of a new class of general protein-based formulations for hydrophobic actives, wherein the linker group imparts the critical properties to the system. Chapter Three will examine conformational changes in peptide-protein conjugate vaccines. The roles of specific methods of covalent modification and linker composition in these conformational changes are emphasized. 1 In memory of Bernard “Saba” Jaffe i Table of Contents Chapter 1: Linker effects on energy transfer interactions in well-defined pigment- protein complexes 1.1 Introduction .................................................................................................................. 2 1.2 Selection and preparation of the protein scaffold .................................................... 2 1.3 Suitability of Mth1491 as a scaffold for pigment-protein complexes .................... 4 1.4 Selection and preparation of pigments ...................................................................... 5 1.5 Determination of pigment mobility ........................................................................... 6 1.6 Effects of pigment mobility on pigment-pigment interactions .............................. 9 1.7 Spectral properties of coumarin 343-Mth1491 conjugates ..................................... 9 1.8 Two-color Mth1491 conjugates ................................................................................11 1.9 Towards homogeneous pigment-Mth1491 conjugates ..........................................12 1.9.1 Synthesis of bifunctional coumarin reagent for chromatography- m mediated bioconjugation ..............................................................................12 1.9.2 Preparation and characterization of pigment-protein complexes using m chromatography-mediated bioconjugation ................................................14 1.10 Effects of linker composition in tobacco mosaic virus-templated pigment- protein complexes ....................................................................................................17 1.11 Conclusions ...............................................................................................................18 1.12 Materials and methods ............................................................................................18 1.12.1 General procedures and methods..............................................................18 1.12.2 Instrumentation and sample analysis .......................................................18 1.12.3 Experimental procedures ............................................................................20 1.12.4 Small molecule synthesis ............................................................................22 1.13 References ..................................................................................................................32 Chapter 2: Development of general protein-based formulations for hydrophobic actives using hydrophobicity modulators 2.1 Introduction ................................................................................................................37 2.1.1 Virus-like particles as drug delivery vehicles .............................................37 2.1.2 Hydrophobicity modulation as a strategy to prepare general protein m nanocarriers ....................................................................................................38 2.2 Interior surface hydrophobicity modulation of MS2 .............................................39 2.2.1 Synthesis of thiol-reactive π-allylpalladium substrate ..............................39 2.2.2 Conjugation and hydrophobicity modulation with allyl phosphate 2.1 42 ii 2.2.3 Synthesis of a photo-induced hydrophobicity modulator ........................45 2.2.4 Hydrophobicity modulation with photocleavable substrate 2.15 ...........47 2.2.5 Characterization of surface hydrophobicity ...............................................49 2.2.6 Improving the biocompatibility of Norrish type II chemistry .................52 2.3 Towards creating a hydrophobic droplet within MS2 ...........................................58 2.4 Towards hybrid hydrophobicity-modulating polymer-protein hybrids .............63 2.5 Conclusions .................................................................................................................66 2.6 Materials and methods ..............................................................................................66 2.6.1 General procedures and methods ................................................................66 2.6.2 Instrumentation and sample analysis ..........................................................67 2.6.3 Experimental procedures ..............................................................................68 2.6.4 Small molecule synthesis ..............................................................................72 2.7 References ....................................................................................................................88 Chapter 3: Elucidating the nature of conformational changes in peptide-CRM197 conjugate vaccines 3.1 Introduction ................................................................................................................95 3.2 Conformational changes in CRM197 conjugates .....................................................96 3.2.1 Effects of the conjugation step on conformational change ......................98 3.2.2 Effects of the activation step on conformational change ..........................99 3.3 Alternative activation-conjugation strategies .......................................................102 3.3.1 Maleimide-NHS ester crosslinking ...........................................................102 3.3.2 CuAAC and SPAAC ....................................................................................104 3.3.3 Disulfide exchange .......................................................................................104 3.4 Conclusions ...............................................................................................................106 3.5 Materials and methods ............................................................................................106 3.5.1 General procedures and methods ..............................................................106 3.5.2 Instrumentation and sample analysis ........................................................106 3.5.3 Experimental procedures ............................................................................107 3.5.4 Small molecule synthesis ............................................................................110 3.6 References ..................................................................................................................111 iii Acknowledgments I would like to thank the following people who have supported, guided, and accompanied me through my stint as a graduate student at Berkeley. I couldn’t have done it without: Matt Francis, thank you for giving me the opportunity to work in your lab. Your constant enthusiasm and wealth of knowledge were inspiring. Thorsten, it has been a pleasure working with you through the CARA program. Thanks for the research and career guidance, and the good times! Anouk, thank you and the Pfizer team for the opportunity to work on such an exciting and challenging project, and for all of your insight and assistance. The Francis lab members who welcomed me as a first year and set me up for success in the world of bioconjugation: Abby, Allie, Amy, Chris, Dan, Gary, Jeff, Jelly, Kareem, Katherine, Kristen, Leah, Michel, Michelle, Mike, Stacy, Troy, Wesley,