De Novo Design and Stabilization of β-Sheet Peptides for Biomimicry of ORR Enzymes

This dissertation presents the de novo design of novel peptide-based materials that mimic the structure and function of the laccase enzyme, with a focus on applications in energy conversion technologies. Laccase is of particular interest due to its ability to catalyze the four-electron reduction of molecular oxygen (O₂) to water (H₂O), a reaction essential for the development of next-generation energy systems such as proton exchange membrane hydrogen fuel cells (PEMFCs). Chapter One introduces the fundamental chemistry of the oxygen reduction reaction (ORR) and highlights how fungal laccases catalyze this transformation efficiently. It also outlines the synthetic strategy for solid phase peptide synthesis employed in the design and construction of peptide models for the purpose of emulating the active site architecture of laccase. Chapter two focuses on efforts to stabilize the β-sheet structural motif that supports the ligand environment surrounding the laccase trinuclear copper cluster (Cu₃ site). Specifically, this chapter explores how tryptophan zippers (TrpZip) mediate β-sheet formation through hairpin folds and other tertiary arrangements. Two newly designed peptides, TZ4H and TZ4H-H3AH10D, are synthesized and characterization for these peptides feature the use of circular dichroism (CD), UV-Vis spectroscopy, isothermal titration calorimetry (ITC), electron paramagnetic resonance (EPR), diffusion-ordered spectroscopy (DOSY), and single-crystal X-ray diffraction (SC-XRD). The peptides are analyzed both in their apo and metalated forms. This work features the first crystallographic evidence for a metallo-β-sheet peptide. Chapter three introduces a macrocyclization strategy to confer maximum stability to the β-sheet motif. Additionally, this work examines how backbone N-methylation, which disrupts hydrogen bonding by installing a methyl group to the peptide backbone at the αN, influences peptide oligomerization. A series of peptides, MC4H, MC4H-MeH, MC4H-MeA, and MC4H-MeAMeH, are synthesized to evaluate these effects. Their secondary, tertiary, and quaternary structure are characterized through CD, DOSY, and SC-XRD. Chapter four explores novel strategies for engineering a dimeric β-sheet sandwich structure, referred to as a β-sandwich. The β-sandwich is a key architectural feature in the ligand environment surrounding the Cu₃ site in laccase. This work culminates in the design and successful synthesis of a novel 25-residue β-sandwich peptide, named Pacman. The structural and functional characterization of Pacman using CD, CV, DOSY, ITC, EPR, and SC-XRD leads to the determination of its metalated structure. This represents a significant step toward functional biomimicry of laccase.