Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemistry

Major Professor

Justin M. Lopchuk, Ph.D.

Co-Major Professor

James W. Leahy, Ph.D.

Committee Member

Jianfeng Cai, Ph.D.

Committee Member

Ernst Schönbrunn, Ph.D.

Keywords

β-sheet mimetics, proline surrogates, reagent development, covalent inhibitors, covalent reactive groups, bicyclobutane

Abstract

Part 1:The initial portion of this dissertation delves into the enhancement of β-sheet secondary structure stability through modification of the peptide backbone while examining the conformational characteristics of peptides featuring N-amino substituents. The investigation consists of two major studies, presented in separate sections. The first study outlines various approaches to achieve stable β-hairpin folds by introducing N-amino substituents to peptide strands. A novel method utilizing on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain was utilized to incorporate pyrazolidinone and tetrahydropyridazinone dipeptide constraints. To evaluate the impact of cyclic N-amino peptide building blocks on β-hairpin stability in water, a model system derived from the β1 immunoglobulin binding domain of protein G (GB1) was employed. The results offer valuable insights into the influence of covalently tethered dipeptide constraints on β-sheet folding. The second study centers around the synthesis and conformational analysis of three derivatives of δ-azaproline and their potential as proline surrogates. These residues, possessing N-amino substituents in the backbone, exhibit distinctive conformational properties resulting from additional hydrogen-bonding, electrostatic, and steric interactions. The investigations include adjusting the stereoelectronic properties of the heterocyclic ring of proline through heteroatom substitution, trans/cis-amide bond conformational preferences, and the barriers to amide isomerization within a series of three unique oxidation state variants. Multiple characterization techniques were employed to elucidate the preferred conformations of each analog; NMR, X-ray diffraction, and density functional theory calculations proved useful to accomplish these studies. The findings indicate that the hybridization and electron density at the δ position plays a significant role in determining the conformational preferences of the δ-azaproline derivatives. Furthermore, both δ-azaproline and γ,δ-dehydro-δ-azaproline exhibit a preference for the trans-amide rotamer, regardless of the conformation (endo vs. exo) of the ring system. In contrast, γ-oxo-δ-azaproline displays rapid kinetic isomerization of the amide bond and isoenergetic amide bond geometries. These properties are significantly influenced by torsional strain and hydrogen bonding interactions present in the system. Introducing the δ-heteroatom in each residue allows for the decoupling of structural effects typically associated with proline and its pyrrolidine-substituted analogs. These δ-azaproline derivatives serve as valuable tools for investigating prolyl cis/trans-amide bond isomerism and hold potential applications in the study of protein and peptide secondary structure, as well as in the design of peptidomimetic drugs. Part 2: In recent years, the pharmaceutical industry has witnessed a resurgence of interest in covalent inhibitors as potential therapeutics for disease intervention. These inhibitors, which interact with nucleophilic residues in target proteins through a covalent interaction, offer advantages such as higher potency and ligand efficiency compared to non-covalent inhibitors. However, the indiscriminate reactivity of existing covalent inhibitors can lead to off-target effects and toxicity, limiting their clinical utility. Additionally, stability issues and rapid reversibility further hinder their effectiveness. This research aims to address these challenges by developing novel covalent reactive groups (CRGs) that exhibit lower overall in vivo reactivity while maintaining high selectivity for specific nucleophiles. The Lopchuk lab has identified strained sulfonyl bicyclobutanes (BCBs) as stable ring systems capable of selectively reacting with thiols under physiological conditions. These promising CRGs have the potential to revolutionize covalent inhibition strategies. To facilitate the translation of this potential into practical applications, proprietary first-generation reagents and reaction conditions have been developed by the Lopchuk lab, providing a toolkit for late-stage installation of sulfinyl, sulfonyl, and sulfonimidoyl BCBs onto nucleophiles. This breakthrough enables the incorporation of these novel CRGs into current medicinal chemistry workflows and represents a significant step toward the development of improved covalent inhibitors with enhanced selectivity and therapeutic potential.

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