Synthetic Platforms for Accessing Tag-free Macrocyclic Peptides Restricted; Files Only

Abstract

Macrocyclic peptides are an increasingly important modality within the pharmaceutical industry. Methods for their discovery are currently limited to platforms that require large encoding tags and few are versatile enough to include a large variety of non-canonical amino acids. In this work, we successfully developed synthetic platforms to access pooled macrocyclic libraries that lack these limitations. First, we utilized CyClick cyclization chemistry in a multi-step pooled library synthesis. We optimized two scavenger-resin purifications steps for the linear and macrocyclization mixtures for selective enrichment of our pooled library. Additionally, we demonstrated the ability to linearize, derivatize, and sequence library members through direct mass spectrometric analysis of each peptide species, allowing us to avoid the use of an encoding tag. As a follow-up, we also explored the use of hydrazone-based cyclization chemistry as a method that sees quantitative peptide conversion and bypasses the purification steps required for CyClick library generation. This strategy was also amenable to a facile linearization and derivatization protocol. Both strategies were employed in affinity selections against protein targets and pooled in vitro permeability assays to demonstrate their potential applications. We discovered a CyClick peptide that perturbs the HIV capsid protein assembly and are in the process of characterizing hydrazone macrocycle binders for the gamma aminobutyric acid type A receptor-associated protein (GABARAP). 

Table of Contents

Introduction 1

1.1 Introduction to Macrocyclic Peptides as Therapeutics 1

1.2 Peptide Macrocyclization Strategies2

           1.2.1 Chemoselective Amide Forming Macrocyclizations2

           1.2.2 Chemoselective Non-Amide Macrocyclizations4

1.3 High Throughput Screening Platforms for Drug Discovery 6

1.3.1 Biologically Synthesized Technologies7

        1.3.2 Organic Synthesis Strategies8

1.4 Novel Cyclization Chemistries for Tag-Free Library Generation 9

 

Development of the Peptide Exploration Platform with Tag-Free Intramolecular Chemistry (PEPTIC) with CyClick Cyclization13

2.1 Filling the Gap in Macrocyclic Peptide Library Technologies with CyClick Chemsitry 14

2.2 Development of Peptide Exploration of Platform with Tag-Free Intramolecular Chemistry14

2.2.1 Linear Library Synthesis and Purification14

2.2.2 One-pot Cyclization and Purification17

2.2.3 CyClick Peptide Linearization, Derivatization, and Stability Studies19

   2.3 PEPTIC Application in Ligand Discovery24

           2.3.1 Identifying a Macrocyclic Peptide Ligand for the HIV-1 Capsid Protein26

  2.4 PEPTIC Application for Pooled In Vitro Membrane Permeability Assay30

Hydrazone Macrocyclization for Rapid PEPTIC Workflow

    3.1 Expanding PEPTIC Library Technologies34

    3.2 Development of Second-Generation PEPTIC Workflow36

3.2.1 Accessing N-terminal Hydrazine Residue37

3.2.2 Exploring Hydrazone Macrocyclization 38

3.2.3 Hydrazone Linearization, Derivatization, and Dimer Reversal43

3.2.4 Hydrazone Macrocycle Stability45

  3.3 Applications of Hydrazone PEPTIC47

3.3.1 Affinity Selection Against Gamma aminobutyric acid type A receptor-associated protein 47

3.3.2 Parallel Artificial Membrane Permeability Assay (PAMPA) 49

Discussion and Future Directions

4.1 Discussion51

4.2 Future Directions

4.2.1 Improving Peptide Limit of Detection52

4.2.2 Focused Libraries Targeting the HIV Capsid FG Binding Site53

4.2.3 Structural Substitutions for Reversable Linkers53

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Chemistry, Analytical Chemistry, Organic
Stichwort	pooled library screening affinity selection-mass spectrometry macrocyclic peptides combinatorial chemistry peptide synthesis
Committee Chair / Thesis Advisor	Monika, Emory University
Committee Members	Christine Dunham, Emory University Katherine M Davis, Emory University

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