Development of Chemical Methods for Labeling Monomethyl Lysine Post-Translational Modification and Serine and their Applications. Restricted; Files Only
Nwajiobi, Ogonna (Summer 2022)
Published
Abstract
Chemoselective modification of peptides and proteins offers a powerful way of studying protein functions and generating hybrid molecules with diverse functions. In this dissertation, I present chemoselective techniques for labeling monomethyl lysine, the novel synthesis of smart macrocyclic peptides, and methods for site-specific modification of serine for the generation of homogeneous biologics.
Lysine monomethylation (Kme) is an impactful post-translational modification (PTM) responsible for regulating biological processes and implicated in diseases, thus there is great interest in identifying these methylation marks globally. Current methods for detecting methyl lysine include Mass Spectrometry (MS) and antibodies, both of which are limited in efficiently characterizing the low abundant monomethyl lysine in a complex mixture. To tackle this, we developed a chemical technology (STaR chemistry) in which an electron-rich diazonium ion covalently labels the monomethyl lysine on peptides/proteins generating a triazene-chromophore in solution-phase or solid support, and the bioconjugate can be released to afford highly pure unmodified peptides from complex mixtures.
The STaR chemistry was further adapted to the synthesis of smart macrocyclic peptides. Myriads of methods exist for the synthesis of macrocyclic peptides, but most of these cannot generate a macrocyclic peptide that is responsive to external stimuli and contains an in-built chromophore. I developed a highly chemoselective, rapid, intramolecular reaction of secondary amines and p-amino phenylalanine that generates an inbuilt chromophoric triazene cyclic peptide. The triazene cyclic peptides can be opened in response to acidic conditions and UV radiations and can be preferentially reclosed when restored to neutral pH.
Lastly, I developed a site-specific modification of serine residues on peptides by enhancing the nucleophilicity of the hydroxyl group on serine residues within the catalytic triad. Through rational finetuning of the microenvironment through spatial positioning of amino acids around serine (HXSXH), serine was made very reactive over other serine residues. 2-aminoethyl) benzene sulfonyl fluoride (AEBSF) was found to react with the peptides containing (HXSXH) motif over other nucleophilic residues on the same peptide. Thus, this strategy can be utilized to preferentially incorporate desired moieties at specific sites on proteins such as in ADCs to generate homogeneous products.
Table of Contents
Table of Contents
CHAPTER ONE: CHEMICAL METHODS FOR TAGGINGMONOMETHYL LYSINE POST-TRANSLATIONAL MODIFICATIONS. 1
1.0. Introduction. 1
1.1. Background. 3
1.1.1. Reported Methods and Limitations. 4
1.2. Secondary Amine Selective Petasis (SASP) Bioconjugation. 6
1.2.1. Development of Secondary Amine Trapping Reagents (STRap) for Tagging Monomethyl Lysine. 7
1.2.1.1. Aliphatic STRap. 8
1.2.1.2. ARYL STRap. 12
1.2.1.3. HETEROARYL STRap. 15
1.3.0. Development of Selective Triazenation Reaction (Star) of Secondary Amines for Tagging Monomethyl Lysine Post-Translational Modifications (PTMs) 18
1.3.1 Chemoselectivity Studies. 21
1.3.2. Pan-Specific Nature of Triazene Reaction. 21
1.3.3. Synthesis of arene diazonium affinity tags for monomethyl lysine. 23
1.3.4. Selective Tagging of Monomethyl Lysine in the Complex Mixture Using Alkyne-Derived Diazonium Ion. 24
1.3.5. Traceless Enrichment of Monomethyl Lysine Using Star 25
1.3.6. Enrichment of Monomethyl Lysine Kme from Complex Mixture. 26
1.3.7. Enrichment on Solid Support 27
1.4. Proteome-wide Profiling of Monomethyl Lysine with ABDz. 31
CONCLUSION.. 35
CHAPTER ONE REFERENCES. 38
CHAPTER TWO: RAPID ARENE TRIAZENE CHEMISTRY FOR MACROCYCLIZATION.. 45
2.0. Introduction. 45
2.1.Peptide-based Drugs and Advantages of Cyclic Peptides. 45
2.2. Synthetic Methods for Generating Macrocyclic Peptide. 47
2.3. Need for Cyclic Peptides for Drug Discovery. 49
2.5.0. Development of a Rapid Arene Triazene Chemistry for Macrocyclization. 50
2.5.1. Initial Studies and Structural Characterization of Triazene Cyclic Peptides. 51
2.5.2. Investigation into the Reactivity of Primary Amines with Diazonium Ion. 53
2.5.3. Scope of Arene Triazene Chemistry. 55
2.5.4. Rate of Arene Triazene Cyclization. 58
2.5.5. Chromophoric Property of Triazene Cyclic Peptides. 60
2.5.6. Response of Triazene Cyclic Peptide to External Stimuli 62
2.5.7. Cyclization in One Pot and Sequencing of Triazene Cyclic Peptides. 64
2.5.8. Stability of Triazene Cyclic Peptides. 65
2.5.9. Synthesis of Bicyclic Peptides. 66
2.5.10. Late-Stage Derivatization of Triazene Cyclic Peptides. 67
2.6. Conclusion. 68
CHAPTER TWO REFERENCES. 70
CHAPTER THREE: ENZYME-INSPIRED SITE-SPECIFIC MODIFICATION OF SERINE.. 76
3.0. Introduction. 76
3.1. Methods for Achieving Site-Specific Modification on Proteins. 76
3.2. Our Approach: Mimicking the Catalytic Triad. 79
3.3. Design of the HB-Hub by Finetuning the Microenvironment of Serine for Site-Specific Modification. 82
3.3.1. Effect of Turn-Inducer 84
3.3.2. Role of Histidine and Aspartic acid. 84
3.3.3. Effect of the Distance between Serine and Histidine in the Peptide Motif 85
3.3.4. Impact of the Amino Acid in-between Serine and Histidine. 85
3.3.5. Selective Modification of Serine in the HB-Hub over other Serine Residues in a Peptide. 86
3.3.6. Confirmation of the Site of Modification. 87
3.4. Screening of Known Protease Inhibitors for Selective Reaction with Peptides with HB-Hub. 89
CHAPTER THREE REFERENCES. 93
List of Tables
Table 1.1: Reaction of Aliphatic STRap (AL-STRap) with AKmeF peptide under different reaction conditions……………………………………………………………………………….10
Table 1.2: Reaction of Aliphatic STRap (AL-STRap) with AAF peptide under different reaction conditions………………………………………………………………………………………...11
Table 1.3: Reaction of Aryl STRap (AR-STRap) with different substrates under different reaction conditions…………………………………………………………………………………….......14
Table 1.4: Reaction of peptides with Aromatic STRap with heteroatom (ArO-STRap) under different reaction conditions……………………………………………………………………..16
Table 1.5: Chemoselective and pan-specific nature of STAR chemistry……………………….22
Table 3.1: Identification of the HB-Hub sequence……………………………………………....83
Table 3.2: Reactions between HB-Hub peptides and AEBSF, a serine protease probe…………90
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