Developing Chemical Tools for Selective Peptide Modification Restricted; Files Only
Adebomi, Victor (Fall 2022)
Abstract
Peptides represent an important class of biomolecules and have shown promise as efficient therapeutics for numerous disease states. However, the real time application of peptides as therapeutics is severely hampered by the low bioavailability, metabolic instability, and random conformations. Through selective peptide modification, we can provide useful tools to circumvent these limitations and improve physiochemical properties to enhance peptide applications in drug discovery. To this end, we have developed several strategies for peptide modifications. Peptide macrocyclization is a method highly touted for selective modification peptides due to their tendency to greatly improve the pharmacokinetic properties of peptides. A major limitation of most macrocyclization methods is that most techniques developed for peptide cyclization often result in the formation of dimers and oligomers at high concentrations. We have successfully developed the first exclusively intramolecular strategy for making cyclic peptides which we have used to make a wide range of cyclic peptides (12- to 23-membered rings) with varying amino acid compositions using mild reaction conditions. We termed this reaction CyClick because it works in an exclusively intramolecular fashion, exhibiting fast reaction kinetics, with high chemo selectivity and stereoselectivity (99% de). In addition, various NMR techniques and modeling studies have revealed that the 4-imidazolodinone moiety generated by the cyclization serves as an endocyclic control element to promote intramolecular hydrogen bonding, leading to the formation of a turn structure. This innovation is highly important as it bring about new ways of synthesizing cyclic peptides in high concentrations leading to higher efficacy of peptide synthesis. This CyClick technology developed in our lab is now being used as a new approach for making large libraries (>106 ) of pure cyclic peptides for screening against various biological targets. In tandem with the project above, I have developed a novel method for selective diversification of amides in polyamides. This has great importance to the scientific community because it incorporates various functional groups, moieties, and markers into peptides. The planarity and resonance character of secondary amides in polyamides lends to high stability making it difficult to selectively modify these locations. Although several advances have been made for the modification of secondary amides, the selective modification of a particular amide in a sea of other amides has never been done. Using Density Functional Theory (DFT) calculations as a guide, we have discovered a two step approach for the selective modification of amides in a polyamide. The first step of the reaction involves the selective introduction of a carbonyl into peptides containing serine or cysteine. This results in the electronic activation of amide bonds thereby allowing new groups such as esters, amides, and other functional groups to be introduced into the peptide.
Table of Contents
Table of Contents Chapter 1: CyClick Chemistry for the Synthesis of Cyclic Molecules.........................................................................15 Introduction.............................................................................................................................................................15
1.1.1 Introduction to Cyclic Peptides .........................................................................................................15
1.1.2 Why Cyclization of Linear Peptides is Important?............................................................................16
1.1.2 Pitfalls of Common Methods for Cyclic Peptide Generation ............................................................16
1.2 Result and Discussion ................................................................................................................................17
1.2.1 Experimental Design for New Cyclization Strategy: CyClick ..........................................................17
1.2.2 CyClick Reaction for Peptide Cyclization.........................................................................................19
1.2.3 Stereoselectivity of the CyClick Reaction.........................................................................................21
1.2.4 Macrocyclization vs Oligomerization ...............................................................................................23
1.2.5 Synthesis of Highly Diverse and Functionalized Cyclic Peptides by CyClick Chemistry ................26
1.2.6 Structural Impact of 4-imidazolidinone.............................................................................................31
1.2.7 Biological Impact of 4-imidazolidinone in cyclic peptides...............................................................32
1.2.8 Reversibility of Cyclic Chemistry and MS/MS sequencing of cyclic peptides.................................33
1.2.9 Mechanistic Investigation into CyClick Chemistry...........................................................................35
1.2.10 Application of CyClick Chemistry for Making Highly Stained Cyclic Peptides .............................44
1.2.11 Application of CyClick chemistry for making Peptide-Peptoid Hybrid............................................47
Chapter 2: Chemical Tools for Selective Modification and Diversification of Secondary Amides ....................54
2.1 Introduction................................................................................................................................................54
2.1.1 Activation of Amide Bonds...............................................................................................................55
2.1.2 Nomenclatures used for Determination of Twisted Amide Reactivity:.............................................57
2.1.3 Recent Advances in Amide bond Distortion and Diversification......................................................59
2.2 Limitations of the Current Approaches for Amide Bond Distortion and Diversification ..........................64
2.3 Metal-Free Selective Modification of Secondary Amides: Application in Late-Stage Diversification of Peptides.......65
2.3.1 Selective Activation of Amides: Design ...........................................................................................65
2.3.2 Key Experiments and Reaction Discovery for Transamidation ........................................................68
2.3.3 Scope of the Transamidation .............................................................................................................69
2.3.4 Esterification of Amides....................................................................................................................70
2.3.5 Site-Selective Transamidation and Esterification of Peptides...........................................................72
2.3.6 Conclusion on Selective Transamidation and Esterification of Secondary Amides..........................75
2.4 Selective Conversion of Unactivated C-N Amide bond to C-C bond in the polyamide via Steric and Electronic Resonance Destabilization .......75
2.4.1 Results and discussion. Discovery of site-selective activation of C-N amide bond for the formation of C-C bond. ...78
2.4.3 Metal-catalyzed C-C bond formation from C-N amide bonds. .........................................................79
2.4.4 Metal-free C-C bond formation by Friedel-Crafts reaction...............................................................83
Chapter 3: Bioinspired Nitroalkylation for Selective Protein Modification and Peptide Stapling ....................91 3.1 Introduction................................................................................................................................................91
3.2 Results and Discussion...............................................................................................................................92
3.2.1 Design of Bioconjugation Reagents..................................................................................................92
3.2.2 Nitroalkanes as Rapid and Aldehyde-Specific Bioconjugation Reagents.........................................93
3.2.3 Fluorination of Nitro-Peptide Conjugates. ........................................................................................96
3.2.4 Substrate Scope of Nitro-Reagents for Peptide Bioconjugation........................................................97
3.2.5 Synthesis of Functionalized Nitro-Reagents .....................................................................................98
3.2.6 Nitro-Reagents for Selective Modification of Proteins .....................................................................99
3.2.7 Fingerprint Pattern of Nitro-Alkylated Proteins..............................................................................103
3.2.8 Protein Structure and Function is Retained After Modification ......................................................103
3.2.9 Myoglobin Bioactivity Assay..........................................................................................................104
3.2.10 Nitro-Reagents for Late-Stage Diversification of Peptides and Stapling of Peptides .....................104
Chapter 4: Supplementary Information 4.1 Experimental Detail for Chapter one………........………………116
4.1.1 Cyclization of Linear Peptides to form Cyclic Tetrapeptides…………………………………....220
4.1.2 Data for Application if CyClick for the Formation of Peptide-Peptoid Hybrids………………...226
4.2 Experimental Detail for Chapter Two……………………………………………………………………..243
4.2.1 Experimental Data for Transamidation of Twisted Amides……………………………………..256
4.2.2 Experimental Detail for Esterification Reaction of Twisted Amides…………………………....272
4.2.3 Experimental Detail for Nucleophilic Addition of Twisted Peptides……………………………283
4.2.4 Experimental Detail for Suzuki Reactions with Twisted Amides……………………………….304
4.2.5 Experimental Detail for Friedel Craft Acylation of Twisted Amides……………………………338
4.3 Supplementary Information for Chapter Three………………………………………………………………………………………378
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File download under embargo until 11 January 2025 | 2022-11-01 13:56:34 -0400 | File download under embargo until 11 January 2025 |
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