Expanding the Toolbox: Development of Novel Chemical Tools for Profiling of Proteins and Dimethyl Post-translational Modification Open Access
Emenike Benjamin (Spring 2024)
Abstract
Protein chemical modification stands as a pivotal technique facilitating the selective functionalization of proteins with small molecule probes or fluorophores to monitor and modulate protein activities within biological systems. However, a prevalent limitation of existing chemical modification strategies lies in their predominant focus on modifying highly reactive yet less abundant amino acid residues, such as lysine, cysteine, tryptophan, and tyrosine. To fully harness the potential of chemical strategies in elucidating biological processes, innovative methods targeting less reactive but highly abundant residues like methionine (Met), asparagine (Asn), and glutamine (Gln) are imperative. In this pursuit, I devised two chemical platforms tailored for profiling hyper-reactive Met and Asn/Gln residues within the human proteome. Firstly, for Met modification, I developed a Copper(I)-Nitrene Platform (CuNiP) enabling the sulfonyl sulfimidation of methionine under mild physiological conditions. This approach facilitated the chemoproteomic profiling of Met in complex whole cell lysates and live cells, revealing previously undiscovered proteins harboring hyperactive methionine residues, thus unveiling novel ligandable sites. Secondly, for Asn/Gln profiling, I introduced the "SMAGIC-Reaction" (Selective Modification of Asparagine and Glutamine via Isohypsic and Carbometallation Reactions), enabling dual functionalization of Asn/Gln sites within proteins and live cells. The integration of the CuNiP and SMAGIC platforms is anticipated to expand the proteome coverage by an additional 11%, enhancing the total coverage to an improved 27%.
Beyond my endeavors in amino acid residue modification, I also developed chemical platforms for profiling dimethyl post-translational modifications of lysine (Kme2) and protein N-termini (Nme2). Protein methylation governs critical biological processes, and dysregulation often underlies various diseases such as cancer and diabetes. Notably, the lack of pan-specific chemical methods for dimethylome profiling presents a significant gap in research. To address this, I devised two innovative chemical strategies. Firstly, for dimethyllysine profiling, I introduced the "Tertiary Amine Coupling by Oxidation" (TACO) chemistry, enabling the conversion of dimethyllysine sites into orthogonal aldehyde handles. Secondly, for N-terminal dimethyl site targeting, I developed the "Oxidative Nitrile Thiazolidination" (OxNiTha) chemistry, leveraging oxidation to generate reactive iminium ions, subsequently trapped with sodium cyanide and enriched with thiol-amine tags. These chemical platforms demonstrated versatility in identifying dimethyllysine (Kme2) and N-terminal dimethylation (Nme2) sites through chemoproteomic approaches, highlighting their efficacy and potential in advancing our understanding of protein post-translational modifications.
Finally, I harnessed TACO chemistry for the synthesis and late-stage diversification of aldehydes. Aldehydes serve as invaluable handles for the site-specific functionalization of biomolecules with various tags, including dyes, drugs, and affinity moieties, via diverse chemical strategies. Current chemical methodologies predominantly focus on introducing them at the N- and/or C-termini of proteins. Motivated by lysyl oxidase's ability to convert lysine side chains to allysine, thereby generating aldehydes, we leveraged TACO chemistry to introduce aldehydes in bioactive small molecules, linear and cyclic peptides, and proteins within cellular environments. This innovative approach offers new avenues for the precise modification and functionalization of biomolecules for diverse applications in chemical biology and drug discovery.
Table of Contents
Chapter One: Chemical Tools for profiling methionine and Asparagine/Glutamine. 13
1. General Introduction to chemical tools for protein modification. 14
1.1 Methionine modification. 16
1.1.1 Result and Discussion. 19
1.1.2 Chemoselectivity studies for CuNiP reaction. 25
1.1.3 Stability of sulfonyl sulfimide adduct 27
1.1.4 Synthesis of of functionalized chloramine-T analogs. 28
1.1.5 Screening of electronic effects on CuNiP reaction. 29
1.1.6 Protein substrate scope. 32
1.1.7 Selective modification of proteins with Choramine-T analogs and fluorophores. 35
1.1.8 Chemoproteomics application of CuNiP for profiling of methionine in cell lysate. 39
1.1.9 Live cell chemoproteomics application of CuNiP on breast cancer cell (T47D) 43
1.1.10 Conclusion. 46
1.2 Asparagine and Glutamine Modification. 47
1.2.1 Result and Discussion. 49
1.2.2 Chemoselectivity studies. 52
1.2.3 Optimization and protein scope for isohysic dehydration of asparagine and glutamine. 53
1.2.4 Activity assay of modified myoglobin. 55
1.2.5 Isohypsic dehydration for Asn / Gln chemoproteomics profiling 56
1.2.6 Live cell chemoproteomics profiling of Asn / Gln in breast cancer cell (T47D) 58
1.2.7 SMAGIC for diversification of proteins. 61
1.2.8 SMAGIC for the synthesis of Antibody-Fluorophore Conjugate (AFC) 63
1.2.9 Activity Assay for SMAGIC modified Trastuzumab antibody. 64
1.2.10 Conclusion. 65
Chapter Two: Chemical Tools for profiling dimethyllysine and N-terminal dimethylation. 67
2 General Introduction to chemical tools for profiling of dimethyl post-translational modification of lysine and N-terminus. 68
2.1 Dimethyllysine (Kme2) Modification. 72
2.1.1 Result and Discussion. 72
2.1.2 Chemoselectivity studies and screening of effects of adducts. 74
2.1.3 Application of TACO for modification and diversification of histone peptides. 76
2.1.4 Fluorosequencing application of TACO chemistry. 80
2.1.5 TACO chemistry mediated modification and labeling of proteins with fluorophores. 82
2.1.6 Enrichment of dimethyl lysine sites from nucleosome. 85
2.1.7 Enrichment and chemoproteomics profiling of dimethyl lysine sites in cell lysates. 86
2.1.8 Conclusion. 89
2.2 N-terminal Dimethylation Modification. 90
2.2.1 Result and Discussion. 90
2.2.2 Mechanistic studies of OxNitha chemistry. 91
2.2.3 Chemoselectivity studies. 93
2.2.4 Selective modification and enrichment of Nme2 over Kme2 and cysteine-nitrile. 95
2.2.5 Stability studies of thiazolidine. 99
2.2.6 Substrate scope with varying N-terminal amino acids. 99
2.2.7 OxNiTha mediated modification and diversification of peptides 100
2.2.8 Selective labeling of Nme2 peptides in complex cell lysate mixture 101
2.2.9 OxNiTha chemistry for modification of Nme2 on proteins. 102
2.2.10 Chemoproteomics profiling of Nme2 proteins in cell lysate. 104
2.2.11 Conclusion. 107
Chapter Three: Bioinspired Synthesis and Late-stage diversification of aldehydes on peptides and proteins. 108
3 General Introduction to bioinspired synthesis and late-stage diversification of peptides aldehydes. 108
3.1 Result and Discussion. 111
3.2 Synthesis of bioactive allysine linear and cyclic peptides, and small molecules. 114
3.3 Late-stage diversification of bioactive allysine peptides. 118
3.4 Solid-support mediated synthesis and diversification of allysine peptides 120
3.5 Delivery of allysine peptide fluorophore in breast cancer cells (T47D) 122
3.6 Chemical introduction of allysine on T47D cells. 122
3.7 Conclusion. 124
Supplementary Information Chapter One. 125
Supplementary Information 1.1 (Methionine modification) 125
Supplementary Information 1.2 (Asparagine and Glutamine modification) 264
Supplementary Information Chapter Two. 457
Supplementary Information 2.1 (Dimethyllysine modification) 457
Supplementary Information 2.2 (N-termini dimethyl modification) 560
Supplementary Information Chapter Three. 690
Supplementary Information 3 (Bioinspired synthesis and diversification of aldehydes) 690
References 788
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