Chemo and Site Selective Modification of Proteins and Chemical Tools for Studying Mono-Methyl Lysine (Kme) Öffentlichkeit
Kuei-Chien Tang (Fall 2022)
Abstract
Site-selective modification of protein is a significant technique that can allow us to modify a single amino acid with biologically active small molecules or fluorophores to track and monitor the enzyme in the living system for biological study. The universal limitation for most of the current modification methods is the low detection sensitivity of the forming product, poor stability and low selectivity of probes for lysine modification, and lack of universal compatibility for the N-terminal modification. In order to circumvent the current limitations, I designed and synthesized various types of azole, azoline, and azolinium thioethers, which are highly selective and specific to our desired targets, and the reactivity and selectivity of those probes are highly tunable by the heteroaromatic ring, and methylation state. Moreover, those probes are able to act as a chemo-selective charge booster to enhance the mass detection sensitivity of the forming product. Our probes 1d-yne, 1c-yne, and N3-1o were applied to the proteomic profiling of lysine and cysteine in HEK293T cell lysate. The probe 1c-yne is highly selective and reactive to the lysine residues. Moreover, 1c-yne shows high permeability and hydrolytic stability and is able to label both cytoplasmic and nuclear proteins in live cells without necrosis those indicating its potential as a probe for rapid live cell labeling and covalent inhibitor. In tandem with the project above, I have developed two novel chemical tools for studying lysine methylation post-translation modification (PTM). Methylation of lysine regulates gene transcription and RNA, DNA binding, and any aberrant change of methylation state and site will cause various diseases such as cancer and diabetes. In the first method, I used triazene cyclization to target the mono-methyl lysine selectively. The forming product indazole is highly stable under basic and acid conditions and easy to modify with different affinity targets. Moreover, this method is the first chemical technique applied to a single-molecule protein sequence to read out unknown Kme1 sites in a single workflow by fluorosequencing. For the second method, I develop a strategy that enables site-selective modification of a high-frequency Lys residue in the mono-methyl lysine containing (Kme) protein for studying, monitoring, and tracking the PPI between the K-me protein and its reader and eraser proteins. The probes I have designed have a diazonium salt warhead with a mask group for selective labeling of mono-methyl lysine. The flexible linker connects with various electrophiles for intramolecular modification and the light-activated group for capturing the reader and eraser proteins. The probe AI showed high intramolecular labeling efficiency with a trace amount of intermolecular side product with different proline, K-me-containing peptides, and Histone-III truncated peptides. Those novel tools offer a chemical platform for identifying and studying the role of monomethyl lysine (Kme) in the whole proteome and a starting point for therapeutic interventions.
Table of Contents
1.1 General Introduction to approaches to site-selective protein modification: ................................. 17
1.1.1 N terminus modification .................................................................................................................. 17
1.1.2 Lysine residue modification: ........................................................................................................... 22
1.1.3 Cysteine side chain modification .................................................................................................... 24
1.2 Results and Discussion ........................................................................................................................ 26
1.2.1 Screening of azoline, azole, and azolinum with standard peptide ............................................... 26
1.2.2 Synthesis of Azoline, Azole, and Azolinum and their Analogs .................................................... 27
1.2.2 One-Step Azolation Strategy for Site-and Chemo-Selective Labeling of Proteins with Mass-Sensitive Probes ......................................................................................................................................... 32
1.2.2.2 Studying Universal Sequence Compatibility of N-terminus Azolztion .................................... 32
1.2.2.3 Protein modification-reaction optimization ............................................................................... 34
1.2.2.4 Synthesis of functionalized Oxazoles for N-terminal modification .......................................... 36
1.2.2.5 Protein Scope for N-terminal Oxazolation ................................................................................. 36
1.2.2.6 Rate and Stability Studies of Oxazolation .................................................................................. 37
1.2.2.7 Labeling of Proteolytic Fragments in the Complex Mixture .................................................... 39
1.2.2.8 Oxazoline as Mass Sensitivity Booster ........................................................................................ 41
1.2.2.9 Unmodified Myoglobin and Modified Myoglobin Bioactivity Assay ....................................... 45
1.2.2.10 Conclusion ................................................................................................................................... 46
1.2.3 Tunable heteroaromatic azoline thioethers (HATs) for cysteine profiling ................................. 48
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1.2.3.1 Rate and Stability Study of HAT Probe ...................................................................................... 48
1.2.3.2 Protein Modification with HAT ................................................................................................... 50
1.2.3.3 Selective tagging of Cys in a complex mixture ........................................................................... 55
1.2.3.4 Reversibility and stability of the HAT–cysteine conjugation .................................................... 57
1.2.3.5 Reactivity inversion by HAT probes ........................................................................................... 60
1.2.3.6 HAT as mass sensitivity booster .................................................................................................. 63
1.2.3.7 HAT probes for gel-based ABPP studies .................................................................................... 68
1.2.3.8 Conclusion ..................................................................................................................................... 70
1.2.4 Tunable amine-reactive electrophiles for selective profiling of lysine ........................................ 71
1.2.4.1 Chemoselectivity Reaction of TARE Probes with Proteins ....................................................... 71
1.2.4.2 Rate, Stability and Reversibility of TARE Probes ..................................................................... 73
1.2.4.3 Chemo-proteomic studies of TAREs ........................................................................................... 80
1.2.4.4 Live cell labe lingand amino-acid selectivity in proteome by probe1c-yne .............................. 86
1.2.4.5 Computational study of probe 1c-yne ......................................................................................... 89
1.2.4.6 Conclusion ..................................................................................................................................... 90
Reference: .................................................................................................................................................. 92
Chapter 2: Chemical Tool for Tagging Mono-Methyl Lysine and K-me-Directed Modification ... 101
2.1 General Introduction of Mono-Methyl Lysine ............................................................................... 101
2.2 Coarctate Cyclization for Selective labeling of Monomethyl Lysine Posttranslational Modifications ........................................................................................................................................... 103
2.2.1 Development of Triazenation Coarctate Cyclization (TCC) ...................................................... 104
2.2.2 Stability Studies .............................................................................................................................. 107
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2.2.3 Chemoselectivity studies for the formation of 2H-indazole-3-carbaldehyde ............................ 108
2.2.4 Tyrosine Vs Kme modification ..................................................................................................... 109
2.2.5 Substrate scope with varying 2-ethyne phenyldiazonium ions. ................................................. 112
2.2.6 Pan-specificity of TCC: Further Diversification ......................................................................... 115
2.2.7 Selective labeling of Kme1 peptides in a complex cell lysate mixture by TCC ........................ 117
2.2.8 Single-molecule sequencing for identification of Kme1 sites by TCC ....................................... 118
3.1 Design of Chemical Probes for Kme-Directed Modification (Kme-DM) ..................................... 125
3.2 Design and synthesis of various probes for Kme-DM .................................................................... 126
3.3 Optimization of intramolecular reaction under UV lamp with different proline containing peptides and chemo selectivity study. .................................................................................................... 137
3.4 Chemoselectivity and control study of AI-I probe ......................................................................... 140
3.5 Screening of AI-III probe with K-2, K-3 and K-4 peptides under UV lamp ............................... 140
3.6 Site-specific study with multiple lysins containing peptide ........................................................... 141
3.7 Intramolecular reaction study of various AI analogs with different affinity tags ...................... 142
3.8 Intramolecular reactivity and chemo selectivity study of unmasked AI-I probe ........................ 143
3.9 Kme-Directed modification of truncated histone peptide: ............................................................ 146
Supporting Information for Chapter One: ........................................................................................... 148
Supplementary Figure 1 : MS/MS of modified peptide ....................................................................... 148
Supplementary Figure 2: General method for the verification of the chemo- and site-selective nature of oxazoline probe with peptides. .............................................................................................. 153
Supplementary Figure 3. Azolation vs 2-PCA method ........................................................................ 161
Supplementary Figure 4. Optimization of the oxazolation on protein Myoglobin Mb. ................... 163
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Supplementary Figure 5. Modification of myoglobin Mb by different oxazoline derivatives. ........ 165
Supplementary Figure 6. Mass intensity enhancement of digested myoglobin by oxazolation. ...... 175
Supplementary Figure 7. Mass intensity enhancement of intact Myoglobin by oxazolation as compared to unmodified myoglobin. ..................................................................................................... 177
Supplementary Figure 8: Rate study of HAT bioconjugation. ........................................................... 178
Supplementary Figure 9. Modification of myoglobin Mb with different heteroaromatic azoline compounds 1a-1c, 1j, and 1n .................................................................................................................. 184
Supplementary Figure 10. Selective cysteine bioconjugation of Insulin a chain and b chain with compound 1o............................................................................................................................................ 192
Supplementary Figure 11. Modification of reduced insulin with 1m ................................................. 192
Supplementary Figure 12. Modification of bovine serum albumin (BSA) and lysozyme with HAT probe 1o ................................................................................................................................................... 193
Supplementary Figure 13. Enrichment of cysteine containing peptides with 1o in mixture of proteolytic fragments. ............................................................................................................................. 195
Supplementary Figure 14. Stability study of Ac-GCF-1o under different pH conditions................ 199
Supplementary Figure 15. Reversible study of Cys-HAT biconjugate with NaBH4 ......................... 202
Supplementary Figure 15. Reversible study of probe 1o ..................................................................... 205
Supplementary Figure 16. Dehydroalanine synthesis from cysteine 2c using 1o at high pH ........... 207
Supplementary Figure 17. Aza-Michael addition and thiol-ene reaction of dehydroalanine .......... 209
Supplementary Figure 18. Aza-Michael addition of dehydroalanine lysozyme ................................ 212 Supplementary Figure 20. Mass sensitivity of 1o-Ac-GCF (low concentration) ............................... 214
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Supplementary Figure 21. Mass intensity enhancement of N3-1o -reduced insulin bioconjugate products. .................................................................................................................................................. 216
Supplementary Figure 24. Rate study of 1c-yne and 1d-yne with peptide Ac-GKF 2c. ................... 225
Supplementary Figure 25. Stability study of 1c-yne, 1d-yne, NHS ester and STPyne ..................... 228
Supplementary Figure 26. Reversibility study of 1e-Cys-conjugate (VCF-1e) and 1d-thio-conjugate with lysine methylester. .......................................................................................................................... 229
Supplementary Figure 27. Chemoproteomic analysis of residue selectivity ...................................... 234
Supplementary Figure 28. TAREs for gel-based ABPP ...................................................................... 237
Supplementary Figure 29. Stability study of GCF-1c-yne conjugate in TCEP, and 1d-thio-conjugate under sodium phosphate buffer. ............................................................................................................ 243
Supplementary Figure 30. Live cell labeling of proteins using different probes. .............................. 244
Supplementary Figure 31. Cell viability studies with 1cyne ............................................................... 247
Supplementary Figure 32 ....................................................................................................................... 247
Supporting Information of Chapter Two: ............................................................................................ 257
Supplementary Figure 1: Synthesis of 2H-indazole-3-carbaldehyde and benzotriazole .................. 257
Supplementary Figure 2: General procedure for the modification of FAP with probes 2a and 3a. 263
Supplementary Figure 3: Stability study of 2H-indazole-3-carbaldehyde group in TFA and pyridine. ................................................................................................................................................... 267
Supplementary Figure 4: Chemoselectivity studies for the TCC reaction ........................................ 269
Supplementary Figure 5: Restriction of TCC side reaction with tyrosine ........................................ 270
Supplementary Figure 6: Selective blocking of tyrosine by 1,3 diphenyl propynone ....................... 272
General procedure for tyrosine labeling by 1,3 diphenyl propynone ................................................ 272
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Supplementary Figure 7: Optimization of triazenation coarctate cyclization with different diazonium salts analogs. ......................................................................................................................... 274
Supplementary Figure 8: Pan-specificity of triazenation coarctate cyclization with truncated histone peptides. ...................................................................................................................................... 291
Supplementary Figure 9: Enrichment of modified peptides by cysteine condensation and oxime chemistry. ................................................................................................................................................. 294
Supplementary Figure 10: Enrichment of modified truncated peptides by cysteine condensation and oxime chemistry. .............................................................................................................................. 296
Supplementary Figure 11: Selective enrichment of Kme1 containing peptides in a complex cell lysate mixture by TCC ............................................................................................................................ 300
Supplementary Figure 12: Single-molecule sequencing for identification of Kme1 sites by TCC .. 302
Supporting Information for Chapter Three: ........................................................................................ 303 Supplementary Figure 1. Synthesis of different K-me-DM probes .................................................... 303 Supplementary Figure 2. Optimization of intramolecular reaction under UV lamp with different proline containing peptides and chemo selectivity study ..................................................................... 339 Supplementary Figure 3. Chemoselectivity and control study of AI-I probe .................................... 342 Supplementary Figure 4. Screening of AI-III probe with K-2, K-3 and K-4 peptides under UV lamp .................................................................................................................................................................. 343 Supplementary Figure 5. Site-specific study with multiple lysins containing peptide ...................... 344 Supplementary Figure 6. Digestion of modified peptide. .................................................................... 346 Supplementary Figure 7. Intramolecular reaction study of various AI analogs with different affinity tags. ........................................................................................................................................................... 346
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Supplementary Figure 8. Intramolecular reactivity and chemo selectivity study of unmasked AI-I probe ........................................................................................................................................................ 348 Supplementary Figure 9. Intramolecular reactivity and chemo selectivity study of unmasked AI-I probe with K-me peptide ........................................................................................................................ 353 Supplementary Figure 10. Chemo-selective study of unmasked AI probe ........................................ 355 Supplementary Figure 11. Kme-Directed modification of truncated histone peptide ...................... 356
NMR for Chapter One ............................................................................................................................ 360
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