O-フタルアルデヒド基を用いたペプチドの高効率マクロ環化とその応用
| Project/Area Number |
22KF0114
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| Project/Area Number (Other) |
22F22334 (2022)
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund (2023)
Single-year Grants (2022) |
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| Section | 外国 |
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| Review Section |
Basic Section 37010:Bio-related chemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
菅 裕明 東京大学, 大学院理学系研究科(理学部), 教授 (00361668)
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| Co-Investigator(Kenkyū-buntansha) |
ZHANG YUE 東京大学, 大学院理学系研究科(理学部), 外国人特別研究員
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| Project Period (FY) |
2023-03-08 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2024: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2023: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2022: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | RaPID / peptide cyclization / OPA / mRNA display |
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| Outline of Research at the Start |
We aim to develop OPA-based cyclic peptide libraries using the FIT system. These libraries will selectively release the free reactive moiety after the translation step for peptide cyclization or PTMs, which will be further applied in the RaPID system to identify potential drug molecules. Additionally, we plan to explore a chemoselective peptide cyclization method for post-translational modifications. Through this, we aim to contribute to the development of chemical biology studies and drug discovery efforts, aimed at identifying peptide candidates against various disease-related proteins.
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| Outline of Annual Research Achievements |
From Oct 2022 to Mar 2023, we aimed to investigate OPA-based technology for constructing nonstandard cyclic peptide libraries using FIT system. Due to OPA's high reactivity, we first explored 3,4-ThDA, a di-aldehyde compound with lower reactivity. Our experiments demonstrated selective cyclization with lysine and thiol-catalyzed thienopyrrole formation. This method yielded good conversion and better stability than the reported OPA chemistry, but the harsh conditions need optimization for in vitro translation.
We are also working on OPA-CME derivatives for flexizyme-mediated OPA-acylated tRNA synthesis, aiming to create non-standard libraries. The OPAon/off method is being developed for controlling OPA reactivity during translation and post-translational cyclization. This strategy helps identify peptide inhibitors targeting protein kinases containing cysteine and lysine residues in the binding pockets.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We've made progress in two areas: 1) 3,4-ThDA-based lysine/lysine cyclization on unprotected peptides: We synthesized various model peptides and optimized cyclization conditions. Although most peptides achieved nearly 90% conversion rate and greater stability compared to traditional OPA chemistry, the harsh reaction conditions are not suitable for in vitro translation systems. We're working on optimizing additives for milder conditions and studying the reaction mechanism involving thiol groups and 3,4-ThDA with lysine side-chains. 2) OPAon/off strategy for post-translational cyclization and nonstandard cyclic peptide library construction: We're synthesizing the crucial OPA-CME derivative for flexizyme-mediated OPA-acylated tRNA preparation. Our current synthetic route starts with N-acetyl-Ltyrosine, followed by a four-step synthesis and CME installation, yielding acetal-protected OPA-CME product.
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| Strategy for Future Research Activity |
1. Synthesis of additional model peptides: Create more peptide models with varying sequences to further demonstrate the utilization of the 3,4-ThDA-based peptide cyclization method.
2. Optimization of cyclization conditions: Minimize the side-reactions of the 3,4-ThDA cyclization and explore milder conditions suitable for the in vitro translation system.
3. Mechanistic investigation: Study the reaction mechanism of how the thiol group participates in the reaction between 3,4-ThDA and amino groups.
4. Synthesis of OPA-CME derivatives: Complete the synthesis of crucial OPA-CME derivatives and assess the acylation efficiency with flexizymes.
5. Application to the FIT system: Demonstrate and incorporate the optimized OPAon/off method into the FIT system, and further construct diverse non-standard peptide libraries.
6. Integration with the RaPID system: Incorporate the developed peptide libraries into the RaPID system to identify peptide candidates against disease-related protein targets.
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Report
(1 results)
Research Products
(2 results)
