| Project/Area Number |
22KF0096
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| Project/Area Number (Other) |
22F22743 (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 37030:Chemical biology-related
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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) |
BINDL DANIEL 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 |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2024: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 2023: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2022: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Bioorthogonal chemistry / masking of polar groups / Cross-coupling chemistry |
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| Outline of Research at the Start |
Model peptide compounds containing suitable functional side chain groups will be synthesized and tested for their bioorthogonal reactivity towards intramolecular amide alkylation. Afterwards, flexizyme catalyzed loading of amino acid analogs bearing these groups onto tRNA and ribosomal in vitro translation will be optimized to enable their usage in RaPID screening for the discovery of novel protein binders.
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| Outline of Annual Research Achievements |
Work has been completed towards developing a new bioorthogonal intramolecular amide alkylation method for the past two years. The goal is to have a reactive amino acid side chain moiety that, upon activation with light or reactants/catalysts, can cyclize onto the neighboring amide nitrogen. This chemistry would aid to expand the chemical space accessible for the use in high throughput screening platforms for peptide drug discovery, and hopefully lead to compounds with enhanced pharmacological properties. Reaction conditions must be bioorthogonal and compatible to be used in in vitro peptide translation and the mRNA display screening platform RaPID. Initially, finding such conditions turned out to be quite challenging. After extensive efforts however, two different reaction methods have crystallized as promising candidates to achieve this endeavor.
First, metal-catalyzed cross-couplings, particularly utilizing Cu(II) containing catalysts in aqueous conditions have proven to yield backbone amide alkylation. However, the scope of this methodology, as of now, seems to be quite limited and only gives satisfactory conversions with glycine as the alkylated, neighboring amino acid. The other technique involves the use of an aspartic acid derived aldehyde side chain. It is known that this functionality is forming a 5-membered hemiaminal cycle with the neighboring backbone amide, and we could show that it can further react with a variety of more distant sidechains to form stable products in model peptides under bioorthogonal reaction conditions.
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| Current Status of Research Progress |
Current Status of Research Progress
4: Progress in research has been delayed.
Reason
As described in the summary of research achievements, two chemical methodologies for intramolecular peptide amide alkylation are currently under investigation. These chemistries are quite different, each posing their individual benefits and downsides.
The copper catalyzed alkylation strategy has been shown to provide clean conversion to the desired products in some cases, however, conversion yields remain in an unsatisfactory range of only up to 50%. Furthermore, it only works on glycine as the neighboring reaction partner with reasonably fast kinetics, severely limiting the chemical space accessibly to this methodology.
The other chemical modification strategy under current investigation involves an aspartate derived aldehyde side chain. Upon acid treatment this reactive group was shown to form a variety of different polycyclic products with the neighboring amide nitrogen and side chain moieties. In most cases of tested model peptides, reactions reach full conversion in a clean manner. However, analytical problems were encountered when expanding this chemistry to ribosomally translated peptide substates, making it difficult to distinguish between hemiaminal intermediate and final polycyclic product.
Despite these challenges, the aldehyde mediated polycyclization strategy remains the most promising candidate so far to achieve the desired backbone amide masking of peptide compounds in a bioorthogonal setting. Continued optimization of reaction conditions and analytical strategies should enable this chemistry to be applicable in mRNA display screening platforms.
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| Strategy for Future Research Activity |
The future research plan aims at optimizing the reaction conditions for the aldehyde cyclization strategy described in the previous paragraphs. The first hurdle for successfully applying this chemistry in high throughput drug discovery endeavors is the proper analysis of reaction products in terms of identity and stereochemical configurations. Once unambiguous assignment is possible, the substrate scope of this chemistry on ribosomally translated peptides can the thoroughly investigated.
In the next step, we also plan to establish a solid phase peptide synthesis (SPPS) approach to obtain this class of post translationally modified peptides on a larger scale for in vitro and in vivo investigation of their biological activities. This involves the synthesis of larger quantities of SPPS precursors and a screening of feasible coupling, resin cleavage and cyclization conditions.
Lastly, we will turn our focus to screening libraries employing the newly developed chemistry against proteins of interest. In the hope that this posttranslational modification will enhance membrane permeability, the focus will lye on intracellular targets which are difficult to reach with conventional cyclic peptide drugs.
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