open access

https://doi.org/10.14943/doctoral.k13941

https://hdl.handle.net/2115/89207

マルチタグ交換可能な新規ジアジリン光アフィニティープロブの創製

英語

2020-03-25

Hokkaido University

93p

In early stage of drug discovery, identifying the molecular interactions between the bioactive compound and its target protein is one of the crucial steps. Over the years, it has been known that identifying target protein at the molecular level is cumbersome for certain diseases. For this purpose, photoaffinity labeling (PAL) has been developed as a quintessential strategy to identify the targets of active biomolecules, owing to its capability to form a covalent bond with its target. Diazirines are one of the important photolabile moieties used in photoaffinity labeling due to their superior photo labeling characteristics. Conventional photoaffinity label consists of three main components; a photophore (ie diazirine), a ligand and detection/purification tags. Although this method has been widely used, target identification is still a challenging task. Herein, we reported on the design and synthesis of nosyl bearing diazirines which consist of photophore and bioactive ligand. In chapter one, general introduction on the concept of photoaffinity labelling was discussed. Types of photolabel and their advantages and disadvantages were also discussed in detail. A brief explanation on some of the successful study of protein identification utilizing photoaffinity labelling approach, followed by literature review on the most recent application of photolabeling concept. The second chapter is consists of preliminary studies on the development of nosyl diazirines in terms of synthesis and identification of the optimum condition for photolabeling. The exchangeable reaction of tag was also discussed in detail using a different fluorophore as the identification tag. Confirmation of the feasibility of this approach was demonstrated using biotin nosyl diazirine as model compound and BSA as model protein. Upon conducting the photolabeling experiment, we successfully proved that the photolabeling efficiency is increased and the insertion of the identification tag was achieved in high yield. This photoaffinity label was further explored using methotrexate (MTX), a widely known inhibitor of dihydrofolate reductase (DHFR) was used as a bioactive ligand. We successfully synthesized the probe via 4 steps. The protein identification experiment was also discussed in detail.

2020-03-25

甲第13941号

博士(生命科学)

(主査) 教授 門出 健次, 教授 比能 洋, 教授 玉置 信之, 助教 村井 勇太

生命科学院（生命科学専攻）

博士論文

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