2004 Fiscal Year Final Research Report Summary
New methods of regulating protein functions
| Project/Area Number |
15350106
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemistry related to living body
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| Research Institution | Toho University |
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Principal Investigator |
FURUTA Toshiaki Toho University, Faculty of Science, Associate Professor, 理学部, 助教授 (90231571)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Caged Compounds / Photochemistry / Regulation of protein functions / Protein kinase C / inhibitors / solid phase synthesis |
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| Research Abstract |
A method of regulating protein function with high spatial and temporal resolution would offer a new tool of investigating cellular chemistry. Caged compounds have been recognized as a powerful tool for elucidating various biological processes with high spatial and temporal resolution. We have reported 6-bromo-7-hydoroxycoumarin-4-ylmethyl (Bhc) group as a novel photoremovable protecting group and found that Bhc-caged neurotransmitters and second messengers have improved photolysis efficiencies. To extend the utility of the Bhc-caged compounds, we report here the synthesis and photochemistry of caged ammo acids, peptides and proteins having the Bhc protecting group on their side chain functional groups. Four types of amino acids (Asp, Tyr, Lys, and Ser) that have different side chain functionality were selected. All of the synthesized Bhc and its 7-methoxy variant Bmc-caged amino acids showed about an order of magnitude larger photochemical efficiencies than those of the conventional 2-nitrobenzyl-caged compounds. The results suggest that the Bhc and Bmc groups would allow us to develop new caged compounds that can generate active proteins and peptides upon UV irradiation with low light intensity to minimize cell damages. Using an in vitro transcription system, lysine-83 in streptavidin was selectively replaced by Bhc-caged Lys. The method could be a general procedure to construct caged proteins whose biological functions can be activated upon photo-irradiation. In addition, Lys residue of octa-peptide (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr), a selective antagonist of εPKC translocation and function, was also replaced by Bmc-caged Lys by utilizing Fmoc solid-phase chemistry. The synthetic caged peptide released the parent octa-peptide on 350-nm irradiation with photolysis quantum yield (Φ) of 0.082 and overall photolysis efficiency (Φε) of 435, which must be high enough for live cell applications.
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[Journal Article] Bhc-cNMPs as either water-soluble or membrane-permeant photo-releasable cyclic nucleotides for both one and two-photon excitation2004
Author(s)
T.Furuta, H.Takeuchi, M.Isozaki, Y.Takahashi, M.Sugimoto, M.Kanehara, T.Watanabe, K.Noguchi, T.M.Dore, M.Iwamura, R.Y.Tsien
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Journal Title
Chem Bio Chem 5
Pages: 1119-1128
Description
「研究成果報告書概要(欧文)」より
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