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Strategic development of fluorescent probes applicable to in vivo dynamic analysis of hydrogen peroxide

Research Project

Project/Area Number 15390012
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionOsaka University

Principal Investigator

MAEDA Hatsuo  Osaka University, Graduate School of Pharmaceutical Sciences, Associate Professor, 大学院・薬学研究科, 助教授 (00229311)

Co-Investigator(Kenkyū-buntansha) HIRATA Kazumasa  Osaka University, Graduate School of Pharmaceutical Sciences, Associate Professor, 大学院・薬学研究科, 助教授 (30199062)
ITOH Norio  Osaka University, Graduate School of Pharmaceutical Sciences, Lecturer, 大学院・薬学研究科, 講師 (60176352)
Project Period (FY) 2003 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥10,200,000 (Direct Cost: ¥10,200,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥6,900,000 (Direct Cost: ¥6,900,000)
KeywordsFluorescent probe / Bioimagine / Hydrogen peroxide / Superoxide / Thiol / Selenocysteine / Selenoprotein / Oxidative stress / セレノール / フローサイトメトリー / 活性酸素種 / 可視化 / コリンエステラーゼ / コリンエステラーゼ阻害薬 / 緑藻 / 好中球
Research Abstract

(1) Hydrogen Peroxide (H_2O_2) Probe : Based on the proposed working hypothesis, we examined the reactivity of various types of benzenesulfonyl fluoresceins toward H_2O_2, and found that 3'-(pentafluorobenzenesulfonyl) 2',7'-difluorofluorescein (1) works as a specific H_2O_2 fluorescent probe. In addition, it was demonstrated that the acetyl derivative of 1 allows fluorometric analysis of H_2O_2 generated inside a green algae Chlamydomonas reinhardtii and human Jurkat T cells by stimulation with Cu^<2+> and butyric acid, respectively. The study on developing these probes, we have been able to propose a novel and general strategy for probe design.
(2) Superoxide (O_2^<-・>) Probe : Based on the proposed strategy, we succeeded in developing 3',5'-bis(2,4-dinitrobenzenesulfonyl) 2',4',5',7'-tetra-fluorofluorescein (2) as a novel O_2^<-・> probe. A further study allowed demonstrating that 3'-(4,5-dimethoxy-2-nitrobenzenesulfonyl) 2',4',5',7'-tetrafluorofluorescein (3) as a more practical O_2^ … More <-・> probe than 2. By loading cells with the acetoxymethyl derivative of 3, flowcytometry and fluorescent microscopy were able to measure O_2^<-・> generated inside human Jurkat T cells stimulated with butyric acid.
(3) Thiol Probe : Based on the proposed strategy, we succeeded in developing 3'-(2,4-dinitrobenzenesulfonyl) 2',7'-dimethylfluorofluorescein (4) as a novel thiol probe. High miscibility of 4 to aqueous media tolerated development of highly sensitive fluorometric assay of cholinesterases useful as a screening method of acetylcholinesterase specific inhibitors.
(4) Selenol Probe : It was demonstrated that at pH 5.8, 4 provides no fluorescent responses toward thiols, while bringing about strong fluorescent augmentation on reaction with selenol, namely, that 4 works as a first selenol fluorescent probe. Thus, the fluorometry with 3 works as a sensitive quantification method for selenocysteine generated from selenocystine by reaction with dithiothreitol, and as an estimating tool for selenocysteine residues in selenoproteins. Less

Report

(4 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (22 results)

All 2006 2005 2004 Other

All Journal Article (21 results) Publications (1 results)

  • [Journal Article] 3'-(2,4-Dinitrobenzenesulfonyl)-2',7'-dimethylfluorescein as a fluorescent probe for selenols2006

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 45

      Pages: 1810-1813

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 保護/脱保護化学に基づく蛍光プローブの設計開発-過酸化水素,スーパーオキサイド,チオールおよびセレノールの可視化計測-2006

    • Author(s)
      前田初男
    • Journal Title

      生物工学 84(in press)

    • NAID

      110004750422

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 必須微量元素セレンをはかる:セレノールの可視化計測2006

    • Author(s)
      前田初男
    • Journal Title

      海洋化学研究 19(in press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 3'-(2,4-Dinitrobenzenesulfonyl)-2',7'-dimethylfluorescein as a fluorescent probe for selenols2006

    • Author(s)
      H.Maeda, K.Katayama, H.Matsuno, T.Uno
    • Journal Title

      Angew.Chem.Int.Ed. 45

      Pages: 1810-1813

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 3'-(2,4-Dintrobenzenesulfonyl)-2',7'-dimethylfluorescein as a Fluorescent Probe for Selenols2006

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 45

      Pages: 1810-1813

    • Related Report
      2005 Annual Research Report
  • [Journal Article] 保護/脱保護科学に基づく蛍光プローブの設計開発:過酸化水素,スーパーオキサイド,チオール及びセレノールの可視化計測2006

    • Author(s)
      前田 初男
    • Journal Title

      生物工学会誌 84(in press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] A design of fluorescent probes for superoxide based on a nonredox mechanism2005

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Journal of American Chemical Society 127

      Pages: 68-69

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman's reagent in thiol-quantification enzyme assays2005

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 44

      Pages: 2922-2925

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 化学的脱保護反応を発蛍光機構とする蛍光プローブの設計開発2005

    • Author(s)
      前田初男
    • Journal Title

      和光純薬時報 73(3)

      Pages: 2-4

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] A design of fluorescent probes for superoxide based on a nonredox mechanism2005

    • Author(s)
      H.Maeda, K.Yamamoto, Y.Nomura, I.Kohno, L.Hafsi, N.Ueda, S.Yoshida, M.Fukuda, Y.Fukuyasu, Y.Yamauchi, N.Itoh
    • Journal Title

      J.Am.Chem.Soc. 127

      Pages: 68-69

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman's reagent in thiol-quantification enzyme assays2005

    • Author(s)
      H.Maeda, H.Matsuno, M.Ushida, K.Katayama, K.Saeki, N.Itoh
    • Journal Title

      Angew.Chem.Int.Ed. 44

      Pages: 2922-2925

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Design of fluorescent probes with fluorogenic mechanisms based on chemical deprotection2005

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      WAKO-Junyaku-Ziho 73(3)

      Pages: 2-4

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman's Regent in Thiol-Quantification Enzyme Assay2005

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 44

      Pages: 2922-2925

    • Related Report
      2005 Annual Research Report
  • [Journal Article] 化学的脱保護反応を発蛍光機構とする蛍光プローブの設計開発2005

    • Author(s)
      前田 初男
    • Journal Title

      和光純薬時報 73

      Pages: 2-4

    • Related Report
      2005 Annual Research Report
  • [Journal Article] A Design of Fluorescent Probes for Superoxide Based on a Nonredox Mechanism2005

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Journal of American Chemical Society 127

      Pages: 68-69

    • Related Report
      2004 Annual Research Report
  • [Journal Article] 2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman's Reagent in Thiol Quantification-Based Enzyme Assay2005

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 44(in press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism2004

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 43

      Pages: 2389-2391

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism2004

    • Author(s)
      H.Maeda, Y.Fukuyasu, S.Yoshida, M.Fukuda, K.Saeki, H.Matsuno, Y.Yamauchi, K.Yoshida, K.Hirata, K.Miyamoto
    • Journal Title

      Angew.Chem.Int.Ed. 43

      Pages: 2389-2391

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Fluorescent Probes for Hydrogen Peroxide Based on a Non-Oxidative Mechanism2004

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Angewandte Chemie International Edition 43

      Pages: 2389-2391

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Design of fluorescent probes based on protection / deprotection chemistry : fluorometry of H_2O_2, O_2^<-・>, thiols, and selenols.

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Seitai-Kougaku Kaishi (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Quantification of essential trace element Selene : visualization of selenols

    • Author(s)
      Hatsuo Maeda
    • Journal Title

      Kaiyou-Kagaku Kenkyu (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Publications] Hatsuo Maeda: "Fluorescent Probes for Hydrogen Peroxide Based on a Non-Oxidative Mechanism"Angewandte Chemie International Edition. 43(in press). (2004)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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