WO2001046209A1 - Sondes fluorescentes - Google Patents

Sondes fluorescentes Download PDF

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Publication number
WO2001046209A1
WO2001046209A1 PCT/US2000/034714 US0034714W WO0146209A1 WO 2001046209 A1 WO2001046209 A1 WO 2001046209A1 US 0034714 W US0034714 W US 0034714W WO 0146209 A1 WO0146209 A1 WO 0146209A1
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WO
WIPO (PCT)
Prior art keywords
complex
probes
fluorescence
lifetime
probe
Prior art date
Application number
PCT/US2000/034714
Other languages
English (en)
Inventor
Zakir Murtaza
Original Assignee
Fluorrx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fluorrx, Inc. filed Critical Fluorrx, Inc.
Publication of WO2001046209A1 publication Critical patent/WO2001046209A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0046—Ruthenium compounds
    • C07F15/0053—Ruthenium compounds without a metal-carbon linkage

Definitions

  • This invention relates generally to fluorescencent materials and to their use in fluorescence lmmunoassavs and more particularly to the use of such materials in fluorescence resonance energy transfer (FRET) based assays and in fluorescence polarization (FP) based lmmunoassavs.
  • FRET fluorescence resonance energy transfer
  • FP fluorescence polarization
  • metal compounds that are fluorescent and also have relatively long fluorescence lifetimes (more than 30 ns)
  • One type includes lanthanide chelate compounds, and other type includes transition metal complexes.
  • the lanthanide compounds have good emission, but they do not show polarized emission and, thus, are not useful as polarization probes.
  • Transition metal complexes show polarized emission and , thus. have potential application as polarization probes as demonstrated recently by Lakowicz.. J. R. et. al; " Recent Development in Fluorescence Spectroscopy. Near Infrared Dyes for High Technology Application. " Edited by Daehne et. al. (eds.) 1998. Kluwer Academic Publishers, page 3-19.
  • the emission lifetimes of these probes are relatively long, in most cases more than 30 ns and in some cases extending up to microseconds. Because emission is due to metal to gand charge transfer (MLCT) in these compounds, delayed emission occurs resulting in longer lifetimes.
  • MLCT metal to gand charge transfer
  • the long lifetime probes make possible new, improved and low cost methods for doing optical sensing using phase modulation and frequency modulation analysis as is described m US Patent 5,281.825. dated Jun. 25, 1994.
  • Lakowicz. J. R., et. rr/j There are several examples of the ruthenium metal complexes as shown by Juris, A. et.al..
  • the selection of the probe is depending upon the size of the analyt. For example, if one wishes to measure larger molecule, such as a large protein, one needs a longer lifetime probe Normally, the larger the protein molecule to be detected in an assay, the longer the desired fluorescence lifetime of the probe. When FP is used as the assay method, a higher value of amsotropy is needed. A useful probe should have 0 3 or higher value of amsotropy.
  • the new invented FP dye is a good to study molecule having molecular weight 10 '-10° range
  • a major problem for commercial success in using fluorescence techniques for lmmunoassavs with conventional probes is that their relatively short lifetimes make their use in assays for larger bio-molecules commercially unattractive because of the relatively expensive high frequency instrumentation needed to measure short fluorescent lifetimes.
  • a further problem with conventional probes is photostabi ty. Many of the conventional dyes decomposes on exposure to high intensity light, creating errors m assays and also creating problems with analysis where longer exposing time is required.
  • Another problem is that, there are very limited number of long lifetime FP probes available, due to that the FP based immunoassav are limited for very low molecular weight antigen.
  • the mam object of the present invention is to circumvent problems described above.
  • the present invention which relates to a fluorescent metal ligand complex having ligands that are capable of attachment to a biomolecule and that have an asymet ⁇ c charge state, the complex being capable of emitting polarized radiation of a sufficient lifetime to be useful in FP assavs with large molecules and as a donor in FRET assays.
  • the present invention provides a probe based on a new ruthenium complex for use in
  • FP lmmunoassavs as well as FRET based assays. As shown below, it provides a 35%, improvement in quantum yield, a 70%) improvement in lifetime values and a 50%> improvement in polarization values when compared to an existing well documented probe that is also based on a ruthenium (II) metal complex with diimme ligands.
  • II ruthenium
  • the present probe is also very photostable as compare to the conventional organic compound based fluorescence probes, which are widely use in fluorescence based lmmunoassavs.
  • FIG. 1 is an absorption and emission spectra of the [Ru(aphen)(phen)(dpphenS)](T_ probe in aqueous solution.
  • SCHEME I is the representation of the fluorescence dye [Ru(aphen)(phen)(dpphenS)]Cl_.
  • the target compound fRu(phen)(aphen)(dpphenS)]Ch was synthesized in three steps In first step compound Ru(phen)C was prepared.
  • the intermediate compound fRu(phen)(aphen)(dpphenS)]Ch was synthesized in three steps In first step compound Ru(phen)C was prepared.
  • Ru(phen)Cl i was obtained by mixing equal molar solution of RuCh and 1, 10-phenathrolme in 3N HC1 (both chemicals were purchased from Aldnch Chemical Co. Inc.). The reaction mixture was stirred at room temperature for seven days after that black color precipitates form, which were separated bv filtration and washed with water and dried in air.
  • Ru(phen)(aphen)Cl'2 was prepared.
  • This intermediate, Ru(phen)(aphen)Cl2 was prepared by reaction of 5-am ⁇ no-l, 10-phenanathrol ⁇ ne (aphen) (purchased from Polysciences Inc.) with Ru(phen)Cl4 in 1.1 ratio respectively m refluxmg DMF for 24 hr.
  • the black color compound was separated from reaction mixture by evaporating solvent and washed with water and dried
  • the third and final step was the synthesis of [Ru(phen)(aphen)(dpphenS)lCb.
  • the target compound. [Ru(phen)(aphen)(dpphenS)]Cb was prepared by reaction of Ru(phen)(aphen)Cl2 With 4 7-d ⁇ phenvld ⁇ sulphon ⁇ c acid- 1, 10-phenanthrolme (dpphenS) in 1.1 ratio respectively in refluxmg 1:1 ethanol water mixture for 24 hrs. After 24-hr. color of the reaction mixture changes from purple brown to orange red At this time the reaction mixture was brought down to room temperature and solvent was reduced to less than half and product was crystallized by adding large amount of acetone. It was further purified by passing through silica column using acetone water mixture.
  • r amsotropy of the analyt.
  • rn amsotropy of dye (m the absence of rotational diffusion), is lifetime of the probe and is rotational correlation time.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention se rapporte à un complexe de ligands métalliques fluorescents présentant un état de champ asymétrique . Ce complexe contient des ligands parmi lesquels un au moins peut se fixer à une biomolécule et émettre une fluorescence polarisée de durée suffisamment longue pour être utilisé en tant que fluorophore dans une analyse FP ou en tant que donneur dans une analyse FRET.
PCT/US2000/034714 1999-12-20 2000-12-20 Sondes fluorescentes WO2001046209A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17268199P 1999-12-20 1999-12-20
US60/172,681 1999-12-20

Publications (1)

Publication Number Publication Date
WO2001046209A1 true WO2001046209A1 (fr) 2001-06-28

Family

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Application Number Title Priority Date Filing Date
PCT/US2000/034714 WO2001046209A1 (fr) 1999-12-20 2000-12-20 Sondes fluorescentes

Country Status (1)

Country Link
WO (1) WO2001046209A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216582B2 (en) 2006-06-23 2012-07-10 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in cancer
US8580257B2 (en) 2008-11-03 2013-11-12 Alethia Biotherapeutics Inc. Antibodies that specifically block the biological activity of kidney associated antigen 1 (KAAG1)
WO2014203067A1 (fr) * 2013-06-18 2014-12-24 Rubipy Scientific, Inc. Complexes métalliques électroniquement neutres en tant que marqueurs biologiques
US8937163B2 (en) 2011-03-31 2015-01-20 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
CN106893108A (zh) * 2017-02-15 2017-06-27 中国科学院福建物质结构研究所 一种利用可见光敏化近红外稀土发光的钌‑钕双金属配位聚合物及其制备方法和用途
CN108732147A (zh) * 2018-04-23 2018-11-02 南京邮电大学 基于fret效应探测细胞凋亡过程的方法
US11084872B2 (en) 2012-01-09 2021-08-10 Adc Therapeutics Sa Method for treating breast cancer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4745076A (en) * 1984-09-17 1988-05-17 Hoffmann-La Roche Inc. Ruthenium complexes useful as carriers for immunologically active materials
US5112974A (en) * 1985-01-18 1992-05-12 The Trustees Of Columbia University In The City Of New York Mixed ligand complexes and uses thereof as binding agents to DNA
US5278043A (en) * 1990-01-25 1994-01-11 Hoffmann-La Roche Inc. Ruthenium-lumazine energy transfer systems

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4745076A (en) * 1984-09-17 1988-05-17 Hoffmann-La Roche Inc. Ruthenium complexes useful as carriers for immunologically active materials
US5112974A (en) * 1985-01-18 1992-05-12 The Trustees Of Columbia University In The City Of New York Mixed ligand complexes and uses thereof as binding agents to DNA
US5278043A (en) * 1990-01-25 1994-01-11 Hoffmann-La Roche Inc. Ruthenium-lumazine energy transfer systems

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216582B2 (en) 2006-06-23 2012-07-10 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in cancer
US8580257B2 (en) 2008-11-03 2013-11-12 Alethia Biotherapeutics Inc. Antibodies that specifically block the biological activity of kidney associated antigen 1 (KAAG1)
US9855291B2 (en) 2008-11-03 2018-01-02 Adc Therapeutics Sa Anti-kidney associated antigen 1 (KAAG1) antibodies
US10597450B2 (en) 2011-03-31 2020-03-24 Adc Therapeutics Sa Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US8937163B2 (en) 2011-03-31 2015-01-20 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US9393302B2 (en) 2011-03-31 2016-07-19 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US9828426B2 (en) 2011-03-31 2017-11-28 Adc Therapeutics Sa Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
US11084872B2 (en) 2012-01-09 2021-08-10 Adc Therapeutics Sa Method for treating breast cancer
WO2014203067A1 (fr) * 2013-06-18 2014-12-24 Rubipy Scientific, Inc. Complexes métalliques électroniquement neutres en tant que marqueurs biologiques
CN105492575A (zh) * 2013-06-18 2016-04-13 联吡啶钌科学公司 作为生物标记物的电中性金属配合物
CN105492575B (zh) * 2013-06-18 2019-01-22 联吡啶钌科学公司 作为生物标记物的电中性金属配合物
US10203333B2 (en) 2013-06-18 2019-02-12 Rubipy Scientific, Inc. Electronically neutral metal complexes as biological labels
CN106893108A (zh) * 2017-02-15 2017-06-27 中国科学院福建物质结构研究所 一种利用可见光敏化近红外稀土发光的钌‑钕双金属配位聚合物及其制备方法和用途
CN106893108B (zh) * 2017-02-15 2019-04-16 中国科学院福建物质结构研究所 一种利用可见光敏化近红外稀土发光的钌-钕双金属配位聚合物及其制备方法和用途
CN108732147A (zh) * 2018-04-23 2018-11-02 南京邮电大学 基于fret效应探测细胞凋亡过程的方法

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