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WO2007017540A1 - Nouveau reactif de biotine, procedes de preparation et de ciblage de celui-ci, et applications correspondantes - Google Patents

Nouveau reactif de biotine, procedes de preparation et de ciblage de celui-ci, et applications correspondantes Download PDF

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Publication number
WO2007017540A1
WO2007017540A1 PCT/ES2006/000451 ES2006000451W WO2007017540A1 WO 2007017540 A1 WO2007017540 A1 WO 2007017540A1 ES 2006000451 W ES2006000451 W ES 2006000451W WO 2007017540 A1 WO2007017540 A1 WO 2007017540A1
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WO
WIPO (PCT)
Prior art keywords
biotin
chloride
preparing
carried out
obtaining
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/ES2006/000451
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English (en)
Spanish (es)
Inventor
Maria José HERNAIZ GOMEZ-DEGANO
Javier MUÑOZ SILES
Maria Fernandez Fernandez
José Vicente SINISTERRA GAGO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad Complutense de Madrid
Original Assignee
Universidad Complutense de Madrid
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 Universidad Complutense de Madrid filed Critical Universidad Complutense de Madrid
Publication of WO2007017540A1 publication Critical patent/WO2007017540A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

Definitions

  • New biotin reagent methods for its preparation and marking, and applications.
  • the invention falls within the area of Organic Chemistry and Biochemistry with application in the Biotechnology and Biomedicine sectors. More specifically, it refers to the development of a new biotin reagent and its use in the preparation of labeled derivatives as well as its binding to biomolecules.
  • Biotin (vitamin H) is a water soluble molecule that is found at low concentrations in the blood and tissues. Its biological function is to act as a transport molecule of the carboxyl group. Biotin is covalently bound to the enzyme pyruvate carboxylase. An activated carboxyl group, derived from a bicarbonate ion, is coupled to biotin through a reaction that requires a supply of energy from the hydrolysis of an ATP molecule. This carboxyl group is then transferred to the pyruvate methyl group forming oxaloacetate.
  • the affinity between avidin and biotin has a large number of applications, for example, the avidin-biotin complex has been used as a detection system where the target molecule is combined with biotin through its terminal carboxyl group, in this way biotinylated molecules can be easily detected and separated from the solution.Biotinylation of the target molecule can be carried out without changing the biological or physicochemical properties of the target molecule and without affecting the binding capacity of biotin to Avidin
  • streptavidin (or avidin) and biotin (Gitlin, G .; Bayer, EA and Wilchek, M. 1987, Biochem. J. 242, 923-926, Gitlin, G .; Bayer, EA and Wilchek, M. 1988, Biochem. J. 250, 291-294) is well known, in fact it is one of the strongest, within non-covalent biological interactions.
  • Biotin-streptavidin affinity is one of the most used in molecular, immunological and cellular assays. Streptavidin can be detected and quantified with a high degree of sensitivity in the complex formed, for example by marking it with an enzyme or a fluorescent or radioactive label. Streptavidin mapping has also been used to detect proteins on the cell surface, to visualize and quantify blots and carry out ELISA-type assays (P. Vincent, Journal of Immunological Methods, ⁇ 65; 177-182, 1993).
  • Streptavidin can also be immobilized on a surface so as to capture biotinylated molecules or cells. These surfaces can be used to detect the molecules of interest from complex mixtures. Therefore, the streptavidin-biotin interaction has been widely used in a large number of separation, purification and isolation processes, such as affinity chromatography, etc. This same approach has been used to carry out studies of interactions between biomolecules through the use of biosensors based on surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • oligonucleotides and nucleic acids are frequently used in many molecular biology procedures and in many other techniques such as sequencing, amplification, cDNA preparation and nucleic acid purification.
  • This methodology has been adapted for use in solid phase, using a support covered with streptavidin.
  • Streptavidin-biotin magnetic microparticles have also been prepared for use in PCR (polymerase chain reaction) (Hultman et al, Nucleic Acids Res. 17: 4937-4946, 1989). This method is of great interest, results in good yields and is easily automated compared to traditional methods of purification based on precipitation and centrifugation.
  • Biotin is a non-aromatic heterocyclic compound with a 4-carboxybutyl tail.
  • biotin binds through 4-carboxybutyl to a linker (spacer) that can be associated with a biomolecule (protein, carbohydrates, DNA, lipid, drug, etc.).
  • linker spacer
  • biomolecule protein, carbohydrates, DNA, lipid, drug, etc.
  • All the procedures described to carry out the reaction between the biotin and the amino group of the molecule under study use the adjuvants typical of the formation of the peptide bond such as NHS and EDC, activators of amino and carboxyl groups respectively.
  • the yields described in the literature for this process are low (30%) and the procedure tedious.
  • New biotin reagent methods for its preparation and marking, and applications.
  • One aspect of the present invention relates to a new biotin reagent, biotin chloride or 5 - ((3a5 r , 45 ⁇ , 6a, S) -2-oxo-hexahydro-lH ' -thieno [3, 4- d] imidazol-4-yl) pentanoyl.
  • another aspect of the present invention refers to the method for the preparation of this biotin reagent that allows its reaction in a quick and simple way with any correctly functionalized biomolecule, as well as its marking with a marker.
  • the method of preparation of biotin chloride described is easy to carry out and, in addition, high yields, high reactivity and great application are obtained with it.
  • the preparation of biotin chloride is a simple procedure that involves a single stage consisting of the addition of thionyl chloride to biotin. A solid immediately precipitates, which is the reaction product that is easily isolated, for example by simple vacuum filtration. The yield obtained is 75%.
  • Another aspect contemplated by the invention is the mapping of biotin chloride.
  • the marking can be done with a fluorescent marker or a radioactive marker.
  • Fluorescent marking consists of a simple procedure that involves a single stage, that is, the addition of fluorophore to a solution of biotin chloride in the appropriate solvent (10% DMF (dimethylformamide) or DMSO (dimethyl sulfoxide) and 90% solvent in which the fluorophore is soluble).
  • the fluorophores 2,6-diaminopyridine, a fluorescent marker that absorbs Ultraviolet-Visible, can be used. After the necessary time, 6 hours, a precipitate forms which is obtained by simple methods such as filtering. 100% yields are obtained.
  • Both the new biotin reagent and the biotinylated marker can be linked to a large number of biomolecules (proteins, enzymes, peptides, oligosaccharides or lipids) which allows their use in studies to analyze their involvement in biological processes or in the preparation of systems Diagnostic
  • This process can be carried out in a simple and consistent way in a single step, that is, the addition of the biomolecule to a solution of the biotin chloride in the appropriate solvent (10% DMF (dimethylformamide) or DMSO (dimethyl sulfoxide) and 90% solvent in which the biomolecule is soluble). 95% yields are obtained.
  • FIG. 1 Fluorescent marking of biotin chloride (2) with 2,6-diaminopyridine (3).
  • thionyl chloride SOCl 2
  • biotin chloride (2) is obtained, which is marked when 2,6-diaminopyridine (3) is added, obtaining the biotinylated marker (4) with a yield of 100 %.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

L'invention concerne un nouveau réactif de biotine, des procédés de préparation et de ciblage de celui-ci, et des applications correspondantes. Cette invention concerne un nouveau réactif de biotine, le chlorure de biotine ou chlorure de 5-((3aSr,4>Sr,6a1S)-2-oxo-hexalhydro-1H-tiène[3,4-d]imidazol-4-il)pentanoil ou, sa préparation en une seule étape, ainsi que son ciblage également en une seule étape. En outre, le chlorure de biotine et le marqueur biotinylé peuvent se lier à un grand nombre de biomolécules (protéines, enzymes, peptides, oligosaccharides ou lipides) ce qui permet son utilisation dans des études en vue d'analyser l'implication de ces molécules dans des procédés biologiques, ainsi que son utilisation dans la préparation de systèmes de diagnostic.
PCT/ES2006/000451 2005-08-02 2006-08-01 Nouveau reactif de biotine, procedes de preparation et de ciblage de celui-ci, et applications correspondantes Ceased WO2007017540A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200501927 2005-08-02
ES200501927A ES2265778B2 (es) 2005-08-02 2005-08-02 Nuevo reactivo de biotina, metodos para su preparacion y marcaje, y aplicaciones.

Publications (1)

Publication Number Publication Date
WO2007017540A1 true WO2007017540A1 (fr) 2007-02-15

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2006/000451 Ceased WO2007017540A1 (fr) 2005-08-02 2006-08-01 Nouveau reactif de biotine, procedes de preparation et de ciblage de celui-ci, et applications correspondantes

Country Status (2)

Country Link
ES (1) ES2265778B2 (fr)
WO (1) WO2007017540A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2519530A (en) * 1947-06-20 1950-08-22 Merck & Co Inc Biotin aliphatic amides and method for their preparation
EP1415995A2 (fr) * 1994-05-11 2004-05-06 Trustees Of Boston University Agents et conjugués photodissociables, utilisés dans la détection et l'isolement de biomolecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2519530A (en) * 1947-06-20 1950-08-22 Merck & Co Inc Biotin aliphatic amides and method for their preparation
EP1415995A2 (fr) * 1994-05-11 2004-05-06 Trustees Of Boston University Agents et conjugués photodissociables, utilisés dans la détection et l'isolement de biomolecules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROTHENBERG B. ET AL.: "Biotinylated diaminopyridine: an approach to tagging oligosaccharides and exploring their biology", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 90, no. 24, 1993, pages 11939 - 11943, XP000918931 *
TOOMRE ET AL.: "Advances in the use of biotinylated diaminopyridine (BAP) as a versatile fluorescent tag for oligosaccharides", GLYCOBIOLOGY, vol. 4, 1994, pages 653 - 663, XP003008858 *

Also Published As

Publication number Publication date
ES2265778A1 (es) 2007-02-16
ES2265778B2 (es) 2007-11-01

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