EP1347985A2 - Biotin-derivate, verfahren zu ihrer herstellung und ihre anwendungen als vectoren - Google Patents

Biotin-derivate, verfahren zu ihrer herstellung und ihre anwendungen als vectoren

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Publication number
EP1347985A2
EP1347985A2 EP01997495A EP01997495A EP1347985A2 EP 1347985 A2 EP1347985 A2 EP 1347985A2 EP 01997495 A EP01997495 A EP 01997495A EP 01997495 A EP01997495 A EP 01997495A EP 1347985 A2 EP1347985 A2 EP 1347985A2
Authority
EP
European Patent Office
Prior art keywords
formula
biotin
bccp
protein
dna sequence
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.)
Withdrawn
Application number
EP01997495A
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English (en)
French (fr)
Inventor
Abdelakim Kharrat
Khalil Bouayadi
Jean-Charles Faye
Emmanuelle Fourcade
Rudi Baron
Daniel Tovar
Casimir Blonski
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.)
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
MilleGen SA
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
MilleGen SA
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Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, MilleGen SA filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1347985A2 publication Critical patent/EP1347985A2/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/557—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
    • C12P17/186—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system containing a 2-oxo-thieno[3,4-d]imidazol nucleus, e.g. Biotin
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the subject of the invention is derivatives of biotin, and their uses as vectors, in particular in the context of the implementation of methods for detecting interactions between biological compounds, as well as in pharmaceutical compositions.
  • Small molecule-large molecule interactions play an important role in biological processes. They include all ligand-receptor, ligand-transport protein, hormone-receptor, enzyme-substrate, enzyme-inhibitor interactions ...
  • Gal4 protein which consists of two independent functional domains: a DNA binding domain and a transactivator domain.
  • the complex formed from these two domains acts on a promoter of the chimeric Gall-LacZ fusion gene, "reporter gene" for the interaction, and thus induces the biosynthesis of ⁇ -galactosidase.
  • reporter gene reporter gene for the interaction
  • transcription does not take place.
  • the DNA binding domain of Gal4 can be fused to a protein X, and the transactivating region of the transcription fused to a protein Y. If X and Y have the capacity to bind, the proximity of the two domains allows activation of the transcription of the Gall-LacZ gene.
  • biotin binds to its carrier proteins covalently via its COOH end, and in particular to a protein designated BCCP (for Biotin Carboxylase Carrier Protein), which. in E. coli, is the only protein that accepts to bind to biotin (under the action of an intracellular ligase designated Bir A).
  • BCCP Biotin Carboxylase Carrier Protein
  • BCCP is thus biotinylated by the formation of an amide bond between the COOH function of biotin (represented above), and the NH2 group of a lysine residue (located approximately 34 to 35 residues from the carboxyterminal end of the amino acid sequence of BCCP).
  • the protein thus biotinylated has an enzymatic role in metabolic carboxylation / decarboxylation reactions.
  • biotin or derivatives whose nitrogen atom located in position I '(NI') is substituted by a determined compound, makes it possible to integrate this compound with inside target cells, without there being:
  • biotinyiés while preserving the property of biotin to bind to its carrier protein, such as BCCP) inside target cells, in particular bacterial cells, advantageously E. cells. coli, or yeast, or cells of the human or animal organism (in particular CHO cells).
  • target cells in particular bacterial cells, advantageously E. cells. coli, or yeast, or cells of the human or animal organism (in particular CHO cells).
  • target cells in particular bacterial cells, advantageously E. cells. coli, or yeast, or cells of the human or animal organism (in particular CHO cells).
  • bacterial cells advantageously E. cells. coli, or yeast
  • cells of the human or animal organism in particular CHO cells.
  • the subject of the invention is the derivatives of biotin substituted with the of general formula (I) below
  • - Xi represents a carbon chain, preferably from 1 to 10 carbon atoms, substituted or not, optionally comprising one or more carbonyl or oxycarbonyl groups. and / or a group -NH-, in particular a chain of formula HN- (CH 2 ) nO-CO- in which n is an integer from 1 to 5, and optionally comprising one or more ether, amide and / or amine.
  • - Y represents a hydrocarbon chain of 1 to 5 carbon atoms, substituted or not, - R-2 represents a hydrogen atom, or a protective group, in particular R? represents an alkyl group of 1 to 5 carbon atoms.
  • a more particular subject of the invention is the biotin derivatives of formula (I) mentioned above in which X ⁇ represents a chain of formula -HN- (CH 2 ) nO-CO in which n represents 2 or 4, Y represents a chain hydrocarbon of 4 carbon atoms, and R.2 is a hydrogen atom or a methyl group.
  • biotin of formula (I) are chosen from the following: the compounds of formula (la) below:
  • Ri represents a radical derived from estradiol hemisuccinate of the following formula
  • Ri represents a radical derived from 1 estradiol of the following formula
  • Ri represents a radical derived from N-acetyl-S-farnesyl-cysteine of the following formula
  • Rj represents a radical derived from N-acetyl-S-geranylgeranylcysteine of the following formula:
  • the invention relates more particularly still to the biotin derivatives of formula (I) described above, in which R2 is a hydrogen atom.
  • a subject of the invention is also the biotin derivatives of formula (II) described below, and their use for the preparation of the derivatives of formula (I) mentioned above, by coupling an appropriate function of a determined compound (this function being, where appropriate, previously grafted onto said determined compound) and the function R of said derivatives of formula (II), in particular according to one of the coupling methods described below.
  • biotin derivatives of general formula (II) above correspond to the following formula:
  • -R represents a function -COOH, -NH2, -N3, or a halogen atom
  • -X represents a carbon chain, preferably from 1 to 10 carbon atoms, substituted or not, optionally comprising one or more carbonyl or oxycarbonyl groups, in particular a chain of formula - (CH2) nO- CO in which n is a whole number from 1 to 5,
  • - Y represents a hydrocarbon chain of 1 to 5 carbon atoms, substituted or not, - R-2 represents a hydrogen atom, or a protective group, in particular
  • R.2 represents an alkyl group of 1 to 5 carbon atoms, in particular a methyl group.
  • a more particular subject of the invention is the biotin derivatives of formula (II) above in which R is a halogen atom such as I or Cl, X represents a chain of formula - (CH 2 ) nO-CO- in which n represents 2 or 4, Y represents a hydrocarbon chain of 4 carbon atoms, and R-2 is a methyl group.
  • biotin of formula (II) are those of the following formulas:
  • biotin derivatives of formula (I) and (II) above are advantageously carried out according to the method comprising the following stages: treatment of a biotin derivative of the following formula:
  • RX-CI in which R is as defined above, in particular R represents a halogen atom such as Cl or I, and X is as defined above, in particular X represents a chain of formula - (CH 2 ) n-0-.C0- in which n represents 2 or 4, advantageously at approximately 65 ° C. for 4 days, which leads to obtaining of the compound of formula (Ha) below:
  • esterase such as pig liver esterase (PLE, for Pig Liver Esterase), advantageously at room temperature for approximately 10 days, or
  • the invention relates to the use of biotin derivatives of formula (II) above, as vectors allowing the introduction of a determined compound inside target cells, when said determined compound is linked to the radical R of said derivatives of formula (II).
  • the invention also relates to the use of analogs of biotin of formula (I), as vectors for introducing a 'compound determined within target cells.
  • the invention relates more particularly to the aforementioned use of biotin derivatives defined above, in the context of the implementation of a method for detecting proteins or other non-protein molecules capable of interacting with said determined compound, or in the context of the preparation of medicaments allowing the introduction of a determined compound of therapeutic interest inside cells of the organism.
  • the object of the invention is to provide new methods for detecting interactions between biological compounds, making it possible to study the interaction between on the one hand a determined compound, proteinaceous or not, and, on the other hand a proteinaceous compound or not, tested for its ability to interact with said determined compound.
  • non-protein compound is meant any compound which cannot be produced by a transcription mechanism of a nucleic acid, as opposed to a protein compound capable of being produced by such a mechanism.
  • the subject of the invention is more particularly any method of detecting proteins capable of interacting with the radical Ri (corresponding to the determined compound) of a biotin derivative of formula (I) as defined above, characterized in that that she understands:
  • biotin derivative of formula (I) into contact with host cells transformed so as to contain: • a DNA sequence encoding a first fusion protein comprising BCCP and a first revealing protein, and
  • a DNA sequence encoding a second fusion protein comprising a protein capable of interacting with the radical Ri mentioned above and a second revealing protein, the two revealing proteins being chosen in such a way that their approximation, following an interaction between the protein capable of interacting with the radical R ] in said second fusion protein, and the radical Ri of said derivative of biotin, generates a reaction process the result of which can be detected, this contacting being carried out for a time sufficient to allow the transport of said biotin derivative inside said host cells, and the binding of said biotin derivative to the BCCP of said first fusion protein,
  • the invention relates more particularly to the application of the aforementioned method to the detection of proteins capable of interacting with a determined non-proteinaceous compound, in particular within the framework of the screening of a bank of nucleic acids coding for proteins capable of interact with said determined compound.
  • the different nucleic acids of said library are coupled with the DNA sequence coding for the second aforementioned revealing protein, and the fusion sequences thus obtained, placed under the control of a transcription promoter, are used to transform host cells. using an appropriate vector.
  • the host cells in which a reaction taking place indicating an interaction between the protein or proteins encoded by one or more nucleidic acids mentioned above and the determined compound occurs, are isolated and the amino acid sequences of said proteins are analyzed.
  • the subject of the invention is also any method of detecting molecules, protein or not, still designated below tested molecules, capable of interacting with the radical Ri of a biotin derivative of formula (I) as described below above, characterized in that it comprises
  • a DNA sequence encoding a first fusion protein comprising the BCCP and a first revealing protein
  • the screening methods described above are applied to the detection of interactions between said proteins or non-protein molecules tested, and a determined non-protein biologically active compound, in particular a compound of therapeutic interest.
  • the two revealing proteins used in the above-mentioned detection methods are chosen in such a way that: - the first revealing protein corresponds to a DNA binding domain (such as the GAL4 binding domain described in the article by M. Johnston et al., A model for fungal gene regulatory mechanism the Gay genes of Saccharomyces cerevisiae, Microbiological Rewiews, 51, 458-476, 1987), while the second revealing protein corresponds to a domain of activation of the transcription of a reporter gene (such as the activation domain of GAL4 activating the transcription of the gene coding for galactosidase, in particular £ GalHis3), so that the detection of the transcription product of the reporter gene corresponds to the detection an interaction between the determined compound and the protein or non-protein molecule tested, or
  • a DNA binding domain such as the GAL4 binding domain described in the article by M. Johnston et al., A model for fungal gene regulatory mechanism the Gay genes of Saccharomyces cerevisiae, Microbio
  • the first revealing protein corresponds to a DNA binding domain (such as the LexA binding domain described in the following articles B.E. Markham et al.,
  • galactosidase corresponds to the detection of an interaction between the determined compound and the protein or non-protein molecule tested, or the first revealing protein corresponds to a first fluorescent protein (such as GFP, for Green Fluorescent Protein, described in the article by A.
  • the host cells used in the context of the implementation of the abovementioned methods are bacterial cells, advantageously E. coli cells, or are yeasts, or else are higher eukaryotic cells.
  • the compounds of formula (I) defined above and more particularly those of formulas (la), (lb), (le), (Id), (le), used in the context of the implementation of the The above-mentioned methods are compounds of formula (I) in which R2 represents a hydrogen atom.
  • the invention also aims to provide new compounds for the implementation of such detection methods.
  • the subject of the invention is thus any nucleotide sequence coding for a fusion protein comprising BCCP fused to a revealing protein as described above, in particular the BCCP of escherichia Coli,
  • the subject of the invention is more particularly:
  • nucleotide sequence containing the DNA sequence coding for the activation domain of GAL4 said DNA sequence being linked to the DNA sequence coding for BCCP, the nucleotide sequence containing the DNA sequence coding for the DNA binding domain of LexA, said DNA sequence being linked to the DNA sequence coding for BCCP,
  • nucleotide sequence containing the DNA sequence coding for BFP said DNA sequence being linked to the DNA sequence coding for BCCP.
  • the subject of the invention is also any vector, in particular plasmid, containing a nucleotide sequence as defined above.
  • the invention also relates to host cells, in particular of E. coli, transformed by a vector defined above.
  • the subject of the invention is also any fusion protein comprising BCCP fused to a revealing protein as described above.
  • the subject of the invention is more particularly: the fusion protein between the DNA binding domain of GAL4, and BCCP, - the fusion protein between the activation domain of GAL4, and BCCP ,
  • the invention also relates to the biotin derivatives of formula (I) mentioned above, in which a fusion protein as defined above, is linked to the carboxyl function carried by Y.
  • nucleotide sequences coding for the fusion proteins described in the abovementioned methods can be replaced by a sequence encoding another protein capable of being recognized by and binding to biotin in the cell host system used.
  • kits or kits capable of being used for the implementation of a detection method as described above of the invention comprising
  • a subject of the invention is also the biotin derivatives of formula (I), as described above, in which the radical R ⁇ represents a compound of therapeutic interest.
  • the invention more particularly relates to the biotin derivatives of formulas (la), (Ib), (le), (Id), (le) above.
  • the invention also relates to pharmaceutical compositions comprising one or more biotin derivatives of formula (I), as described above, in which the radical R ⁇ represents a compound of therapeutic interest, in association with a pharmaceutically acceptable vehicle .
  • a subject of the invention is also the use of biotin derivatives of formula (I), as described above, in which the radical Ri represents a compound of therapeutic interest, for the preparation of medicaments allowing the introduction of said compound of therapeutic interest inside cells of the organism, said drugs being intended for the treatment of pathologies against which said compound of therapeutic interest is active.
  • the invention more particularly relates to the use of compounds of formulas (la), (Ib), (le), (Id) and (le) above for the preparation of a medicament intended for the treatment of breast cancer, atheroma, osteoporosis, or usable as part of wound healing.
  • the invention relates more particularly to the use of compounds of the above-mentioned formula (Ie), in particular of the compound (10) described below, as regulators of the cell proliferation, in particular for the preparation of a medicament intended for the treatment of atheroma, or usable as a healing agent.
  • the invention relates more particularly to the use of compounds of formula (Id) and (the) above, in particular compounds (20) and (13) described below, as inhibitors of cell proliferation, in particular for the preparation of a medicament for the treatment of cancers, such as breast cancer.
  • the invention also relates to the use of biotinyied compounds consisting of biotin, or derivatives thereof, whose nitrogen atom in position l (NI ') is directly or indirectly linked to a determined compound, as as vectors allowing the introduction of a determined compound inside target cells.
  • biotin derivatives any molecule derived by substitution of one or more atoms of biotin other than NI ', in particular by substitution of the carbonyl group adjacent to the atom NI' by a group -NH-, or by substitution of the atom of S by
  • said determined compound is directly linked to the atom NI 'by one of its functions capable of reacting with the atom NI'.
  • said determined compound is modified so as to carry this function capable of reacting with the hydrogen atom carried by NI '.
  • the nitrogen atom NI 'of biotin, or derivatives thereof is substituted by a function capable of forming a covalent bond with a function of said determined compound, the latter being if necessary modified so as to carry this other function.
  • the invention also relates to the use of biotin, or biotin derivatives, mentioned above, in which the nitrogen atom NI 1 is directly or indirectly substituted by a determined compound for the implementation of methods for detecting proteins or other non-protein molecules capable of interacting with said determined compound, and more particularly of methods as described above.
  • the invention also relates to the use of biotin, or biotin derivatives, mentioned above, whose nitrogen atom NI 'is directly or indirectly substituted by a determined compound of therapeutic interest, for the preparation of drugs allowing the introduction of said compound of therapeutic interest inside cells of the organism, said drugs being intended for the treatment of pathologies against which said compound of therapeutic interest is active.
  • the compounds used come from Sigma-Aldrich.
  • d-biotin methyl ester (2) The d-biotin (1) (10 mmol, 2.44 g) is dissolved in 75 ml of anhydrous methanol. 2.6 g of Amberlite 1R120 ion exchange resin, previously washed with 2N hydrochloric acid, rinsed with water until neutral and then dried, are added. The solution is brought to reflux (70 °) for 7 hours under an inert atmosphere. After returning to room temperature, the resin is filtered and rinsed several times with hot methanol. The filtrate is recovered and the methanol evaporated under reduced pressure. 2g (yield 79.5%) of product (2) are obtained in the form of a white powder.
  • N-l ⁇ (4-chloro-l-butoxycarbonyl) -d-biotin methyl ester (3) The d-biotin methyl ester (2) (Immole, 258mg) is dissolved in 5ml of hot anhydrous chloroform, l ⁇ mmoles (3, lg, 2.46ml) of 4-chlorobutyl chloroformate are added and the solution is brought to reflux (65 ° C) for 4 days. After returning to ambient temperature, the chloroform is evaporated under reduced pressure and the yellow solution obtained added dropwise to 250 ml of petroleum ether at 4 ° C. The mixture is kept at 4 ° C. for approximately 15 minutes, until a pale yellow precipitate appears, which is recovered by filtration.
  • the d-biotin methyl ester (2) (Immoles, 258mg) is dissolved in 5ml of hot anhydrous chloroform, l ⁇ mmoles (2,57g, 1,86ml) of 2-chloroethyl chloroformate are added and the solution is brought to reflux (65 C ) during 4 days. After returning to ambient temperature, the chloroform is evaporated under reduced pressure and the yellow solution obtained added dropwise to 250 ml of petroleum ether at 4 C. The mixture is kept at
  • N-acetyl-S-farnesyl cysteine (8) (synthesized according to the method of L. Zhang and P. Casey, The Journal of Biological Chemistry, vol269, N ° 23, ppl5973-15976, 1994) are dissolved in 5 ml of anhydrous dimethylformamide.
  • the solution is placed under an inert atmosphere. 107.2 mmoles (40 mg) of Nl '- (4-amino-1-butoxycarbonyl) -d-biotin methyl ester (S) are dissolved in 4 ⁇ l of anhydrous dimethylformamide to which 214.4 mmoles (30 ml) of triethylamine have been added.
  • the biotin solution is added to the N-acetyl-S-farnesyl cysteine solution, and the mixture placed under an inert atmosphere is stirred at room temperature for 4 days. The solution becomes dark yellow. Dimethylformamide is evaporated under reduced pressure by formation of an azeotrope with toluene.
  • Immoles (28.25 mg) of oleic acid (11) are dissolved in 5 ml of anhydrous dimethylformamide. 0.15 mmoles (16.5 ⁇ l) of N-methylmorpholine are added, 0.2 mmoles (88 mg) of BOP (benzotriazolyl hexafluorophosphate (dimethylamino) phosphonium) and 0.2 mmoles (27 mg) of BOH (N-hydroxybenzotriazole) . The solution is placed under an inert atmosphere.
  • BOP benzotriazolyl hexafluorophosphate (dimethylamino) phosphonium
  • 0.37mmoles (lOOmg) of 17 i-oestradiol (14) and 0.37mmoles (30 ⁇ l) of anhydrous pyridine are dissolved in 3ml of anhydrous tetrahydrofuran.
  • a solution containing 0.37 mmol (571) of 1,2,2,2-tetrachloroethyl chloroformate dissolved in 2 ml of anhydrous tetrahydrofuran is added.
  • a yellow precipitate immediately appears.
  • the solution is stirred at room temperature for 16 hours.
  • the precipitate is filtered through a b ⁇ chner and rinsed with 2 ml of tetrahydrofuran.
  • the filtrate is recovered and the tetrahydrofuran evaporated under reduced pressure.
  • the residue is purified by Flash Chromatography (MeOH: CH2Cl2, 1:99). 25 mg (14.4% yield) of product (15) are obtained in the form of a yellow oil.
  • N-l '- (4- (l 7 i-oestradioI-l 7-oxy-amido) -l-butoxycarbonyl) -d-biotin methyl ester (16) 41.5 ⁇ moles (20mg) of 17y6-oestradiol-l7carbonate of 1, 2,2,2-tetrachloroethyl (15) and 41.5 ⁇ moles (15 mg) of N- - (4-amino-1-butoxycarbonyl) -d-biotin methyl ester (5) are dissolved in 6 ml of tetrahydrofuran and 100 ⁇ l of water. 41.5 ⁇ moles (3.5 ⁇ l) of pyridine are added. A precipitate forms immediately.
  • the biotin solution is added to the N-acetyl-S-geranylgeranylcysteine solution, and the mixture placed under an inert atmosphere is stirred at room temperature for 4 days.
  • the solution becomes dark yellow.
  • Dimethylformamide is evaporated under reduced pressure by formation of an azeotrope with toluene.
  • the residue is dissolved in 10 ml of dichloromethane and the solution obtained washed 4 times with water, then 1 time with an aqueous solution saturated with NaCl.
  • the organic phase is then dried over MgSO 4, then purified by Flash chromatography (MeOH: CH 2 Cl 2 , 3: 97). 44.5 mg (70% yield) of product (19) are obtained in the form of a yellow oil.
  • the BCCP gene was amplified by PCR (Polymerase Chain Reaction) from genomic DNA from Escherichia coli.
  • GAL4 DNA binding domain - BCCP the BCCP gene is inserted (Ncol / BamHI sites) in the eukaryotic expression vector pAS1-CYH2, in fusion with the gene corresponding to the binding domain has GAL4 DNA (amino acids 1 to 147 of GAL4).
  • the fusion gene is under the control of a constitutive promoter.
  • the BCCP gene is inserted (Apal / BamHI sites) in the prokaryotic expression vector pTTQ19CAT, in fusion with the gene corresponding to the binding domain DNA of LexA (amino acids 1 to 81 of LexA) itself inserted by the SphI / Apal sites.
  • the fusion gene is under the control of an IPTG-inducible ptac promoter.
  • a cDNA library of human placenta cells is fused with the GAL4 activator domain gene. Fragments of 500 to 1500 base pairs cloned into Pact2 in fusion with GAL4-ad, marketed by Clontech.
  • the double-hybrid test is developed according to the method of Chang Bai and Stéphen J. Elledge, Methods in Enzymology, 283, ppl41-156, 1997.
  • the plasmids used are vectors of eukaryotic expression: pAS1-CYH2-BCCP which contains the gene of the Biotin Carboxylase Carrier Protein (BCCP) in fusion with the gene of the DNA binding domain of GAL4, and the gene of l 'auxotrophy vis-à-vis tryptophan; pACTII-RhoGDB which contains the RhoGDB gene in fusion with the gene for the activating domain of GAL4, and the gene for auxotrophy with respect to leucine.
  • BCCP Biotin Carboxylase Carrier Protein
  • the yeast strain Y 190 (operator of GAL4-gene for b-galactosidase, deficient in tryptophan and leucine) is transformed by pASl-CYH2-BCCP and pACTII-RhoGDB. spread on a dish containing medium a medium deficient in Leu and Trp: SC Leu-Trp- and incubated for 3 days at 30 C.
  • the transformed yeasts are cultured on SC medium Leu-Trp- deficient in biotin for 24 hours at 30 ° C., then on SC Leu-T ⁇ - medium deficient in biotin and supplemented with InM biotin-ligand for 6 h at 30 ° C.
  • a ⁇ -galactosidase activity test is carried out.
  • biotin derivatives in accordance with the invention find a particularly advantageous application in the following fields, without limitation:
  • Application B development of hormonal assays in vivo (allowing the assay of a global activity) marketable in the form of ready-to-use kits.
  • Application D Identification of primary protein targets (search for targets for new molecules) and secondary (determination of side effects) of small non-peptide molecules in Escherichia coll.
  • the compounds according to the invention can be administered in pharmaceutical preparations at doses of between 0.1 mg to 10 mg per day and per kilo of the person's weight.

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EP01997495A 2000-11-22 2001-11-21 Biotin-derivate, verfahren zu ihrer herstellung und ihre anwendungen als vectoren Withdrawn EP1347985A2 (de)

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FR0015049 2000-11-22
FR0015049A FR2816943B1 (fr) 2000-11-22 2000-11-22 Derives de la biotine, leurs procedes de fabrication et leurs utilisations en tant que vecteurs
PCT/FR2001/003669 WO2002042311A2 (fr) 2000-11-22 2001-11-21 Derives de la biotine, leurs procedes de fabrication et leurs utilisations en tant que vecteurs

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JP2002097197A (ja) * 2000-07-18 2002-04-02 Yanaihara Kenkyusho:Kk エストラジオール誘導体およびこれを使用した測定法
US8338648B2 (en) * 2004-06-12 2012-12-25 Signum Biosciences, Inc. Topical compositions and methods for epithelial-related conditions
EP2315802A4 (de) * 2008-07-31 2012-04-18 3M Innovative Properties Co Azidzusammensetzungen sowie verfahren zu ihrer herstellung und verwendung
US8288005B2 (en) 2008-07-31 2012-10-16 3M Innovative Properties Company Fluoropolymer compositions and method of making and using thereof
WO2018122204A1 (en) 2016-12-27 2018-07-05 F. Hoffmann-La Roche Ag Novel biotin-specific monoclonal antibody and use thereof
EP3562845B1 (de) 2016-12-27 2025-10-01 F. Hoffmann-La Roche AG Neuartiger biotinspezifischer monoklonaler antikörper und verwendung davon
WO2018122043A1 (en) * 2016-12-27 2018-07-05 F. Hoffmann-La Roche Ag Novel biotin-specific monoclonal antibody and use thereof

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FR2601956B1 (fr) * 1986-07-22 1989-11-03 Pasteur Institut Nouveaux derives de desoxy-2' adenosine, leur procede d'obtention par voie de synthese et leurs applications biologiques
US5252743A (en) * 1989-11-13 1993-10-12 Affymax Technologies N.V. Spatially-addressable immobilization of anti-ligands on surfaces
WO1991007087A1 (en) * 1989-11-13 1991-05-30 Affymax Technologies N.V. Spatially-addressable immobilization of anti-ligands on surfaces
US5128476A (en) * 1991-02-20 1992-07-07 The Midland Certified Reagent Company Biotinylated oligonucleotides and reagents for preparing the same
US5874239A (en) * 1993-07-30 1999-02-23 Affymax Technologies N.V. Biotinylation of proteins
FR2717499B1 (fr) * 1994-03-17 1996-05-24 Ulp Fragments d'anticorps recombinants synthétisés et biotinylés dans E. coli, leur utilisation en immunodosages et en purification par immunoaffinité.
GB9900298D0 (en) * 1999-01-07 1999-02-24 Medical Res Council Optical sorting method
US6242610B1 (en) * 1999-05-27 2001-06-05 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Derivatized biotin compounds and methods of use
DE50014159D1 (de) * 1999-12-22 2007-04-26 Dade Behring Marburg Gmbh Lösungen von humanem Procalcitonin

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WO2002042311A3 (fr) 2002-09-19
AU2002222004A1 (en) 2002-06-03

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