EP1907404A1 - Monomeres electropolymerisables solubles en solution aqueuse et sondes electroactives susceptibles d'etre obtenues avec de tels monomeres - Google Patents

Monomeres electropolymerisables solubles en solution aqueuse et sondes electroactives susceptibles d'etre obtenues avec de tels monomeres

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Publication number
EP1907404A1
EP1907404A1 EP06778815A EP06778815A EP1907404A1 EP 1907404 A1 EP1907404 A1 EP 1907404A1 EP 06778815 A EP06778815 A EP 06778815A EP 06778815 A EP06778815 A EP 06778815A EP 1907404 A1 EP1907404 A1 EP 1907404A1
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EP
European Patent Office
Prior art keywords
sequence
monomer according
monomer
monomers
formula
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.)
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Application number
EP06778815A
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German (de)
English (en)
French (fr)
Inventor
Aurélie BOUCHET
Carole Chaix
Bernard Mandrand
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Biomerieux SA
Centre National de la Recherche Scientifique CNRS
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Biomerieux SA
Centre National de la Recherche Scientifique CNRS
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Publication of EP1907404A1 publication Critical patent/EP1907404A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
    • C08G73/0605—Polycondensates containing five-membered rings, not condensed with other rings, with nitrogen atoms as the only ring hetero atoms
    • C08G73/0611—Polycondensates containing five-membered rings, not condensed with other rings, with nitrogen atoms as the only ring hetero atoms with only one nitrogen atom in the ring, e.g. polypyrroles
    • 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
    • C07F17/00—Metallocenes
    • C07F17/02—Metallocenes of metals of Groups 8, 9 or 10 of the Periodic Table
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/04—Polymerisation in solution
    • C08F2/10—Aqueous solvent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/12—Polymerisation in non-solvents
    • C08F2/16—Aqueous medium
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/46—Polymerisation initiated by wave energy or particle radiation
    • C08F2/52—Polymerisation initiated by wave energy or particle radiation by electric discharge, e.g. voltolisation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G61/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G61/12—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule
    • C08G61/122—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule derived from five- or six-membered heterocyclic compounds, other than imides
    • C08G61/123—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule derived from five- or six-membered heterocyclic compounds, other than imides derived from five-membered heterocyclic compounds
    • C08G61/124—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule derived from five- or six-membered heterocyclic compounds, other than imides derived from five-membered heterocyclic compounds with a five-membered ring containing one nitrogen atom in the ring

Definitions

  • the invention relates to the technical field of electropolymerization.
  • the present invention relates to soluble electropolymerizable monomers in aqueous solution.
  • Electroactive polymers are used in many fields of application. For example, it is possible to use electroactive polymers to detect the interaction of a probe biological ligand with a target biological ligand.
  • the specific interaction of a probe ligand with a target ligand results in a substantial and selective variation of the electrochemical properties of the electroactive polymer, such as a decrease in the electroactivity of said polymer.
  • This variation which depends on the concentration of target ligand bound on a probe ligand, is observed, possibly measured, and directly correlated to the amount of bound ligand target.
  • One of the essential applications of this technique lies in the detection, identification, and possibly the determination of a ligand, present in a biological sample.
  • the aforementioned variation is of the potentiometric type, and corresponds, for example, to a variation of the oxidation potential of the electroactive polymer before and after interaction, or of amperometric type, and corresponds, for example, to a variation of the oxidation current or of reducing the polymer before and after hybridization, determined at a determined potential.
  • To precisely characterize the electrochemical response of the polymer it must have high electroactivity.
  • Polymers obtained by electropolymerization for example in the form of a homopolymer or copolymer of pyrroles and comprising an electron-donor group, such as a ferrocene, making it possible to improve its electroactivity and its conductivity have therefore been developed and described in particular in the application for WO 01/81446.
  • the electropolymerization reactions are generally carried out in organic solvent, the monomers used being hydrophobic.
  • the systems used up to now require electrolytic polymerization in an organic solvent because the monomers used, for example ferrocenylated, are rather hydrophobic (Synthetic Metals, 2001, 119, 265-266).
  • manipulations in the middle organic are not compatible with the use of biomolecules. The latter are not soluble in such media and / or, very often, are denatured and their properties are altered. In the case of proteins, there is most often a loss of active conformation.
  • Polymers that can be used in this strategy are, for example, described in WO 95/29199 and in WO 01/81446 which describes polymers having improved electroactivity, obtained by electropolymerization, for example in the form of a homopolymer or copolymer of pyrroles and comprising an electron-donating group, such as a ferrocene.
  • This "multi-layer" strategy is not entirely satisfactory because it is tedious because of the implementation of several organic solvent-aqueous medium transitions with the need each time to flush the chip several times.
  • post-functionalization it consists in the covalent post-polymerization fixation of biomolecules, in aqueous medium, thanks to reactive functions located on the polymer layer.
  • This post-functionalization strategy does not allow, for its part, the addressing of biomolecules.
  • it has a lack of stud plot reproducibility related to the variability of the coupling efficiency of the biomolecule on the polymer.
  • biomolecule electrochemically is a promising principle of detection whose main advantage is the absence of prior labeling of biomolecules, which is mandatory for fluorescence detection, for example.
  • equipment needed for a measurement of electrochemical potential are compact and suggest a great practicality of use.
  • the inventors propose to provide new monomers which are perfectly compatible with an electropolymerization reaction in an aqueous medium and which thus make it possible to dispense with the use of organic solvent.
  • the monomers according to the invention are perfectly compatible for carrying biological ligands.
  • the subject of the present invention is firstly an electropolymerizable monomer, intended to be polymerized in aqueous solution, comprising a single electropolymerizable unit and an electron-donor group, as well as at least one ionizable arm in aqueous solution.
  • the electropolymerizable monomer as defined above has one or more of the following characteristics: it is soluble in distilled water, at least up to a concentration of 1 mM, preferably at least up to at a concentration of 10 mM, and preferably at least up to a concentration of 30 mM,
  • the ionizable linker comprises an ionizable function in aqueous solution chosen from the functions: amine, carboxylic acid and phosphate,
  • the biological ligand is a polynucleotide
  • the electropolymerizable unit is chosen from acetylene, pyrroles, thiophenes, indoles, anilines, azines, p-phenylenevinylenes, p-phenylenes, pyrenes, furans, selenophenes, pyrridazines and carbazoles; , acrylates, methacrylates and their derivatives; preferably the electropolymerizable unit is a pyrrole, the linkage to the ionizable link is preferably provided in position 3 of the pyrrole,
  • the electron-donor group is chosen from metallocenes, quinone and their derivatives; preferably the electron donor group is a ferroocene.
  • the monomers according to the invention because of the presence of an ionizable arm in aqueous solution, will be soluble in aqueous solution, thus allowing their polymerization in such aqueous media.
  • electropolymerizable monomer a monomer having a single electropolymerizable unit, said monomer being capable of reacting by electrochemical polymerization with other monomers to form a polymer.
  • An electropolymerizable pattern has alternating single bonds and double bonds.
  • an electropolymerizable unit mention may be made of acetylene, pyrroles, thiophenes, indoles, anilines, azines, p-phenylenevinylenes, p-phenylenes, pyrenes, furans and selenophenes. pyridazines, carbazoles, acrylates, methacrylates and their derivatives.
  • the monomers comprising a single electropolymerizable unit chosen from acetylene, pyrroles, thiophenes, indoles, anilines, azines, p-phenylenevinylenes, p-phenylenes, pyrenes, furans, selenophenes, pyridazines, carbazoles and their derivatives which lead to a conductive polymer will be preferred.
  • ionizable arm in aqueous solution is meant a hydrophilic chemical group capable of forming a cation or anion in aqueous solution.
  • the ionized form in aqueous solution is obtained according to a first embodiment without carrying out a chemical reaction, hydrolysis or degradation type.
  • the ionized form is, for example, obtained by exchange of a proton or in the form of a pair of ions in solution from a salt.
  • Such ionizable arms include in particular a amino group, polyamine, carboxylic acid (-COOH) or phosphate or sulfonate.
  • An ionizable arm is in ionic form when it is placed in an aqueous solution which has a pH of between 5 and 8.
  • the ionizable arm is in ionized form in distilled water.
  • the ionized form in aqueous solution is obtained by a chemical reaction.
  • the ionized monomer is obtained by an alkylation reaction starting from the monomer Ia, Ha or IHa, according to the scheme below: Ia
  • a 2 , A 3 , Li, R, R 6 , R 7 , Z 1 are as defined below.
  • the alkylation reaction is a bis-methylation reaction.
  • soluble monomer in aqueous solution is meant a soluble monomer in aqueous solution under the polymerization conditions, namely under the conditions of temperature, pH and ionic strength used during its implementation in an electrochemical polymerization reaction. .
  • the electropolymerization will, in general, be carried out in an aqueous solution whose pH is between 3 and 8 and at a temperature of the order of 20 to 30 ° C.
  • the solubility of a monomer according to the invention such that the introduction of said monomer into the distilled water at a temperature of 25 ° C, at least up to a concentration of 1 mM, preferably at least up to a concentration of 10 mM, and preferably at least up to a concentration of 30mM, lead to a homogeneous solution and transparent to the naked eye, without precipitation.
  • Polymerization is understood to mean a reaction by chemical or electrochemical means of units of the same chemical nature allowing the assembly of a certain number of monomers to form a polymer ( r ⁇ M ⁇ (M) r with r greater than or equal to 2 ).
  • the term “polymerization” embraces copolymerization and homopolymerization. This is advantageously in the context of the invention of the condensation of pyrrole units to form a polypyrrole.
  • copolymerization means the simultaneous polymerization of different monomers, such as, for example, the simultaneous polymerization of a mixture of a substituted monomer carrying a biological ligand and a soluble monomer containing no biological ligand.
  • electrochemical polymerization denotes an electrochemical polymerization.
  • Electropolymerization processes are well known to those skilled in the art. Examples include cyclic voltammetric techniques, chronopotentiometry (imposed current) and chronoamperometry (imposed potential). In a particular embodiment of the invention, the polymerization will be carried out by chronoamperometry deposition or controlled potential deposition. This method consists of imposing a potential jump from the equilibrium potential (zero current) to a fixed value at which the reaction to the electrode takes place and the current is measured as a function of time.
  • Polymerization conditions refer to the pH, temperature, and ionic strength of the aqueous solution used in the polymerization.
  • the electropolymerization is carried out by the Diaz mechanism (Sadki et al., Chem Soc Rev., 29: 283-293, 2000) leads to the formation of polypyrrole.
  • This polymerization takes place at the 2 and 5 positions of the pyrrole monomers.
  • a pyrrole substituted at the 3 or 4 position of the pyrrole ring is therefore capable of polymerizing or copolymerizing with other pyrroles at the 2 and 5 positions.
  • the 3-substituted pyrrole units are preferred.
  • conducting polymer is understood to mean a polymer whose electrons are strongly delocalised, most often along a sequence of single and double bonds (conjugated bonds), which leads it to behave as a micro-semiconductor. electronic.
  • Electrode group means a chemical group corresponding to a redox couple having a narrow oxidation wave, rapid and reversible, such as metallocenes such as ferrocene, quinone and their derivatives.
  • acyl As a group protecting the alcohols, there may be mentioned, by way of example, the acyl, trityl, silyl and tetrahydrofuranyl groups.
  • esters in particular alkyl esters, acyl esters, silyl esters or thioesters.
  • a protective group of amines there may be mentioned, for example, trifluoroacetyl, te / t-butoxycarbonyl and 9-fluorenylmethoxycarbonyl groups.
  • activating group of carboxylic acids there may be mentioned, for example, succinimidyl or phthalimidyl groups, or any other group for forming an activated ester.
  • an activating group for alcohols or amines mention may be made, by way of example, of the groups which, in the case of alcohols, lead to a phosphodiester, phosphotriester, H-phosphonate or phosphoramidite and, in the case of amines, to a phosphoramidate monoester, phosphoramidate diester, H-phosphoramidate or phosphoramidite.
  • groups are in particular chosen from:
  • biological ligand is meant a compound which has at least one recognition site allowing it to react with a target molecule of biological interest.
  • ligands polynucleotides, antigens, antibodies, polypeptides, proteins, haptens, biotin, oligosaccharides ...
  • a ligand / anti-ligand pair capable of interacting specifically to form a conjugate is also named, in the present invention probe ligand / target ligand.
  • polynucleotide means a sequence of at least 2 deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nudeotide, for example at least one nucleotide comprising a modified base such as inosine, methyl-5-deoxycytidine, dimethylamino- 5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization.
  • a modified base such as inosine, methyl-5-deoxycytidine, dimethylamino- 5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization.
  • This polynucleotide can also be modified at the level of the internucleotide linkage, for example phosphorothioates, H phosphonates, alkylphosphonates, at the backbone level, for example alpha-oligonucleotides (FR 2 607 507), or NAPs (Egholm). M. et al., J. Am. Chem., Soc., 1992, 114, 1895-1897), or 2-O-alkyl ribose, or LNA (Loked Nucleic Acids), described in particular in US Pat. published patent application WO 00/66604). Each of these modifications can be taken in combination.
  • the polynucleotide may be an oligonucleotide, a natural nucleic acid or its fragment such as a DNA, a ribosomal RNA, a messenger RNA, a transfer RNA, a nucleic acid obtained by an enzymatic amplification technique.
  • polypeptide is meant a sequence of at least two amino acids.
  • amino acids we mean the primary amino acids that code for proteins, amino acids derived after enzymatic action such as and natural amino acids but not present in proteins such as norvaline, N-methyl-L-leucine, Stalin (Hunt S. in Chemistry and Biochemistry of amino acids, Barett GC, ed., Chapman and Hall, London, 1985), amino acids protected by chemical functions that can be used in solid or liquid phase synthesis and non-natural amino acids.
  • hapten refers to non-immunogenic compounds, i.e. incapable by themselves of promoting an immune reaction by production of antibodies, but capable of being recognized by antibodies obtained by immunizing animals in animals.
  • hapten-protein conjugate generally have a molecular mass of less than 3000 Da, and most often less than 2000 Da and may be, for example, glycosylated peptides, metabolites, vitamins, hormones, prostaglandins, toxins, antibiotics or various medicaments, nucleosides and nucleotides.
  • antibodies includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination and antibody fragments.
  • antigen refers to a compound capable of being recognized by an antibody for which it has induced synthesis by an immune response.
  • protein includes holoproteins and heteroproteins such as nucleoproteins, lipoproteins, phosphoproteins, metalloproteins and both fibrous and globular glycoproteins.
  • the subject of the present invention is the different series of monomers comprising a pyrrole unit and a metallocene unit as defined hereinafter: a) firstly the monomers of formula (I):
  • - M is a transition metal, preferably Fe, Ru or Os
  • - Ai is a sequence:
  • (Ci-C 4) alkyl eg methyl or ethyl
  • R 6 , R 7, which independently of one another represents a (C 1 -C 4 ) alkyl group
  • R represents a hydrogen atom or a group protecting the amine function, for example chosen from monomethoxytrityl, dimethoxytrityl, tosyl, triisopopylsilyl, tert-butoxycarbonyl, 9-fluorenyloxycarbonyl, benzyloxycarbonyl, triphenylmethanesulfenyl and acetyl groups, and b) then monomers of formula (II):
  • a ⁇ represents a spacer arm defined as follows: o when Ai is a sequence -A 2 -NR ⁇ A 3 - or -A 2 -N + R 6 R 7 -A 3 -, in which R 1 , A 2 , A 3 , R 6 and R 7 are as defined for the monomers of formula (I) and
  • a 2 represents - (GH 2 ) m i- or - (CH 2 ) m 2 -O- [(CH 2 ) 2 O] m 3 - (CH 2 ) 2 - and A 3 represents - (CH 2 ) n i - or - [(CH 2 ) 2 O] n 2 - (CH 2 ) n 3 -
  • Ae represents a sequence -A 2 -NR 1 -, with ml, m2, m3, ni, n2, n3 and
  • a 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 - and A 3 represents - (CH 2 ) n i- or - [(CH 2 ) 2 O] n 2 - (CH 2 ) n 3 -
  • a 6 represents a sequence - (CH 2 ) m 4 -C (O) O-, or -A 2 -NR 1 -, with m 4, R 3 , m5, ni, n2, n3 and R 1 as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 - and A 3 represents - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -
  • a 6 represents a sequence - (CH 2 WC (O) O-, -A 2 -NR 1 - or
  • a 2 represents - (CH 2 ) m r or - (CH 2 ) m 2 -O- [(CH 2 ) 2 O] m 3 - (CH 2 ) 2 - and A 3 represents - (CH 2 ) n i- or
  • a 6 represents a sequence -A 2 -Y-, with ml, m2, m3, n, n 2, n 3 and Y as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 - and A 3 represents - (CH 2 ) n - or
  • a 6 represents a sequence - (CH 2 ) m 4 -C (O) O- or - A 2 -Y-, with m 4, R 3 , m5, n1, n2, n3 and Y as defined for the monomers of formula (I),
  • a 6 represents a sequence -A 2 -Y- or -A 2 -YP (O) (OR 2 ) -O - [(CH 2 ) 2 O ] n 4 - (CH 2 ) 2 -NR 4 -, with ml, m 2, m 3, n 4, n 5, Y, R 4 and R 2 as defined for the monomers of formula (I),
  • a 6 represents a sequence
  • a 4 represents - (CH 2 ) p 2 -C (O) -
  • a 5 represents - (CH 2 ) q i-
  • a 6 represents a - (GH 2 ) p 2 - C (O) O- or -A 4 - [NH (CH 2 ) 2 ] n '-NH-
  • a 5 represents -NR 5 -C (O) - (CH 2 ) q 2 -
  • a 6 represents a sequence
  • a 6 represents a sequence - (G ⁇ b) p i- [NH (CH 2 ) 2 ] n -NH- with n 'an integer in the range from 1 to n -1, and if A 5 represents -NR 5 -C (O) - (CH 2 ) q 2 -, then A 6 represents a sequence - (CH 2 ) p i- [NH (CH 2 ) 2 ] n - NR 5 or - (CH 2 ) p i- [NH (CH 2 ) 2 ] n '-NH-, with pi, ql, q2, n and R 5 as defined for the monomers of formula (I) and n' integer in the range of 1 to n-1,
  • Z 1 represents a hydrogen atom, or a protecting group or activator of the alcohols, amines or carboxylic acids, as a function respectively of the amine, alkoxy or carboxy terminal function of the spacer arm A 6 to which Z 1 is bonded, the monomers of formula (III): in which :
  • a 6 represents a spacer arm defined as follows: where A 1 is a sequence -A 2 -NR 3 -A 3 or -A 2 -N + R 6 R 7 -A 3 -, in which R 1 , A 2 , A 3 , R 6 and R 7 are as defined for the monomers of formula (I) and
  • a 2 represents - (CH 2 ) m i- or - (CH 2 ) m 2 -O- [(CH 2 ) 2 O] m 3 - (CH 2 ) 2 - and A 3 represents - (CH 2 ) n i - or
  • a 6 represents a sequence -A 2 -NR 1 -, with ml, m2, m3, n, n 2, n 3 and R 1 as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 - and A 3 represents - (CH 2 ) n i-, or
  • a 6 represents a sequence - (CH 2 ) m 4 -C (O) O-, or -A 2 -NR 1 -, with m 4, R 3 , m 5 , n 1 , n 2 , n 3 and R 1 as defined for the monomers of formula (I), when A 2 represents - (CH 2 ) m i- or - (CH 2 ) m 2 -O-
  • a 6 represents a sequence -A 2 -NR 1 - or -A 2 -NR 1 - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 -, with ml, m2, m3, n4, n5, R 4 and R 1 as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 WC (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) r and A 3 represents - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 - NR 4 -C (O) - (CH 2) n 5 -
  • a 6 represents a
  • a 6 represents a sequence -A 2 -Y-, with ml, m2, m3, ni, n2, n3 and Y as defined for the monomers of formula (I), • when A 2 represents - (CH 2 WC (O) - NR 3 -
  • a 6 represents a sequence - (CH 2 WC (O) O- or - A 2 -Y-, with m 4, R 3 , m 5, n, n 2, n 3 and Y as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 ) m i- or - (CH 2 ) m 2 -O-
  • a 6 represents a sequence -A 2 -Y- or -A 2 -YP (O) (OR 2 ) -O- [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 -, with ml, m 2, m 3, n 4, n 5, Y, R 4 and R 2 as defined for the monomers of formula (I),
  • a 6 represents a sequence - (CH 2 WC (O) O-, -A 2 -Y- or -ArY-P (O) (OR 2 ) -O - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 -, with m 4, R 3 , m 5, n 4, n 5, R 2 , R 4 and Y as defined for the monomers of formula (I), when A 1 is a sequence -A 4 - [NH (CH 2 ) Z] n -A 5 -, in which A 4 , n and A 5 as defined for the monomers of formula (I), when A 1 is a sequence -A 4 - [NH (CH 2 ) Z] n -A 5 -, in which A 4 , n and A 5 as defined for the monomers of formula (I), when A 1 is a sequence -A 4 - [NH (CH 2 ) Z] n -A 5 -, in which A 4
  • a 6 represents a sequence - (CH 2 ) p 2 -C (O) O-, -A 4 - [NH (CH 2 ) 2 ] n -NR 5 - or
  • a 6 represents a sequence - (CH 2 ) p i-
  • n ' is an integer in the range of 1 to n-1, and if A 5 is -NR 5 -C (O) - (CH 2 ) q 2 -, then
  • a 6 represents a sequence - (CH 2 ) pI- [NH (CH 2 ) 2 ] n -NR 5 - or -A 4 - (CH 2 ) P i- [NH (CH 2 ) J n -NH-, with pi, ql, q2, n and R 5 as defined for the monomers of formula (I) and n 'an integer in the range 1 to n-1 - a 7 represents a linker arm, such that a polymer or an alkyl chain, or a direct bond, and
  • Li represents a biological ligand.
  • the ionizable arms (binding or free) present on these monomers make it possible to obtain a soluble monomer in aqueous solution, despite the presence of a metallocene group, typically a ferrocene group, with a hydrophobic nature.
  • the presence of this ionizable arm does not alter the properties of the electropolymerizable monomer present, the polymerization can be carried out in the aqueous phase to form polymer layers which will preferably be conductive.
  • the sequence A 6 corresponds partially to the arm A 1, that is why the value that A 6 takes is directly related to the value of A 2 and A 3 in particular.
  • a 2 In order not to weigh down the definition of the A ⁇ arms, in the monomers (II) and (III), although it is specified at each point at which A 2 corresponds, the name A 2 has been kept to define A ⁇ . For example, even if it is specified that A 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 -, we will have kept for A 6 the definition -A 2 -Y-, while of course this corresponds to - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 -Y. It is the same for A 4 .
  • the monomers (Ia) have the following formula (Ia):
  • the monomers (Ic) correspond to the following formula (Ic):
  • the monomers (Ib) correspond to the following formula (Ib)
  • the monomers (Ib) have one or more of the following characteristics:
  • - R 2 represents a hydrogen atom
  • the monomers of formula (II) are preferred.
  • These monomers of formula (II), (IIa), (IIc) and (IIb) correspond to the monomers of formula (I), (Ia), (Ic) and (Ib) respectively, modified to bear on the other cyclopentadiene metallocene, an ionizable free arm.
  • the latter may carry a reactive function (F) of the hydroxyl amine or carboxylic acid type, optionally in protected or activated form, for the subsequent fixation of a biological ligand in particular.
  • the monomers (IIa) have the following formula (IIa):
  • a 2 represents - (CH 2 W-, - (CH 2 ) m2 -O - [(CH 2 ) 2 O] m3 - (CH 2 ) 2 - or - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 -, with ml to m 5 and R 3 as defined for the monomers of formula (I),
  • a 3 is - (CH 2) n i-, - [(CH 2) 2 O] n2 - (CH 2) n3 - or - [(CH 2) 2 O] n4 - (CH 2) 2 -NR 4 -C (O) - (CH 2 ) n 5 -, with n to n and R 4 as defined for the monomers of formula (I),
  • a 6 represents a spacer arm defined as follows:
  • a 6 represents a linking -A 2 -NH-, with ml, m2, m3, ni, n2 and n3 as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) r and A 3 represents - (CH 2 ) n i-, or - [(CH 2 ) 2 O] n 2 - (CH 2 ) n 3 -
  • a 6 represents a sequence - (CH 2 ) m 4 -C (O) O-, or -A 2 -NH-, with m 4, R 3 , m5, n1, n2 and n3 as defined for the monomers of formula (I),
  • a 2 represents - (CH 2 ) m i- or - (CH 2 ) m 2 -O- [(CH 2 ) 2 O] m 3 - (CH 2 ) 2 - and A 3 represents - [(CH 2 ) 2 O] n4 - (CH 2 ) 2 -
  • a 6 represents a sequence -A 2 -NH- or -A 2 -NH - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 -, with ml, m 2, m 3, n 4 , R 4 and n 5 as defined for the monomers of formula (I), "when A 2 represents - (CH 2 ) m 4 -C (O) -NR 3 -
  • a 3 represents - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 - NR 4 -C (O) - (CH 2 ) n 5-
  • a 6 represents a sequence - (CH 2 ) m 4 -C (O) O-, -A 2 -NH- or -A 2 -NH - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 - NR 4 -, with m 4, R 3 , m 5, n 4, n 5 and R 4, such as for the monomers of formula (I), and
  • Z 1 is a hydrogen atom or a protecting or activating group of the amines or acids as a function respectively of the amine or carboxy terminal function of the spacer arm A 6 to which Z 1 is bonded.
  • These monomers can be used in the supported synthesis oligonucleotides, as described for example in the patent application WO 03/068787.
  • the monomers of formula (Ia) are preferred the monomers of formula (IIa), as defined above, in which at least one of the sequences A 2 and A 3 comprises a unit - [(CH 2 ) 2 O] m with m which represents m3, m5, n2 or n4 as defined for the monomers (I).
  • Z 1 is a hydrogen atom or a protective group of the amines, for example chosen from trifluoroacetyl, tert-butoxycarbonyl and 9-fluorenylmethoxycarbonyl groups, or Z 1 represents an activating group of the amine function forming with the amine function to which it is bound a phosphoramidate monoester, a phosphoramidate diester, an H-phosphoramidate or a phosphoramidite.
  • the monomers (Ile) correspond to the following formula (Ile):
  • a 2 represents - (CH 2 ) m i-, - (CH 2 ) m 2 -O - [(CH 2 ) 2 O] m 3- (CH 2 ) 2 - or - (CH 2 ) m 4 -C (O) - NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2-, with ml to m 5 and R 3 as defined for the monomers of formula (I),
  • a 3 is - (CH 2) n R, - [(CH 2) 2 O] n2 - (CH 2) n3 or - [(CH) 2 ISO 4 - (CH 2) 2 -NR 4 -C (O) - (CH 2 ) n 5 -, with n to n and R 4 as defined for the monomers of formula (I),
  • a 6 represents a spacer arm defined as follows:
  • a 6 represents a sequence -A 2 -NH-, with ml, m2, m3, ni, n 2 and n 3 as defined for the monomers of formula (I),
  • a 6 represents a sequence - (CH 2) m 4-C (O) O-, or -A 2 -NH-, with m4 , R 3 , m 5 , n 1, n 2 and n 3 as defined for the monomers of formula (I), • when A 2 represents - (CH 2 ) m r or - (CH 2 ) m 2 -O- [(CH 2 ) 2 O] m 3 - (CH 2 ) r and A 3 represents - [(CH 2 ) 2 O] n4- (CH 2 ) 2 -RN 4 -C (O) - (CH 2 ) n 5
  • a 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - (CH 2 ) 2 - and A 3 represents - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 - NR 4 -C (O) - (CH 2 ) n 5 -
  • a 6 represents a sequence - (CH 2 ) m 4 -C (O) O-, -A 2 -NH- or -A 2 -NH - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -
  • Z 1 is a hydrogen atom or a protecting or activating group of the amines or acids as a function respectively of the amine or carboxy terminal function of the spacer arm A 6 to which Z 1 is bonded.
  • a 6 represents a sequence -A 2 -NH- or -A 2 -NH - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 - and Zi represents an activating group of the amine function forming, with the amine function to which it is bonded, a phosphoramidate monoester, a phosphoramidate diester, an H-phosphoramidate or a phosphoramidite, constitute a particular aspect of the invention.
  • These monomers may be used in the supported synthesis of oligonucleotides, as described for example in the patent application WO 03/068787.
  • the monomers of formula (Ic) preferred are the monomers of formula (IIc), as defined above, in which at least one of the chains A 2 and A 3 comprises a unit - [(CH 2 ) 2 O m with m which represents m3, m5, n2 or n4 as defined for the monomers (I).
  • Particularly preferred are the monomers of formula (IIc) as defined previously in which: - A 2 represents - (CH 2 ) m i-, with ml as defined for the monomers of formula (I), and in particular ml 1,
  • Z 1 is a hydrogen atom or a protective group of the amines, for example chosen from trifluoroacetyl, tert-butoxycarbonyl and 9-fluorenylmethoxycarbonyl groups, or Z 1 represents an activating group of the amine function forming with the amine function to which it is bound a phosphoramidate monoester, a phosphoramidate diester, an H-phosphoramidate or a phosphoramidite.
  • the monomers (Hb) have the following formula (Hb):
  • a 6 represents a spacer arm defined as follows:
  • a 6 represents a sequence -A 2 -Y-, with ml, m2, m3, n, n 2 , and Y as defined for the monomers (I ) - when A 2 is - (CH 2) m4 -C (O) -NR 3 - [(CH 2) 2 ⁇ ] m 5 - (CH 2) 2 - and A 3 is - (CH 2) n or i- - [(CH 2) 2 O] n 2 - (CH 2) n3 -, then a 6 represents a sequence - (CH 2) m4 -C (O) O-, -A 2 -Y-, with m
  • a 6 represents a sequence - A 2 -Y- or -A 2 -YP (O) (OR 2 ) -O- [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 -, with ml, m2, m3, n 4, R 4 , n 5, Y and R 2 as defined for the monomers (I), - when A 2 represents - (CH 2 ) m 4 -C (O) -NR 3 - [(CH 2 ) 2 O] m 5 - ( CH 2 ) 2 - and A 3 represents - [(CH 2 ) 2 O] n 4 - (CH 2 ) 2 -NR 4 -C (O) - (CH 2 ) n 5 -, then A 6
  • the monomers of formula (III) the monomers of formula (HIa), (IHc) and (IHb) as defined below are preferred.
  • monomers of formula (III), (HIa), (IIIc) and (IHb) correspond to the monomers of formula (II), (IIa), (IIc) and (IIb) on which a biological ligand has been covalently coupled , optionally via a spacer arm, on the reactive function (F) present on the ionizable free arm substituting the metallocene.
  • the monomers (HIa) correspond to the formula (HIa):
  • a 7 represents a linking arm or a direct bond
  • Li represents a biological ligand
  • a 2 , A 3 , A 6 and R are as defined for the compounds of formula (IIa) and for which the preferred values indicated during of the definition of compounds of formula (IIa) also apply.
  • the monomers (IIIc) correspond to formula (IIIc): in which A 7 represents a linking arm or a direct bond, U represents a biological ligand and A 2 , A 3 , A 6 , R 6 , R 7 and R are as defined for the compounds of formula (II) and for which preferred values indicated in the definition of the compounds of formula (IIc) also apply.
  • the monomers (HIb) correspond to the formula (HIb):
  • the monomers (I), (Ia), (Ib), (Ic), (II), (IIa), (Hb), (IIc), (III), (HIa), (IIb) and (HIc) have one or more of the following characteristics:
  • R is a hydrogen atom
  • This monomer (1) will then be reacted with the corresponding pyrrole substituted by a chain A 3 bearing a terminal hydroxyl function, one of the hydroxyl functions, preferably the one located on the ferrocene side, having previously been activated in the form of a phosphoramidite.
  • the ferrocene (2) thus obtained can then be coupled, when X represents -NR 1 , with the corresponding pyrrole substituted by a chain bearing a terminal halogen atom, said chain being chosen to obtain the desired arm A 3 , or else when X represents -NH-P (O) (OR 2 ) -O-, the terminal amine function of ferrocene (2) will be activated in the form of a phosporamidite to be able to couple to a pyrrole bearing a terminal hydroxyl on the arm A 3 .
  • a 3 represents - (CH 2 ) n- or - [(CH 2 ) 2 O] n 2 - (CH 2 ) n 3 -
  • pyrrole substituted with a - (CH 2 ) n -Br or - (CH 2) group ) n 3 - [O (CH 2 ) 2 ] n 2 -Br can be obtained from the pyrrole substituted with a - (CH 2 ) n -OH group (prepared according to Synth Commun., 1996, 26, 1289) or - (CH 2 ) n 3 - [O (CH 2 ) 2] n 2 -OH (prepared according to FR 2849038) respectively, for example in the presence of CBr 4 and triphenyl phosphine in methanol at 0 ° C.
  • This pyrrole substituted with a polyamine will be obtained conventionally by coupling a polyamine (15) on an alkyl halogenated pyrrole (16), as shown in FIG. 4.
  • Monomers (II), (IIa), (IIc) and (IIb) have a metallocene group with a 1-1 'substitution. Metallocene substitutions show some symmetry found in Ai and Ae.
  • a 1 - (CH 2 ) 3 - OPO- (CH 2 ) r + TBDMSiCl
  • the monomers (III) (HIa) 7 (HIc) and (HIb), for their part, are obtained by coupling a ligand on the corresponding monomer (II), (IIa), (IIc) or (IIb).
  • a reactive function Fi (Z 1 H) of amine, hydroxyl or carboxylic acid type.
  • this coupling will be via a spacer arm A 7, for example of the polymer or alkyl chain type.
  • Spacer arms that can be used to move the ligand Biological chain polymer finally obtained are well known to those skilled in the art. Any spacer arm which will not alter the solubility or the electroactive properties of the monomer may be used.
  • the spacer arm is coupled to the reactive function Fi, then the ligand is coupled with another reactive function F ⁇ , for example of the activated ester type carried by the spacer arm.
  • the arm (II) is used for the supported synthesis of the oligonucleotides, that is to say in the cases defined above where Zi represents an activating group of the amino or alkoxy function of the spacer arm Ae to which Z 1 is bonded respectively forming with the amine function to which it is attached a phosphoramidate monoester, a phosphoramidate diester, an H-phosphoramidate or a phosphoramidite or with the alkoxy function to which it is attached a phosphodiester, a phosphotriester, an H-phosphonate or a phosphoramidite, then the arm
  • a 6 bonded to the phosphorus atom may be an oxygen atom or a nitrogen atom, -NH- OR - NR 4 -.
  • the use of the monomers according to the invention allows an addressing of the biomolecules on a pad of an electrode.
  • this addressing can be done in a single step.
  • another object of the invention is to provide electroactive probes and electrodes, at least partially covered with such a probe, which are simpler to produce, and which allow a more direct measurement of the ligand probe / target ligand interaction.
  • electroactive probe is meant a probe whose electrochemical response is modified when a target ligand interacts specifically with the probe ligands carried by the probe. Thus, a modification of the electrochemical signal is observed following the specific interaction with the analyte.
  • target ligand is intended to mean any molecule capable of interacting specifically with a probe ligand attached to the monomer according to the invention and therefore capable of being detected with a copolymer or polymer according to the invention, obtained from such a polymer. monomer.
  • This target ligand can be, for example, a biomolecule such as, for example, a nucleotide, in particular an oligonucleotide, a protein, an antibody, an antigen, a peptide, a lipid, a steroid, a sugar or a nucleic acid.
  • the probe ligand carried by the polymer is specific for the target ligand to be detected.
  • the present invention therefore also relates to electroactive probes that can be obtained by electropolymerization of at least two soluble monomers according to the invention each carrying a biological ligand forming a conductive homopolymer.
  • at least two monomers of formula (III), (IHa), (HIb) or (HIc) as previously defined will be used.
  • a monomer of formula (II), (IIa), (Hb) or (Ile) as previously defined will be used.
  • the subject of the present invention is electroactive probes in the form of a conductive copolymer capable of being obtained by copolymerization between two different monomers of which at least one is in accordance with the invention, at least one, and preferably one, monomers carrying a biological ligand
  • the copolymerization will be carried out starting from a monomer of formula (III), (HIa), (HIb) or (IIIc) and a monomer (I), (Ia), (Ib) or (Ic) as previously defined. This gives a spacing of biological ligands probe which improves the sensitivity.
  • the electroactive probes in the form of a conductive copolymer that can be obtained by electropolymerization of at least one, and preferably a single, soluble monomer according to the carrier invention. of an amine, hydroxyl or carboxylic acid reactive functional group (F), optionally in protected form, followed by a coupling of said reactive function (F) with a biological ligand, form an integral part of the invention.
  • the copolymerization will be carried out starting from a monomer of formula (II), (IIa), (Hb) or (Ile) and a monomer (I), (Ia), (Ib) or (Ic) as previously defined.
  • a pair of monomers (II) / (I) or (IIa) / (Ia) or (IIb) / (Ib) or (IIc) / (Ic) in which M, Al and R are identical will be used.
  • the present invention also relates to an electropolymerization process carried out in aqueous solution, implementing at least one of the monomers according to the invention, and in particular at least one of the monomers carrying a biological ligand.
  • This electropolymerization may be a homopolymerization.
  • the electropolymerization will be a copolymerization carried out between two different monomers of which at least one is in accordance with the invention.
  • at least one, and preferably only one, monomer carries a biological ligand.
  • the copolymerization will be carried out starting from a monomer of formula (III), (IHa) 7 (HIb) or (HIc) and a monomer (I), (Ia), (Ib) or (Ic) as previously defined.
  • a pair of monomers (III) / (I) or (IIIa) / (Ia) or (IIIb) / (Ib) or (IIIc) / (Ic) in which M, Al and R are identical will be used. .
  • copolymerization of at least two different soluble monomers according to the invention each carrying a reactive function (F) amine, hydroxyl or carboxylic acid, optionally in protected form.
  • This copolymerization reaction in aqueous phase will advantageously be followed by coupling of said reactive function (F) with a biological ligand.
  • the copolymerization will be carried out starting from a monomer of formula (II), (IIa), (Hb) or (Ile) and a monomer (I), (Ia), (Ib) or (Ic) as previously defined.
  • the subject of the present invention is also the polymers that can be obtained by such polymerization reactions, optionally followed by coupling with a biological ligand.
  • the present invention also relates to electrodes comprising a conductive support of which all or part of the surface is coated with an electroactive probe as defined above.
  • the present invention also relates to a method for detecting a target ligand in a biological sample, in which the sample is brought into contact with an electroactive probe as defined. previously, carrying a probe ligand, under conditions suitable for the probe ligand / target ligand interaction and the difference in potential or current emitted by the probe before and after being placed in contact with, and possibly quantifying, the potential and current difference with the sample.
  • the polymers obtained from the monomers according to the invention can be used in particular in all applications in which biological ligands are addressed and immobilized on a solid support.
  • these polymers can be obtained in the form of self-supported films or in the form of an electrode film.
  • the electrode makes it possible to control, by measuring the current delivered during the reaction, the evolution of the polymerization reaction.
  • the electrode also makes it possible to measure the subsequent electrochemical responses of the polymer.
  • the present invention therefore also relates to an electrode comprising a conductive support coated on the surface with at least one electroactive conductive polymer functionalized with biological ligands according to the invention, that is to say an electroactive probe according to the invention.
  • the state of the art is known of conductive supports for electrodes, in particular substrates made of metal or of carbon derivatives.
  • the polymer is generally deposited on the conductive support.
  • the electrochemical polymerization is advantageously carried out at the surface of the electrode to obtain an electrode comprising a conductive support coated on the surface of a polymer according to the invention.
  • the electrode is obtained by depositing a layer of polymer on the surface of a support in gold or platinum.
  • the monomers according to the present invention allow the immobilization and addressing of biological ligands on small surfaces.
  • This electrocopolymerization addressed makes it possible to produce a matrix of miniaturized and ordered points, each of the points carrying a defined biological ligand.
  • the invention thus also relates to an electrode matrix.
  • the invention therefore also relates to an electrode matrix comprising at least one electrode according to the invention.
  • electrode matrices may be in the form of a card or analysis chip comprising a series of wells, each well corresponding to an electrode.
  • the different electrodes of the matrix carry different biological ligands.
  • the invention relates to a plurality of electrodes or microelectrodes fixed on a solid support, these electrodes are coated with a copolymer according to the invention and advantageously carry different biological ligands.
  • Such matrices of electrodes may advantageously be obtained by electropolymerization of monomers according to the invention, and in particular by copolymerization of at least two monomers, at least one of which carries a biological ligand, such as a monomer of formula (III), (HIa), (HIb) or (IIIc) and a non-functionalized monomer with a ligand, such as a monomer of formula (I), (Ia), (Ib) or (Ic).
  • a biological ligand such as a monomer of formula (III), (HIa), (HIb) or (IIIc)
  • a non-functionalized monomer with a ligand such as a monomer of formula (I), (Ia), (Ib) or (Ic).
  • the electrodes and the matrices of electrodes according to the invention are particularly useful for the detection of analytes likely to be present in a sample and likely to react specifically with the biological ligands carried by the polymer.
  • the present invention makes it possible to detect a target ligand in any type of sample.
  • the sample is a biological sample.
  • this sample may have been taken from a patient for diagnostic purposes.
  • the sample may be, for example, urine, blood, serum, plasma, cell extracts or body fluid.
  • the probe Since the probe is electroactive, its electrochemical response will be modified when a target ligand interacts specifically with the probe ligand carried by the polymer.
  • the electroactive conductive polymer according to the invention therefore translates the interaction with the target ligand into an electrochemical signal.
  • the specific interaction of a ligand target with the oligonucleotides carried by the polymer causes a change in the electrochemical response of the polymer studied compared to that obtained before introduction of the target ligand.
  • the detection of the target ligand is therefore performed by an electrical measurement.
  • electrical measurement is meant the measurement of a potentiometric type variation such as the variation of the oxidation potential of the polymer or the measurement of an amperometric type variation by variation of the oxidation current observed at a given potential.
  • the electrical measurement consists of measuring a potential variation or a current variation.
  • cyclic voltammetry is used. It is an electroanalytical method that consists in sweeping a range of potential in one direction then in the other, at constant speed. The voltammogram obtained gives the current response of the electrochemical system studied and allows its characterization.
  • the detection of the specific interaction between the target ligand and the probe ligand carried by the polymer can be done with the electrode which was used for the electropolymerization of the polymer.
  • the hybridization of a nucleic acid complementary to oligonucleotides carried by the polymer can be detected by electrical measurement on the electrode which supports the polymer according to the invention.
  • Hybridization of oligonucleotides can be directly monitored by measuring the variation of the detected electrochemical signal or via an enzymatic reaction.
  • the target oligonucleotide is, for example, carrying a biotin.
  • the detection can be done either at the level of the substrate, or at the level of the electrochemical signal.
  • the monomer Ia.1 is prepared according to the scheme below
  • reaction medium After stirring for 6 hours at this temperature, the reaction medium is cooled to ambient temperature and left stirring overnight.
  • 40 mg (1.06 mmol, 1.10 eq.) Of sodium borohydride (Aldrich) is added directly to the reaction medium. Allowed to react for 2 hours with stirring at room temperature.
  • the ethanol is then evaporated, the medium taken up in dichloromethane then purified on a silica column with a mixture of dichloromethane-methanol-triethylamine 80-18-2.
  • ferrocene (10-amino-5,8-dioxa-2-azadedyl) (0.26 mmol, 1 eq.) are introduced into a flask and dissolved in 1 ml of acetonitrile / water mixture. 10. 0.26 mmol (1 eq) of phthalimidyl or succinimidyl activated pyrrole ester obtained in paragraph I.2.b previously dissolved in 1 ml of solvent are added slowly. The mixture is stirred at room temperature for 30 minutes (phthalimidyl ester) at 4 hours. (succinimidyl ester). The reaction medium is then purified on a silica column with a 90-10 dichloromethane-methanol mixture. 15 mg of a yellow solid (0.03 mmol, 13%) which is soluble in water to a concentration of at least 30 mM is obtained.
  • the monomer (Here) is obtained from the monomer (Ia.1) by a methylation reaction.
  • Step 1 Synthesis of 1- [3-O- (2-cyanoethyl-N, N-diisopropylphosphoramide) propyl] ferrocene:
  • Ferrocene monopropanol (53.3 mg, 0.218 mmol) is coevaporated three times in 1 ml of anhydrous acetonitrile. After taking the orange oil in ImL anhydrous acetonitrile under argon the DIPEA (120 ⁇ L, 0.480mmol), then chlorophosphine (42 ⁇ L, 0.240mmol) are added. The progress of the reaction is monitored by TLC (cyclohexane-ethyl acetate-triethylamine, (49.5: 49.5: 1)), on premigrated plates in the same eluent: after 10 min of reaction there is a more starting material, the reaction medium is concentrated by half.
  • TLC cyclohexane-ethyl acetate-triethylamine, (49.5: 49.5: 1)
  • the crude reaction product obtained is purified on silica gel, neutralized with a cyclohexane-triethylamine mixture (99: 1); eluent ethyl acetate-cyclohexane (50:50).
  • the oil is taken up in ImL of acetonitrile and then filtered on 0.45 ⁇ m PVDF filter, the product is concentrated.
  • Step 2 Synthesis of [3-Ferrocenylpropyl-2-cyanoethyl-2- (3-pyrrole) ethyl] phosphate triester: 1) Tetrazole
  • Pyrrole-3-ethanol (26 mg, 0.233 mmol) is coevaporated twice in 1 ml of anhydrous acetonitrile and then taken up in 1 ml of anhydrous acetonitrile.
  • the 0.45M terazole solution in acetonitrile (1 mL, 0.436 mmol) is added in the presence of a few grains of molecular sieve (3 angstroms).
  • the ferrocene phosphoramidite derivative obtained at step 1 (97 mg, 218 mmol) is added in solution in anhydrous acetonitrile (ImL). The solution becomes orange.
  • reaction is monitored by TLC in the eluent cyclohexane-ethyl acetate-triethylamine (49.5: 49.5: 1). After one hour of reaction, no more starting material remains, the oxidation solution (butanone peroxide 0.67% in dichloromethane) (2 ml) is added. The solution becomes brown.
  • the product is purified on silica gel previously neutralized with a dichloromethane-triethylamine mixture (99: 1), eluent: dichloromethane-methanol (90:10).
  • Step 3 Synthesis of [3-ferrocenylpropyl-2- (3-pyrrole) ethyl] phosphate diester rib.11
  • reaction crude thus obtained is purified on silica gel, conditioned with a mixture of dichloromethane-triethylamine (99: 1). For this purification an elution gradient is necessary: dichloromethane-methanol (100: 0) to (85:15). After purification, 21.1 mg (0.05 mmol, 48%) of a yellow-orange oil are obtained. at
  • reaction medium is then purified on a silica column with an 80-20 dichloromethane-methanol mixture. 18 mg (0.02 mmol, 17%) of a brown-yellow oil which is soluble in water are obtained, at least up to a concentration of 100 mM.
  • This monomer is obtained from the monomer (Ha.1). It suffices to carry out a deprotection of the primary amine (removal of the trifluoroacetyl group) as illustrated in the diagram below.
  • the monomer (Hb.1) is prepared according to the scheme below
  • silica (prior silica neutralization with TEA) with a gradient of
  • Monomer IIa.2 (1 eq) is dissolved in water.
  • DiSuccinimidylGIutaryl 200 eq is dissolved in the minimum amount of DMF such that the final proportion of DMF does not exceed 10%. Allowed to react 2h at 37 ° C with stirring. Then a reverse phase silica column is made by eluting with a water-acetone mixture. The desired product is recovered which can be identified by its yellow color. Acetone is evaporated.
  • the coupling is allowed to proceed for 1 h at 37 ° C. with stirring and then the conjugate can be purified by HPLC.
  • Figure 9 Percent decrease in current intensity at the peak of oxidation of ferrocene as a function of time; (") control (polymer prepared with the monomer Ib.1 and 3-ethanol pyrrole) + addition of the HBV complementary target (A) + addition of the non-complementary target HIV (4) + addition of the complementary target HBV .
  • the readings were carried out with a buffer already used for detecting biological interactions.
  • the MICAM oligonucleotide hybridization buffer (Apibio, Grenoble, France) was used. This last buffer contains: 9.5 mM phosphate buffer, 0.515 M NaCl, 2.6 mM KCl, 0.048% Tween, Denhardt IX, salmon sperm DNA 10 ⁇ g / mL
  • Lb- A copolymer layer with the monomer (Ib.1) and pyrrole-3-ethanol was also carried out.
  • the following formulation is used: 50 mM pyrrole-3-ethanol and 20 mM monomer (Ib.l).
  • the deposits are made at 0.60 V with a load of 21.6 mC / cm 2 in 10 mM sodium acetate / acetic acid buffer, pH 4.2, 0.2 M LiClO 4.
  • the voltammogram obtained at 200 mV / s in the deposition buffer clearly shows the reduction and oxidation of ferrocene.
  • the voltammogram of the polymer formed in this same buffer is shown in Figure 2 and shows the oxidation and reduction of ferrocene.
  • the voltammogram obtained is rather dissymmetrical for scanning rates higher than 50 mV / s, which reflects the fact that the polymer layer obtained is sterically constrained and therefore the electron exchanges are less easy.
  • IXLb- A layer of copolymer with the monomer (Ila.l) and 3-pyrrole-3-ethanol was also carried out.
  • the advantage of making a copolymer lies in the fact that it will make it possible to space, through the pyrrole-3-ethanol, the positive charges on the surface of the layer.
  • a concentration of 50 mM pyrrole-3-ethanol and 20 mM monomer (IIa.l) is used to make each deposit on an electrode chip.
  • Two deposit buffers yielded good results:
  • the monomer was electropolymerized at a concentration of 2 mM in a
  • Electropolymerization was carried out by 3-chronoamperometry
  • sequences is derived from the HIV virus (SEQ ID No. 1). The others are from the HIV virus.
  • HBV-105C virus (SEQ ID NO: 2 and SEQ ID NO: 3).
  • the grafting of pyrrole is
  • oligonucleotide modified at the 5 'position by a hexylamine.
  • a copolymer was obtained from 50 mM pyrrole-3- monomer
  • oligonucleotides derived from the HBV virus SEQ ID No. 2 and SEQ ID No. 3, each at
  • the hybridization buffer contains sodium acetate / acetic acid
  • sequence SEQ ID No. 4 is a 33-mer.
  • the HBV capture probe oligonucleotide (SEQ ID No. 3) modified at the 5 'position
  • HBV-105C pyrrole
  • the hybridization reaction is monitored over time by measuring the
  • HBV or HIV HBV or HIV
  • the white is made on a polymer pad that does not contain
  • the monomer (Here) is copolymerized at 0.60 V and 21.6 mC / cm 2 with pyrrole monomers functionalized with oligonucleotides (SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, provided by Biomérieux Polytech).
  • the concentrations of the monomers are respectively 15 mM and 3.75 ⁇ M.

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EP06778815A 2005-07-11 2006-07-07 Monomeres electropolymerisables solubles en solution aqueuse et sondes electroactives susceptibles d'etre obtenues avec de tels monomeres Withdrawn EP1907404A1 (fr)

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FR2888240B1 (fr) 2007-09-07
WO2007006944A1 (fr) 2007-01-18
US7812180B2 (en) 2010-10-12
US20090053826A1 (en) 2009-02-26
JP2009500507A (ja) 2009-01-08

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