EP1899434A1 - Polymeres fluorescents solubles en solution aqueuse et procede de preparation de polymeres fluorescents solubles en solution aqueuse - Google Patents
Polymeres fluorescents solubles en solution aqueuse et procede de preparation de polymeres fluorescents solubles en solution aqueuseInfo
- Publication number
- EP1899434A1 EP1899434A1 EP06778734A EP06778734A EP1899434A1 EP 1899434 A1 EP1899434 A1 EP 1899434A1 EP 06778734 A EP06778734 A EP 06778734A EP 06778734 A EP06778734 A EP 06778734A EP 1899434 A1 EP1899434 A1 EP 1899434A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polymer
- fluorophores
- mol
- monomer
- fluorophore
- 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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Classifications
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- 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
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
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- 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
- C08F2/38—Polymerisation using regulators, e.g. chain terminating agents, e.g. telomerisation
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- 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
- C08F8/00—Chemical modification by after-treatment
- C08F8/34—Introducing sulfur atoms or sulfur-containing groups
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B69/00—Dyes not provided for by a single group of this subclass
- C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
- C09B69/103—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds containing a diaryl- or triarylmethane dye
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B69/00—Dyes not provided for by a single group of this subclass
- C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
- C09B69/109—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds containing other specific dyes
Definitions
- the present invention relates to the field of fluorescent polymers. More particularly, the present invention relates to novel polymers
- the invention also relates to the process for preparing soluble fluorescent polymers in aqueous solution.
- Synthetic polymers have been used for a long time both in the therapeutic field for vectorizing active molecules or genes, and in the field of diagnosis. In the latter case, biological ligands are attached to the polymers by complexation, covalence or specific recognition, and the conjugates thus formed are used in molecular detection tests.
- Fluorescent polymers can also find applications in different methods of labeling, detection, imaging, including fluorescence microscopy and confocal microscopy. For applications in the
- polystyrene sulfonate derivative for water solubility
- fluorescent coumarin styrene derivative and a methacrylate-PEG-COOH derivative for covalent coupling of proteins.
- the polymers obtained have a broad polymolecularity (Ip).
- Ip polymolecularity
- the relative quantum yield of these immobilized fluorophores on the polymer is 0.67 (in relative relation to the coumarin alone), which corresponds to a fluorescence amplification factor of 9 for the 38 000 g / mol polymer. , that is, an amplification factor of the fluorescence based on the molar mass of the polymer of 0.24 per Kg / mol of polymer, and a fluorescence enhancement factor of 40 for the polymer of 150000 g / mol, that is to say at a factor of amplification of the fluorescence based on the molar mass of the polymer of 0.27 per Kg / mol of polymer.
- the polymer obtained carries 22 lateral fluorophores per chain and a coumarin at one end of the chain.
- This polymer has a certain number of drawbacks: it is soluble in water only when it is ionized, in the example given at pH 11, which greatly limits its use in biological media which have a pH close to 7 ; the lateral fluorophores are grouped on the hydrophobic block, which is not favorable to good fluorescence of these lateral fluorophores; the 22 fluorophores are all hydrophobic, which leads to a tendency of the polymer to aggregate into a micelle type structure: it is therefore not really soluble in water but dispersible in water; this polymer is not very fluorescent: only the coumarin located at the end of the chain emits fluorescence, the lateral fluorophores serving as a light storage antenna.
- the amplification of the fluorescence obtained, and in particular, the amplification of the fluorescence relative to the mass of the polymer is low.
- some of these polymers are labeled with fluorescein and are therefore very sensitive to pH and poorly photostable. In all these prior art techniques, the polymers exhibit a limited fluorescence amplification factor.
- the polymers are most often obtained by a polymerization process leading to a broad chain size distribution, which requires purification by fractionation, to achieve a narrow size distribution. Polydispersity in size is, for example, a problem for the analysis of intercellular communication and size exclusion phenomena such as the permeability of membranes.
- the Applicant proposes to provide new polymers, soluble in aqueous solution, which have a high fluorescence.
- the subject of the present invention is a soluble fluorescent polymer in aqueous solution which carries at least 5 fluorophores distributed over the polymer, said fluorophores having the following properties:
- the fluorophores are water-soluble, the fluorophores do not form a self-association in water, up to a concentration of 10 -4 mol / l, preferably up to a concentration of 10 3 mol / l,
- the free fluorophores in aqueous solution have a molar extinction coefficient greater than 1000 M -1 cm -1 , preferably greater than 5000 m -1 cm -1 , the free fluorophores in aqueous solution have a higher quantum yield at 0.3, preferably greater than 0.6,
- the polymer according to the invention has a fluorescence amplification factor greater than or equal to 0.35 per Kg / mol of polymer, preferably greater than 0.45.
- the polymers according to the invention have a characteristic below or a combination of the characteristics below, where they do not exclude each other: the fluorophores comprise or are linked via a spacer arm, comprising at least one sequence -CH 2 -CH 2 - located between the fluorescent portion of the fluorophore and the polymer;
- the polymer is obtained by using a process of controlled radical polymerization or living ionic polymerization;
- the polymer is obtained by using a controlled radical polymerization process based on reversible addition / fragmentation chain transfer (RAFT);
- RAFT reversible addition / fragmentation chain transfer
- the polymer has a polymolecularity index of less than 1.5, preferably less than 1.3;
- the fluorophores comprise at least one polar or ionizable group in aqueous solution
- the polymer has less than 2.4 kg / mol, preferably less than 2 kg / mol of polymer per fluorophore; said fluorophores having a relative fluorescence quantum yield of at least 0.7, preferably at least 0.75;
- the fluorophores are chosen from: N- (5-aminopentyl) -4-amino-3,6-disulfo-1,8-naphthalimide, 3,6-diamino-9- (2-methoxycarbonyl) phenyl, 9 - (2,4-disulfophenyl) -2 5 3,6,7,12,13,16,17-octahydro-lH, 5H, llH, 15h- xanthéno [2,3,4-ij: 5,6,7 1-diquinolizin-18-ium, 9- (2,4-disulfophenyl) -3,6-bis (ethylamino) -2,7-dimethyl-xanthylium, 3,6-bis (diethylamino) - 9- (2,4-disulfophenyl) -xanthylium and their derivatives;
- the fluorophores are identical and are N- (5-aminopentyl) -4-amino-3,6-disulpho-1,8-naphthalimide;
- the fluorophores are insensitive to variations in pH
- the fluorophores are photostable
- the polymer is in the form of a random copolymer, comprising at least two distinct repeating entities, one carrying the fluorophore and at least one other hydrophilic entity;
- the polymer comprises, at one of its ends, a compound of interest such as a covalently bound biological ligand; preferably, the compound of interest is either bound to the polymer by a thioether function at its end ⁇ , is bound to the polymer by an amide function at its end ⁇ .
- the invention proposes to provide processes for the preparation of new fluorescent polymers, soluble in aqueous solution, which have a high fluorescence. The subject of the invention is therefore the processes as defined in the claims
- the subject of the invention is therefore also a first process for the preparation of soluble fluorescent polymers in aqueous solution and which exhibit a fluorescence amplification factor greater than or equal to 0.35 per Kg / mol of polymer, preferably greater than 0 45 per Kg / mol of polymer comprising the following steps:
- the fluorophores do not form a self-association in water, up to a concentration of 10 -4 mol / l, preferably up to a concentration of 10 3 mol / l,
- the free fluorophores in aqueous solution have a molar extinction coefficient greater than 1000 M -1 cm -1 , preferably greater than 5000 Iv T 1 X m -1 ,
- the free fluorophores in aqueous solution have a quantum yield greater than 0.3, preferably greater than 0.6.
- the subject of the invention is also a second process for the preparation of soluble fluorescent polymers in aqueous solution and which has a fluorescence amplification factor greater than or equal to 0.35 per Kg / mol of polymer, preferably greater than 0, 45 per Kg / mol of polymer implementing a polymerization step by copolymerization of a functionalized monomer carrying a fluorophore, with a hydrophilic monomer, or with a monomer, which after treatment can lead to a hydrophilic entity, said fluorophores having the following characteristics:
- the fluorophores are water-soluble, the fluorophores do not form a self-association in water, up to a concentration of 10 -4 mol / l, preferably up to a concentration of 10 mol / l,
- the free fluorophores in aqueous solution have a molar extinction coefficient greater than 1000 M -1 . cm "1, preferably greater than 5000 M -1 Xm '1,
- the free fluorophores in aqueous solution have a quantum yield greater than 0.3, preferably greater than 0.6.
- the functionalized monomer carrying a fluorophore may be obtained by coupling a fluorophore, optionally via a spacer arm, to a functionalized monomer bearing a reactive function X1.
- the monomer bearing a reactive functional group X1 is chosen from the following functional monomers: N-acryloxysuccinimide, N-methacryloxysuccinimide, 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, 2-hydroxyethyl acrylate, 2-aminoethyl acrylate, maleic anhydride and, preferably, N-acryloxysuccinimide, N-methacryloxysuccinimide, maleic anhydride, and more particularly N-acryloxysuccinimide ( ⁇ AS).
- N-acryloxysuccinimide N-methacryloxysuccinimide
- maleic anhydride and more particularly N-acryloxysuccinimide ( ⁇ AS).
- the monomer carrying a reactive function X1 is chosen from derivatives of a sugar, preferably 6-O- (2-vinyloxyethyl) - ⁇ -D-galactopyranose, 6-O-acryloyl- ⁇ - D-galactopyranose, 6-Oacryloylamino-6-deoxy- ⁇ -D-galactopyranose and 6-O- (8-acryloylamino-3,6-dioxaoctyl) - ⁇ -D-galactopyranose.
- a sugar preferably 6-O- (2-vinyloxyethyl) - ⁇ -D-galactopyranose, 6-O-acryloyl- ⁇ - D-galactopyranose, 6-Oacryloylamino-6-deoxy- ⁇ -D-galactopyranose and 6-O- (8-acryloylamino-3,6-dioxaoctyl) - ⁇ -D-galactopyranose.
- the fluorophores used in these processes have the additional characteristics as defined above for the polymers.
- the coupling step is carried out in such a way that the fluorescent part of the fluorophores is removed from the polymer by a spacer arm comprising at least minus one concatenation -CH 2 -CH 2 -; for example, the fluorophores comprise a spacer arm, comprising at least one sequence --CH 2 --CH 2 -, located between the fluorescent portion of the fluorophore and the polymer;
- the polymerization step is carried out by controlled radical polymerization or living ionic polymerization, preferably by a controlled radical polymerization process based on reversible addition / fragmentation chain transfer (RAFT);
- RAFT reversible addition / fragmentation chain transfer
- the number of fluorophores attached to the polymer is adjusted, so as to obtain less than 2.4 kg / mol, preferably less than 2 kg / mol of polymer per fluorophore;
- the polymerization step is carried out by copolymerization, preferably a statisitic one, between a functionalized monomer, optionally carrying a fluorophore, and a hydrophilic monomer;
- the hydrophilic monomer is chosen from hydrophilic derivatives of acrylate, methacrylate, acrylamide, methacrylamide, N-vinylpyrrolidone, hydrophilic derivatives of saccharide monomers and, preferably, from: N-vinylpyrrolidone, N N-dimethylacrylamide and N-acryloylmorpholine;
- the polymerization step is carried out by random copolymerization between a monomer carrying a reactive function X1, optionally in protected form, and a hydrophilic monomer with the exception of unprotected hydrophilic saccharide monomers, or a monomer which, after treatment can lead to a hydrophilic entity; preferably, a monomer bearing a reactive functional group Xl, advantageously chosen from the following functional monomers: N-acryloxysuccinimide, ⁇ -methacryloxysuccinimide, 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, acrylate 2-hydroxyethyl, 2-aminoethyl acrylate, maleic anhydride and, preferably, N-acryloxysuccinimide, N-methacryloxysuccinimide, maleic anhydride, etc.
- N-acryloxysuccinimide after the coupling step, a treatment of the remaining Xl functions on the polymer is advantageously carried out, either by deactivation or by coupling with non-fluorescent water-soluble molecules; the polymerization step is carried out by random copolymerization between a monomer carrying a reactive function X1, optionally in protected form, and a hydrophilic monomer; a hydrophilic monomer chosen from hydrophilic acrylate, methacrylate, acrylamide, methacrylamide and N-vinylpyrrolidone derivatives is preferably used, and preferably from: N-vinylpyrrolidone, N, N-dimethylacrylamide and N-acryloylmorpholine;
- the polymerization step is carried out by homopolymerization of a monomer carrying a reactive function X1 in protected form, said function being deprotected before coupling of the fluorophores; preferably, the monomer bearing a reactive function X1 in protected form is chosen from derivatives of a sugar, preferably 1,2,2,4-di-O-isopropylidene-6-O- (2- vinyloxyethyl) - ⁇ -D-galactopyranose, 6-O-acryloyl-1,2,2,4-di-O-isopropylidene- ⁇ -D-galactopyranose, 6-O-acryloylamino-6-deoxy-1,2 3,4-di- (3-isopropylidene- ⁇ -D-galactopyranose and 6-O- (8-acryloylamino-3,6-dioxaoctyl) -1,2,3,4-di-O-isopropylidene- ⁇ ; -D-galact
- the polymerization step is carried out by homopolymerization of a monomer bearing a reactive functional group X1, advantageously chosen from the following functional monomers: N-acryloxysuccinimide, ⁇ -methacryloxysuccinimide, 2-hydroxyethyl methacrylate, methacrylate, 2-aminoethyl, 2-hydroxyethyl acrylate, 2-aminoethyl acrylate, maleic anhydride and, preferably, N-acryloxysuccinimide, N-methacryloxysuccinimide, maleic anhydride and, more particularly, N-acryloxysuccinimide ( ⁇ AS); after the fluorophore coupling step, a treatment of the remaining Xl functions on the polymer is advantageously carried out, either by deactivation or by coupling with non-fluorescent water-soluble molecules;
- the reactive functional group X1 is chosen from hydroxyl, amine, aldehyde, anhydride and activated carboxylic acid functions in the form of an activated ester, for example N-hydroxysuccinirnide; the activated carboxylic acid function in the form of N-hydroxysuccinimide ester being preferred;
- a compound of interest such as a biological ligand
- a compound of interest is coupled in a covalent to an E reactive function, present at the end of the chain of the fluorescent polymer obtained; in the case where the polymer is obtained by RAFT method 5 the compound of interest is coupled, at the end ⁇ of the polymer, to form a thioether function;
- the biological ligands are chosen in particular from polynucleotides, antigens, antibodies, polypeptides, proteins, haptens, and biotin.
- the present invention also relates to soluble fluorescent polymers in aqueous solution, obtainable by such methods.
- the term "molar mass” means the number-average molar mass, Mn, of the polymer chains formed. In the present case, it is obtained after analysis of the samples by size exclusion chromatography, by using a refractometer type detector coupled to a light scattering apparatus, which makes it possible to have access to absolute molar mass values.
- the light scattering apparatus is a miniDawn (Wyatt Technology) and the absolute molar masses are determined with ASTRA software (Wyatt Technology).
- the polymolecularity index is the molar mass distribution index well known to those skilled in the art.
- the polymolecularity index is Ip, with
- Ip MwZMn, Mn being as defined above and Mw being the mass average molecular weight of the polymer chains. In this case, it has also been determined with ASTRA software.
- copolymer is to be understood as a polymer formed by at least two different repeating entities and, in particular, block copolymers and random copolymers.
- random copolymer refers to polymers formed by at least two different repeating entities, in which either the entities are statistically distributed along the macromolecular chain, or the entities successively succeed one another in a general structure (Bn'Cm ') p' in where n ', m' and p 'are identical or different integers.
- random copolymer therefore encompasses alternating copolymers.
- monomer is meant a polymerizable entity.
- functionalized monomer is meant a monomer carrying a reactive function Xl (reactive monomer), optionally in protected form, or a fluorophore (fluorescent monomer).
- soluble polymer in aqueous solution a polymer which, introduced into an aqueous solution at 25 0 C 5 at a concentration by weight equal to 1%, allows to obtain a solution having a maximum transmittance value of the light at a wavelength at which the polymer does not absorb, through a sample 1 cm thick, at least 70%, preferably at least 80%.
- water-soluble fluorophore is meant a fluorophore which, introduced at 25 ° C. in an aqueous solution to a concentration of at least 10 -3 mol / L, gives a homogeneous and transparent solution.
- the polymer or fluorophores can be tested is, for example, pure water or a buffer solution of pH between 5 and 10.
- the fluorescence quantum yield of a fluorophore is the ratio between the number of photons emitted by this fluorophore and the number of photons absorbed by this fluorophore. It is always less than or equal to 1. The closer it is to 1, the more the fluorophore considered has a high quantum yield.
- the relative fluorescence quantum yield of a fluorophore immobilized on a polymer is the fluorescence quantum yield of the immobilized fluorophore divided by the fluorescence quantum yield of the free fluorophore in solution.
- This fluorescence amplification factor is related to the molar mass of the polymer by dividing it by the molar mass of the polymer expressed in Kg / mol.
- the quantum yield of a free fluorophore in solution or immobilized on a polymer is determined by a method using as standard a dilute solution of rhodamine 101 in ethanol having a quantum yield equal to 0.92 at 25 ° C.
- the fluorescence spectra of the free fluorophore in solution and of the fluorophore immobilized on the polymer are obtained under the same experimental conditions (excitation wavelength, bandwidth of the excitation and emission monochromators, optical geometry) using SPEX Fluorolog F112A spectrofluorometer.
- the number of fluorophores immobilized on a polymer chain is determined by dividing the molar extinction coefficient of the fluorescent polymer by the molar extinction coefficient of the free fluorophore in solution.
- the molar extinction coefficient ⁇ of a free fluorophore in solution is determined from the slope of the curve representing the absorbance (optical density or OD) of the fluorophore (or the polymer respectively) depending on the concentration (C) of the fluorophore (or polymer) according to the well-known law of Beer-Lambert:
- the absorbance is determined at the maximum absorbance wavelength with a JASCO V-650 UV / Vis spectrophotometer.
- the phenomenon of self-association of a free fluorophore in aqueous solution is determined from the UV absorption spectra of solutions of increasing concentration.
- concentration at which the self-association phenomenon occurs is determined to be the concentration where the Beer-Lambert curve deviates from linearity, and this for an absorbance value of less than 1.0 using path cells appropriate optics (from 0.1 cm to 1 cm depending on the fluorophore concentration).
- the molar extinction coefficient and the fluorescence quantum yield are measured in an aqueous solution, under conditions, in particular pH and ionic strength, where these parameters take maximum values. Most often, the aqueous solution used is a solution pH buffer between 5 and 10.
- compound of interest is meant any type of molecular or macromolecular compound, or solid support, which it is advantageous to couple to the end of a polymer, for this or that application, in particular for an application in biology, therapeutic or diagnostic.
- compounds of interest mention may be made of biological ligands, mono- or disaccharides, lipids, fluorescent molecules, dyes, polymer chains and solid supports.
- the only necessary condition for the compound of interest is that it carries a reactive function capable of reacting with a function E present at one of the ends of the polymer or, in the case of the RAFT process, at one of the ends. reversible chain transfer agent or at one end of the polymer.
- this function E is a thiol or activated ester function, as detailed later in the description.
- the reactive function is respectively chosen as an example from the functions maleimide and iodoacetamide and among the functions amine, hydrazine, hydrazide, azide, alkoxyamine, hydroxy, thiol.
- the compound of interest can be bonded to the polymer according to the invention by means of a spacer arm which then carries the reactive function.
- biological ligand is meant a compound which has at least one recognition site allowing it to react with a target molecule of biological interest.
- biological ligands of polynucleotides, antigens, antibodies, polypeptides, proteins, haptens, labiotin, etc.
- polynucleotide means a sequence of at least 2 deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, 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 may also be modified at the level of the internucleotide linkage, for example phosphorothioates, H-phosphonates or alkylphosphonates, at the level of the backbone such as, 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 changes 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, the amino acids derived after enzymatic action such as trans-4-hydroxyproline 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 for use in solid support synthesis or liquid phase and unnatural 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 immunization of animals in animals. known conditions, in particular by immunization with a hapten-protein conjugate. These compounds generally have a molecular mass of less than 3000 Da, and most often less than 2000 Da and can be for example glycosylated peptides, metabolites, vitamins, hormones, prostaglandins, toxins or various drugs, 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.
- monosaccharide there may be mentioned, for example, glucose, galactose, mannose, fructose, and as “disaccharide”, for example, sucrose, cellobiose, lactose, maltose.
- lipid there may be mentioned, for example, dipalmitoylphosphatidylcholine.
- die mention may be made of methylene blue, bromocresol green, methyl red, safranine O.
- fluorescent molecules there may be mentioned for example fluorescein, rhodamine, pyrene, phenanthrene, anthracene, coumarin.
- polymer chain is meant a natural or synthetic polymer having been modified to bring at least one reactive function vis-à-vis the activated ester function.
- a natural polymer mention may be made, for example, of polysaccharides such as cellulose, dextran, chitosan and alginates.
- Synthetic polymers that may be mentioned include, for example, polyethylene oxide, polypropylene oxide, polyvinyl chloride, polyethylenes, polypropylenes, polystyrenes, polyacrylates, polyacrylamides, polyamides, polymethacrylates, polymethacrylamides, polyesters, or copolymers with vinyl aromatic monomers, alpha-beta unsaturated acid alkyl esters, unsaturated carboxylic acid esters, vinylidene chloride, dienes or compounds having nitrile functions (acrylonitrile), copolymers of vinyl chloride and propylene, vinyl chloride and vinyl acetate, copolymers based on styrenes or substituted derivatives of styrene
- solid support includes all materials for use in diagnostic tests or in therapeutics, affinity chromatography and separation processes. Natural materials, synthetic, modified or not chemically, can be used as a solid support, including polymers such as polyvinyl chloride, polyethylene, polystyrenes, polyacrylates, polyamides, polymethacrylates, polyesters, or copolymers based on vinyl aromatic monomers, alkyl esters unsaturated alpha-beta acids, unsaturated carboxylic acid esters, vinylidene chloride, dienes or compounds having nitrile functions (acrylonitrile); copolymers of vinyl chloride and propylene, vinyl chloride and vinyl acetate; copolymers based on styrenes or substituted styrene derivatives; synthetic fibers such as nylon; inorganic materials such as silica, glass, ceramic, quartz; latexes; magnetic particles; metal derivatives.
- polymers such as polyvinyl chloride, polyethylene, polystyrenes, polyacrylates, poly
- the solid support according to the invention may be, without limitation, in the form of a microtiter plate, a sheet, a cone, a tube, a well, beads, particles or the like, of a plane support like a silica wafer or silicon.
- the material is either hydrophilic or intrinsically hydrophobic or as a result of a chemical modification such as a hydrophilic support made hydrophobic.
- Solid supports capable of reacting by covalent bond formation with a thiol function carried by the polymer advantageously have maleimide functions on the surface.
- maleimide functions for example, it is possible to chemically introduce maleimide functions, at the surface of a silica wafer by silanization, using an aminoalkylsilane and then a heterobifunctional spacer arm carrying an N-hydroxysuccinimide function at one end (which reacts on the amine function ) and a maleimide function at its other end.
- Solid supports capable of reacting by covalent bond formation with an activated ester function for example, carried the polymer obtained by implementing the RAFT process, advantageously have amine functions on the surface.
- amine functions for example, it is possible to chemically introduce amine functions at the surface of a silica wafer by silanization using an aminoalkylsilane such as aminopropyldimethylchlorosilane, aminopropylmethyldichlorosilane or aminopropyltrichlorosilane.
- an aminoalkylsilane such as aminopropyldimethylchlorosilane, aminopropylmethyldichlorosilane or aminopropyltrichlorosilane.
- functionalisation by amine functions can be obtained by copolymerization of styrene with aminomethylstyrene or with aminoethyl methacrylate.
- alkyl is meant, when not more precise, a saturated hydrocarbon group, linear or branched having from 1 to 18, preferably from 1 to 6, carbon atoms.
- alkyl group there may be mentioned methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and the like.
- Alkoxy means an O-alkyl group, alkyl being as defined above.
- halogen is meant a chlorine, bromine, iodine or fluorine atom.
- alkenyl and alkynyl correspond to a hydrocarbon group of 2 to 18, and preferably 2 to 6 carbon atoms, comprising respectively at least one double or one triple bond.
- alkenyl or alkynyl are, for example, vinyl, allyl, isopropenyl, 1-, 2- or 3-butenyl, pentenyl, hexenyl, ethynyl, 2-propynyl, butynyl.
- cycloalkyl denotes an alkyl, alkenyl or alkynyl group, monocyclic or polycyclic, for example bicyclic, comprising from 3 to 10 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bridged cycloalkyl groups such as adamantyl, bicyclo [3.2.1] octyl.
- heterocycloalkyl means cycloalkyl as defined above, comprising one or more heteroatoms selected from nitrogen, oxygen and sulfur.
- Aryl groups denote mono-, bi- or polycyclic carbocycles comprising at least one aromatic group.
- heteroaryl refers to an aryl group as defined above comprising at least one atom selected from nitrogen, oxygen or sulfur.
- Aryl or heteroaryl include phenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, biphenyl, benzocycloalkyl, i.e. 5-triene, benzodioxolyl, such as pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, pyridyl, pirazinyl, pyrimidyl, tetrazolyl, thiadiazolyl, oxadiazolyl, triazolyl, pyridazinyl, indolyl, pyrimidyl.
- cycloalkylalkyl group means that the group consists of an alkyl group itself substituted with a cycloalkyl group.
- alkylcycloalkyl group means that the group consists of a cycloalkyl group itself substituted by an alkyl group.
- substituted group is meant a group bearing one or more substituents.
- substituents is meant a group chosen from: halogens, cyano, alkyl, trifluoroalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, amino, alkylamino, dialkylamino, hydroxy, alkoxy, aryloxy, an optionally substituted phenyl group, an aromatic group.
- alkoxycarbonyl or aryloxycarbonyl (-COOR 0 ), carboxy (-COOH), acyloxy (-O 2 CR 0 ), carbamoyl (-CONR ° 2 ), isocyanatoalkylcarbonyl, alkylarylcarbonyl, arylcarbonyl, arylalkylcarbonyl, phthalimido, maleimido , succinimido, amidino, guanidino, allyl, epoxy-SR °, groups having a hydrophilic or ionic character such as the alkaline salts of carboxylic acids, alkaline salts of sulphonic acid, polyalkylene oxide chains (PEO, PPO) 5 cationic substituents (quaternary ammonium salts) where R 0 represents an alkyl or aryl group.
- PEO polyalkylene oxide chains
- the invention relates to soluble polymers in aqueous solution and having a high fluorescence.
- the polymers according to the invention have a fluorescence enhancement factor relative to the polymer weight greater than or equal to 0.35 per Kg / mol of polymer, preferably greater than 0.45 per Kg / mol of polymer. Therefore, even for relatively small polymers, it is possible within the scope of the invention to obtain a rather high fluorescence amplification factor.
- the polymers according to the invention have a large number of fluorophores, namely at least 5, preferably at least 10 fluorophores, distributed over the polymer chain. The distribution of fluorophores along the chain is achieved through statistical functionalization of the polymer and / or random copolymerization as explained below. Given this distribution, the fluorescence quenching of the fluorophores is low.
- the fluorophores used in the context of the invention have a certain number of characteristics making it possible to obtain the desired properties.
- the fluorophores used are water-soluble and exhibit, when they are free in aqueous solution, a molar extinction coefficient greater than 1000 M -1 cm -1 , preferably greater than 5000 m 4 ⁇ 1 m -1 , and a higher quantum yield. at 0.3, preferably greater than 0.6,
- the fluorophores selected form few self-associations in aqueous solution, so as to further reduce the quenching of fluorescence.Freferentially, the selected fluorophores will not form self-associated in water to a concentration of 10 "4 mol / 1, preferably to a concentration of 10" 3 mol / 1.
- the aqueous phase solubility of the polymer according to the invention results, on the one hand, from the hydrosoluble nature of the fluorophores and, on the other hand, from the presence in the polymer chain of hydrophilic monomeric entities.
- the solubility of the polymer may of course be related to the pH or the ionic strength of the aqueous solution in which it is dissolved.
- the polymers according to the invention will be soluble in an aqueous solution having a pH of between 5 and 10.
- the fluorophores used carry polar or ionizable groups, which make them water-soluble. Ionizable fluorophores in aqueous solution will preferably be used.
- Nonionic polymers the solubility of which does not depend on pH, are a preferred variant for certain applications.
- the fluorescent portion of the fluorophores is removed from the polymer by a spacer arm having at least one sequence -CH 2 -CH 2 -. That is, the fluorescence emitting entity is separated from the polymer chain by at least two successive atoms.
- the polymer has less than 2.4 Kg / mol, preferably less than 2 Kg / mol of polymer per fluorophore.
- the subject of the invention is a polymer soluble in the aqueous phase, carrying at least 5 fluorophores, as previously defined, said fluorophores having a significant relative fluorescence quantum yield, that is to say say greater than or equal to 0.7, preferably at least 0.75.
- the fluorophores used will be insensitive to variations in pH, and / or thermostable and / or photostable. Nevertheless, it is possible to envisage using environmentally sensitive fluorophores such as pH or temperature, or capable of transferring fluorescence energy.
- fluorophores used are all identical.
- fluorophores that may be used in the context of the invention, mention may be made of the following fluorophores:
- N- (5-aminopentyl) -4-amino-3,6-disulpho-1,8-naphthalimide including the dipotassium salt of formula:
- Rhodamine 123 is marketed under the name Rhodamine 123;
- N- (5-aminopentyl) -4-amino-3,6-disulfo-1,8-naphthalimide is a particularly preferred fluorophore.
- This fluorophore is particularly advantageous because, in addition to being water-soluble, non-sensitive to pH, it does not form a self-association up to a concentration of 10 -3 mol / L in aqueous solution, to exhibit a function -NH Since it has a covalent bond with the polymer, it has on the one hand a spacer arm - (CH 2 ) 5 - which makes it possible to move the fluorescent part away from the polymer backbone and on the other hand to obtain an emission wavelength. It will be close to the emission wavelength of fluorescein, which implies that the filters usually used for fluorescein will be directly usable.
- the fluorophores are all identical and are N- (5-aminopentyl) -4-amino-3,6-disulfo-1,8-naphthalimide.
- the polymer can then be used, for example, to establish an identification code of the polymer chain.
- the covalent immobilization of 2 or 3 different fluorophores as defined above, on the same polymer chain and in defined proportions may be used to establish an identification code of the polymer chain insofar as the fluorophores chosen have a maximum emission wavelength sufficiently distinct.
- n 1 to 5
- the polymers according to the invention are obtained using a controlled radical polymerization technique (K. Matyjazewski, Controlled
- polymerization techniques make it possible to control the architecture of the polymers obtained and thus to synthesize polymers with well defined macromolecular characteristics (molecular weight, polymolecularity index and chain architecture).
- these techniques make it possible to control the molar mass of the polymers obtained, a molar mass that is quite predictable, and to obtain polymers that are very homogeneous in size and, in particular, that have a polymolecularity index of less than 1.5. and preferably less than 1.3.
- the number of fluorophores per chain is also very homogeneous.
- these various controlled polymerization techniques make it possible to obtain at the ends of the polymer at least one reactive function, which will serve as an attachment entity for a compound of interest, and in particular a biological ligand.
- the polymers according to the invention are biological tools, particularly interesting.
- controlled radical polymerization techniques are preferred.
- Radical polymerization has three stages: initiation (creation of free radicals and reaction with the first monomer unit), propagation (successive additions of monomer units on the growing ((macro) radical chain) and termination ( stopping the chain) by coupling or disproportionation between two growing chains or by transfer of a proton on a growing chain.
- the termination and transfer reactions affecting the (macro) radicals are responsible for the loss of control of the polymerization (polymer chains of unpredictable mass, high polymolecularity). To obtain a polymerization controlled radical, it is therefore necessary to strongly reduce these termination and irreversible transfer reactions.
- the general principle consists in reversibly deactivating the active centers by forming dormant (non-reactive) species, in order to have a very low concentration of (macro) radicals in the medium throughout the polymerization (K. Matyjazewski, Controlled Radical Polymerization, American Chemical Society Symposium Series, 768, Washington DC, USA, 2000).
- RAFT Reversible Addition Fragmentation Chain Transfer
- the patent application WO99 / 31144 describes the RAFT polymerization process in which the transfer agent is chosen from xanthates and dithiocarbamates. Since then, other organosulfur transfer agents have been described: either xanthates, as described in particular in patent applications WO00 / 75207, WO01 / 42312 and FR 2 809 829, or dithiocarbamates, as described in particular in patent applications.
- WO99 / 35177, FR 2 809 829, or trithiocarbonates as described in particular in patent applications WO98 / 58974, WO01 / 60792, WO02 / 070571, WO 03/066685, or thioetherthiones, as described in particular in the patent application FR 2 794 464, or dithiocarbazates, as described in particular in US Pat. No. 6,380,335 and US Pat. No. 6,395,850, or dithiophosphoroesters as described in particular in patent application FR 2 812 293, or tetrathiophosphates as described in US Pat. in particular in the patent application FR 2 816 311.
- the RAFT technique will preferably be used, since it is particularly applicable to any family of monomers, unlike the SFRP method, which is not applicable to the family of methacrylates, and the ATRP hardly applicable to acrylamide derivatives.
- the RAFT technique does not involve metals, unlike the ATRP process for which metal residues, even in very small quantities, may be a contraindication for biomedical applications.
- the RAFT technique makes it possible to introduce a compound of interest, in particular, via a binding entity, either at the beginning (end ⁇ ) or at the end of the chain (end ⁇ ) of the polymer, unlike the ATRP process which does not allow an easy introduction at the beginning of the chain via the polymerization initiator molecule.
- the RAFT polymerization process with a reversible chain transfer agent is carried out, according to standard techniques well known to those skilled in the art, from identical or different monomers, in the presence of a source of initiator radicals.
- Controlled radical polymerization reactions are generally carried out from one or more ethylenically unsaturated monomers. It will be possible to refer for the polymerization conditions to the documents of the prior art previously mentioned and to WO 2004/055060 in particular.
- the polymerization leads to polymers of the homopolymer type. In the opposite case, it leads to polymers of the copolymer type, so as to obtain, in the end, in the context of the invention, either random copolymers, for example alternating, or block copolymers, at least one of the blocks. being a random copolymer.
- random copolymers for example alternating, or block copolymers, at least one of the blocks. being a random copolymer.
- biocompatible polymers that is to say that do not disturb the biological properties of the biological ligand attached to the polymer, in terms of molecular recognition.
- the polymerization can be carried out in different ways.
- the first route consists of using a functionalized monomer B1 bearing a reactive function X1, optionally in protected form. This is followed by either a homopolymerization reaction of this monomer B1 or a reaction of copolymerization of Bl with a hydrophilic monomer B2 with the exception of unprotected hydrophilic saccharide monomers, or with a monomer B2 'which, after treatment, can lead to a hydrophilic entity.
- hydrophilic monomer is meant a monomer whose polymer has an expanded structure in the aqueous phase, corresponding to a Mark-Houwink Sakurada coefficient greater than or equal to 0.5.
- the hydrophilic monomer is, for example, chosen from hydrophilic derivatives of acrylate, methacrylate, acrylamide, methacrylamide and N-vinylpyrrolidone, unprotected saccharide monomers and their derivatives.
- N-vinylpyrrolidone ( ⁇ VP), N, N-dimethylacrylamide and N-acryloylmorpholine ( ⁇ AM) are preferred in the context of the invention.
- hydrophobic monomer is meant a non-hydrophilic monomer.
- hydrophobic monomer mention may be made of hydrophobic derivatives of methacrylate, acrylate, acrylamide, methacrylamide and styrene, advantageously n-butyl acrylate, t-butyl acrylate and t-butylacrylamide. styrene.
- a monomer in protected form can be chosen, such as protected saccharide monomers, for example 1,2,2,4-di- ⁇ -isopropylidene.
- a hydrophobic monomer carrying a reactive function can also, after deactivation of the reactive functional group or after coupling of the latter with a water-soluble molecule, lead to a hydrophilic entity.
- examples of such monomers include N-acryloxysuccinimide, ⁇ -methacryloxysuccinimide, maleic anhydride and more particularly, N-acryloxysuccinimide ( ⁇ AS).
- the monomer B1, which carries a reactive function X1, optionally in protected form, can be a hydrophilic or hydrophobic monomer. Of course, this monomer must be polymerizable with the selected polymerization technique.
- the reactive function X1 must be capable of reacting with a reactive function X2 carried by the fluorophore or the spacer arm that is to be grafted.
- the reactive function X1 is chosen, for example, from amine, hydrazine, hydrazone, azide, isocyanate, isothiocyanate, alkoxyamine, aldehyde (optionally a masked aldehyde, as in the case of saccharide monomers), epoxy, nitrile, maleimide, haloalkyl, hydroxy, thiol, anhydride, carboxylic acid activated in the form of N-hydroxysuccinimide ester, pentachlorophenyl, trichlorophenyl, p-nitrophenyl, carboxyphenyl.
- the reactive function X1 is chosen from amino, aldehyde, anhydride or activated carboxylic acid functions in the form of N-hydroxysuccinimide ester.
- the Xl function will advantageously be of the activated ester, aldehyde or anhydride type.
- hydrophilic monomer B1 having a reactive function X1 there may be mentioned 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, 2-hydroxyethyl acrylate, 2-aminoethyl acrylate and saccharide monomers, such as 6-0- (2-vinyloxyethyl) - ⁇ -D-galactopyranose polymerizable by living cationic polymerization, 6-Oacryloyl- ⁇ -D-galactopyranose, 6-O-acryloylamino-6-deoxy- ⁇ -D-galactopyranose, the 6-O (8-acryloylamino-3,6-dioxaoctyl) - ⁇ -D-galactopyranose, these three monomers being polymerizable by controlled radical polymerization, in particular by the RAFT method.
- 2-hydroxyethyl methacrylate 2-aminoethyl methacrylate
- 2-hydroxyethyl acrylate
- hydrophobic monomer B1 carrying a reactive functional group X1 there may be mentioned N-acryloxysuccinimide, ⁇ -methacryloxysuccinimide, maleic anhydride and, preferably, N-acryloxysuccinimide ( ⁇ AS).
- the monomer B1 carries a function reactive XI in protected form. This is, for example, the case of protected saccharide monomers as defined above.
- the monomers B1, B2, B2 ' are chosen according to the selected polymerization technique.
- a coupling reaction of the fluorophore on the reactive functions X1 is carried out, so as to immobilize the number of desired fluorophores on the polymer.
- the coupling reaction will be preceded by an appropriate deprotection reaction.
- the coupling of the fluorophores is carried out in such a way that the fluorescent portion of the fluorophores is removed from the polymer by a spacer arm comprising at least one -CH 2 -CH 2 - chain.
- either the fluorophore comprises a spacer arm and carries an X2 function and is therefore directly coupled with the reactive function X1, ie the fluorophore used does not have a spacer arm or is not a carrier.
- X2 reactive function in this case, the fluorophore will be modified to include the spacer arm, if necessary, and the reactive function X2. It may also be provided to perform the coupling in two steps, a first consisting of coupling a spacer arm on the reactive function X1, the second of coupling the fluorophore on the spacer arm.
- the reactive function X 2 situated at the end of the fluorophore or of the spacer arm, capable of reacting with the function X1, is preferably a primary or secondary amine function.
- a particularly stable amide function is obtained, so that the fluorescent polymers obtained will be chemically stable.
- the monomer B2 ' is preferably carrying a reactive function Xl' in protected form to avoid, after polymerization, competitive coupling reactions of the fluorophore on the monomers B1 and B2 '.
- the distribution of the fluorophores along the polymer chain is obtained on the one hand, by the fact that the coupling is done statistically on the reactive functions Xl present on the polymer and secondly, in the case where a copolymerization is carried out, by obtaining a random copolymer.
- a masking reaction of the residual reactive functions is then carried out, either by deactivation (for example a hydrolysis) or by coupling with a non-fluorescent water-soluble compound, which makes it possible to on the one hand, to eliminate residual reactive functions along the polymer chain and, on the other hand, to provide additional hydrophilicity to the polymer.
- the reactive functional group is an anhydride or an activated carboxylic acid in the form of N-hydroxysuccinimide ester
- an excess of a water-soluble amine such as aminoethylmorpholine, may be used.
- the polymerization will preferably be carried out between a monomer carrying a reactive function X1, optionally in protected form, and a hydrophilic monomer.
- a hydrophobic monomer B having a reactive function such as N-acryloxysuccinimide, ⁇ -methacryloxysuccinimide, maleic anhydride and preferably N-acryloxysuccinimide ( ⁇ AS)
- ⁇ AS N-acryloxysuccinimide
- hydrophilic monomer such as the hydrophilic derivatives of acrylate, methacrylate, acrylamide, methacrylamide and ⁇ -vinylpyrrolidone, and preferably N-vinylpyrrolidone ( ⁇ VP), N, N-dimethylacrylamide and the
- N-acryloylmorpholine ( ⁇ AM). which does not require any deprotection reaction of the reactive functional groups Xl and makes it possible to obtain a polymer having a satisfactory hydrophilicity, after treatment of any remaining reactive functions X1 after coupling of the fluorophores.
- Another preferred variant consists in carrying out a homopolymerization reaction of a protected sugar, for example 1,2,2,4-di-O-isopropylidene-6-O- (2-vinyloxyethyl) - ⁇ -D-galactopyranose.
- a protected sugar for example 1,2,2,4-di-O-isopropylidene-6-O- (2-vinyloxyethyl) - ⁇ -D-galactopyranose.
- a second route which can also be envisaged, although it is not preferred, is to carry out the coupling of the fluorophore (or of the spacer arm) with the monomer B1 bearing the function X1 and to carry out the polymerization with this new monomer.
- This route therefore consists in using a monomer B3 already carrying a fluorophore.
- a copolymerization reaction of this monomer B3 is then carried out with another hydrophilic monomer B4, or a monomer B4 'which, after treatment, can lead to a hydrophilic entity.
- the monomer B3 is either commercially available or obtained from a monomer B1 described above, by coupling, as previously described, of a function X2 carried by the fluorophore or the spacer arm, on the reactive function X1 of the monomer.
- the monomer B1 carries a protected reactive function, it will of course be deprotected beforehand.
- the monomers B4 and B4 ' correspond, respectively, to the hydrophilic monomers (B2), and to the monomers which, after treatment, can lead to a hydrophilic entity (B2'), mentioned above.
- a hydrophilic monomer B4 will preferably be used.
- the polymer has at its end ⁇ , the function -SC (S) -Z " terminal and at its end ⁇ the group R ".
- transfer agents already carrying a compound of interest such as RAFT reversible chain transfer agents (II), belonging to the family of dithioesters, xanthates or trithiocarbonates, which comprise a group of formula:
- R ' comprises at least one -amide-L function, with L selected from biological ligands, mono- or disaccharides, lipids, dyes, fluorescent molecules, polymer chains and solid supports.
- L selected from biological ligands, mono- or disaccharides, lipids, dyes, fluorescent molecules, polymer chains and solid supports.
- the -amide-L function corresponds to a function:
- X which represents a hydrogen atom, an optionally substituted group chosen from the following: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, or a covalent bond, so as to forming an amine ring with the compound L, and L which is as defined above.
- the group -R 'of the transfer agents (II) is chosen from the following groups: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, heteroaralkyl, alkylheteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, alkylcycloalkyl , alkylheterocycloalkyl, or a polymer chain, and said group being, on the one hand, carrying at least one function:
- organosulfur reversible chain transfer agents described in the prior art have the following pattern: Z C-S -...
- the group Z of the transfer agents (II) is advantageously chosen so that the transfer agent belongs to the family of dithioesters (Z containing an H, C atom,
- Z represents a hydrogen atom, a chlorine atom, -COOH, -CN, or a group chosen from the following optionally substituted groups: alkyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, -ORa, -SRa, -COORa , -O 2 CRa, -CONRaRb, -CONHRa, -P (O) ORaRb, -P (O) RaRb, -O-CRcRd-P (O) (ORa) (ORb), -S-CReRf-COOH, - 0-CReRf-COOH or a polymer chain,
- Z ' is a derivative of an optionally substituted alkyl, an optionally substituted aryl or a polymeric chain, its bond with the carbonylthio groups, occurring via an aliphatic carbon, an aromatic carbon or a sulfur atom, or oxygen,
- R ' is selected from the following groups: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, heteroaralkyl, alkylheteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, alkylcycloalkyl, alkylheterocycloalkyl, or a polymer chain, and said group being on the one hand, carrying at least one function:
- X represents a hydrogen atom, an optionally substituted group chosen from the following: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, or a covalent bond, so as to form an amine ring with the compound L,
- p is an integer greater than 1
- Ra and Rb represent, each independently of one another, an optionally substituted group chosen from the following: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl,
- Rc and Rd represent, each independently of one another, a hydrogen or halogen atom, a group -NO 2 , -SO 3 H, -SO 3 Rg, -NCO, -CN, -Rg , -OH, -ORg, -SH, -SRg, -NH 2 , -NHRg, -NRgRh, -COOH, -COORg, -O 2 CRg, -CONH 2 , -CONHRg, -CONRgRh, -NHCORg, -NRgCORh, C m F 2m + 1 with m between 1 and 20, preferably equal to 1,
- Rf represent, each independently of one another, an optionally substituted alkyl or aryl group
- Rg and Rh represent, each independently of one another, an optionally substituted group chosen from the following: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl,
- R ' represents a group
- - A represents an optionally substituted group chosen from the following: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, heteroaralkyl, alkylheteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, alkylcycloalkyl, alkylheterocycloalkyl, or a polymer chain,
- L and X are as defined above, and
- n is an integer greater than or equal to 1, preferably equal to 1.
- the group A of the group R ' is a branched aliphatic chain, optionally substituted, having a carbon atom secondary or tertiary, alpha of the sulfur atom.
- Z represents a group chosen from: alkyl, cycloalkyl, aryl, -ORa, and -SRa, said groups being optionally substituted and Ra being as defined above for (IIa), (IIb) and (IIc).
- transfer agents (II), (IIa), (IIb) and (IIc) are obtained by coupling of corresponding transfer agents (I) carrying at least one activated ester function with an amine compound of interest.
- activated ester is well known to those skilled in the art.
- An activated ester function can be defined as an ester whose "alcohol" part is a good leaving group with respect to nucleophilic substitution reactions, that is to say a leaving group which makes it possible to carry out a reaction of nucleophilic substitution between 0 and 100 ° C, preferably between 0 and 60 ° C, more preferably between 0 and 40 ° C.
- Such activated esters have, for example, been described by W. Anderson et al, in American Society, 1964, 46, 1839-1842 and by R. Arshady in Advances in Polymer Science, 1994, 111, 1-41.
- the functionalization can therefore be carried out by reacting a compound of interest comprising a nucleophilic function, and in particular a primary or secondary amine function, or an ammonium function which will make it possible to generate a reactive amine function in situ on the ester function. activated.
- This coupling reaction is easy, fast, quantitative and in a single step, which makes it possible to obtain a bond with the compound of interest, with a very high yield (close to 100%).
- the activated amine / ester function coupling leads to an amide bond, which in turn makes a very stable compound with respect to, for example, a more fragile and hydrolysable ester type function obtained in the prior art (Stenzel et al., in J. Mater Chem 2003, 13, 2090 and Chen et al., in Chem. Comm., 2002, 2276-2277).
- Such transfer agents (I) belong to the family of dithioesters, xanthates, or trithiocarbonates and comprise a group of formula:
- R is a group which comprises an activated ester function.
- this group R comprises at least one activated ester function -C (O) OY, -OY being a leaving group, Y being, for example, chosen from the following groups: N-succinimidyl, 1-benzotriazole, pentachlophenyl, 2 , 4,5-trichlorophenyl, 4-nitrophenyl, 3-pyridyl, 2-methoxycarbonylphenyl, N-phthalimidyl, and 2-carboxyphenyl.
- Y is the N-succinimidyl group:
- any type of group R described in the prior art can be used; it will be chosen according to the group R 'that one wishes to obtain.
- chain transfer agents (I) comprise a group R which is chosen from the following groups: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, heteroaralkyl, alkylheteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, alkylcycloalkyl, alkylheterocycloalkyl, or a polymer chain, and said group carrying at least one activated ester function as defined above and being optionally substituted by one or more other substituents.
- R is -A - (- C (O) -OY) n wherein: - A represents a group optionally substituted selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, heteroaralkyl, alkylheteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, alkylcycloalkyl, alkylheterocycloalkyl, or a polymer chain, - Y is as defined above and,
- n is an integer greater than or equal to 1, preferably equal to 1.
- transfer agents of formula (Ia), (Ib) or (Ic) will be used.
- Z represents a hydrogen atom, a chlorine atom, -COOH, -CN, or a group chosen from the following optionally substituted groups: alkyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, -ORa, -SRa, -COORa , -O 2 CRa, -CONRaRb, -CONHRa, -P (O) ORaRb, -P (O) RaRb, -O-CRcRd-P (O) (ORa) (ORb), -O-CReRf-COOH, - S-CReRf-COOH or a polymer chain,
- Z ' is a derivative of an optionally substituted alkyl, an optionally substituted aryl or a polymeric chain, its bond with the carbonylthio groups, occurring via an aliphatic carbon, an aromatic carbon or a sulfur atom, or oxygen
- R is selected from the following groups: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, heteroaralkyl, alkylheteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, alkylcycloalkyl, alkylheterocycloalkyl, or a polymer chain, and said group bearing at least one activated ester function as defined above and being optionally substituted by one or more other substituents,
- p is an integer greater than 1
- Ra and Rb represent, each independently of one another, an optionally substituted group chosen from the following: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl, - Rc and Rd represent, each independently of one another, a hydrogen or halogen atom, a group -NO 2 , -SO 3 H, -SO 3 R 8, -NCO, -CN, -Rg, -OH, -ORg, -SH, -SRg, -NH 2 , -NHRg, -NRgRh, -COOH, -COORg, -O 2 CRg, -CONH 2 , -CONHRg, -CONRgRh, -NHCORg, -NRgCORh, C m F 2m + 1 with m between 1 and 20, preferably equal to 1,
- - Re and Rf represent, each independently of one another, an optionally substituted alkyl or aryl group
- - Rg and Rh represent, each independently of one another, an optionally substituted group chosen from the following: alkyl , alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, alkylaryl.
- the transfer agents of formula (Ia) are preferred, and in particular those in which R is substituted by a single activated ester function.
- the group R of the transfer agents (I), (Ia), (Ib) and (Ic) as defined above have a secondary or tertiary carbon atom, located in alpha of the sulfur atom.
- the group A of the R group is a branched aliphatic chain having a secondary or tertiary carbon atom, alpha to the sulfur atom.
- Z is chosen from the groups: alkyl, cycloalkyl, aryl, -ORa, and -SRa, said groups being optionally substituted and Ra being as defined above for (Ia), (Ib) and (Ic).
- succinimido-6-phenyl-6-thioxo-5-thia-4-cyano-4-methylhexanoate succinimido-4-phenyl-4-thioxo-3- thia-2-methylbutanoate.
- the transfer agents of formula (I), (Ia), (Ib) and (Ic) are prepared according to techniques well known to those skilled in the art.
- a transfer agent carrying an acid function is obtained for example according to the reference Dupont WO98 / 01478 if it is a dithioester or a trithiocarbonate, or for example according to Rhodia reference WO98 / 58974 if is a dithiocarbonate also called xanthate.
- the acid function of this transfer agent is converted into function "activated ester" for example by addition of NHS (N-hydroxysuccinimide) in the presence of DCC (dicyclohexylcarbodiimide).
- the coupling between the activated ester function and a compound carrying a nucleophilic function is carried out directly on the transfer agent, which is then used in the RAFT polymerization and thus allows the synthesis of functionalized polymers at their ⁇ -ends.
- the coupling on the activated ester with a compound of interest (carrying a nucleophilic function) is thus preferably carried out before the RAFT polymerization.
- the RAFT polymerization of B1 (homo or copolymerization) is carried out in the presence of the transfer agent (I); a homopolymer (or a copolymer) carrying at least one activated ester function at its ⁇ -end is thus obtained,
- the compound of interest that is attached to the ⁇ or ⁇ end of the polymer will preferably be a biological ligand.
- the biological ligands that can be attached to the polymer of the present invention are, for example, those used in the field of diagnosis in detection tests for target molecules, for example, or in the therapeutic field, in particular for vectorizing active molecules or of genes.
- the biological ligand is capable of forming a ligand / anti-ligand capture complex.
- said anti-ligand may constitute the target molecule.
- those skilled in the art will choose the nature of the biological ligand to bind to the polymer.
- the biological ligand may be a biotin.
- the anti-ligand will be a streptavidin immobilized on the target via a nucleic acid labeled with a biotin and sufficiently complementary to the target for to hybridize specifically according to the reaction conditions and in particular the temperature or the salinity of the reaction medium.
- the fluorescent polymer then directly allows the detection of the target molecule.
- a polynucleotide is synthesized by a solid support chemical method having a reactive function at any point in the chain, such as, for example, 5 'end or 3' end or on an internucleotide phosphate base or on the 2 'position of sugar (see Protocols for Oligonucleotides and Analogs, Synthesis and Properties edited by S.
- a biotin, a sugar, an oligonucleotide, or a lipid that promotes membrane insertion may be coupled to the end of the polymer.
- the fluorescent polymer may then be fixed by one of its ends and specifically, on any support having, on the surface, the entity recognized by the selected compound of interest (polymer, latex, silica, lipid vesicle, protein , DNA or RNA target ).
- the fluorescent polymer may be immobilized on a given surface coated with streptavidin.
- the polymers according to the invention are very resistant to photodegradation, hence an interest in following kinetics by fluorescence microscopy, or by confocal microscopy with laser scanning to analyze different levels.
- the highly fluorescent polymers according to the invention may be used in diagnostic tests to enhance the detection of the capture of a biological target, such as a nucleic acid per molecule.
- a biological target such as a nucleic acid per molecule.
- the polymers according to the invention may also be used for marking materials (polymers, hybrid compounds, other) in optical microscopy applications (confocal, multiphoton, SNOM).
- FIG. 1 shows, in a comparative manner, the evolution over time of the fluorescence intensity between a polymer according to FIG. the invention (poly (N AM-LY)) and an R-Phycoerythrin-Streptavidin (RPE) conjugate.
- N-acryloylmorpholine ( ⁇ AM, sold by ALDRICH, reference 44.827-3) is distilled prior to use in polymerization.
- the N-acryloxysuccinimide ( ⁇ AS, sold by ACROS, reference 40030) is purified by chromatography on a silica column before use in polymerization.
- the dioxane (polymerization solvent) (sold by SDS, reference 27,053-9) is distilled on LiAlH 4 before use.
- 2,2'-Azobis-isobutyronitrile AIB ⁇ (polymerization initiator) (Fluka, reference 11630) is recrystallized from ethanol.
- the tert-butyl dithiobenzoate (chain transfer agent (ATC) of the RAFT polymerization) is synthesized according to the method described by A. Favier et al. in Macromolecule, 2002, 35, 8271-8280 Trioxane (internal reference for 1 H NMR monitoring) (JANSSEN-CHIMICA, reference 14.029.61) is used as it is.
- a copolymerization is carried out between the NAM and the NAS, in the dioxane, in the presence of AIBN and of fert-butyl dithiobenzoate.
- the various reagents are introduced into a Schlenk type reactor at room temperature, and the mixture is degassed by a succession of freeze / vacuum / thawing cycles, and then put under nitrogen.
- the reaction mixture is brought to 90 ° C. and left stirring for 2 hours.
- the polymer obtained, named poly (NAM-st-NAS) is precipitated in ether, several times if necessary, that is to say until the residual monomers are completely removed, then is recovered by centrifugation and dried under empty of the vane pump.
- Nuclear Magnetic Resonance (NMR) analysis conditions 1 H The kinetic monitoring of the monomer consumption is carried out by 1 H NMR (Nuclear Magnetic Resonance) with a Bruker AC 200 MHz spectrometer.
- the samples to be analyzed are prepared by mixing 300 ⁇ L of each sample with 300 ⁇ L of deuterated solvent: CDCl 3 .
- the fluorophore Lucifer Yellow Cadaverine, (N- (5-aminopentyl) -4-amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt) (LY, sold by Molecular Probes, reference A-1340) is used as what.
- 4- (2-Aminoethyl) morpholine (sold by ALDRICH, reference A5,500-4) is used as it is.
- Dimethylformamide (solvent) (sold by Merck, reference 822275) is dried over CaH 2 and distilled before use.
- the poly (NAM-st-NAS) polymer obtained in paragraph a) and the fluorophore LY are introduced with the solvent into a glass flask.
- the reaction mixture placed under argon is heated to 45 ° C. and left stirring in the dark for 96 hours.
- the temperature is lowered to 30 ° C. and 4- (2-aminoethyl) morpholine is introduced into the reaction medium.
- the reaction mixture is left for 4 days under argon, protected from light.
- the resulting polymer, named poly (NAM-LY) is precipitated in ether from a dichloromethane solution (several times), recovered by centrifugation and dried under vacuum from the vane pump. Operational conditions of the fluorophore coupling reaction and masking of the
- Characteristics of the fluorescent poly (NAM-LY) polymer in aqueous solution The absorption and emission maxima of the LY fluorophore are not modified by the coupling of LY molecules on the poly (NAM-st-NAS) polymer.
- the exact amount of LY bound to the polymer is determined by NMR and confirmed by comparing the absorption and fluorescence emission spectra of the fluorescent polymer and free LY: 36 fluorophore molecules are attached per chain, which corresponds to a coupling efficiency of 58% and 1.56 Kg / mol of polymer per fluorophore.
- the quantum yield of free LY is 0.61 (free ⁇ FLY) and the coupled quantum yield of LY on the polymer is 0.48 (immobilized ⁇ FLY).
- the relative quantum yield of LY fluorophores immobilized on the polymer is therefore 0.79.
- the fluorescence amplification factor of the poly [NAM-LY] fluorescent polymer is determined by the following formula:
- Fluorescence amplification factor number of LY / chain x (immobilized ⁇ FLY) / (free ⁇ FLY)
- the fluorescent polymer obtained has a fluorescence amplification of 28 compared with a free fluorophore in aqueous solution. If this value is reported to the molar mass of the polymer which is 56.2 Kg / mol, a fluorescence amplification factor of 0.5 per Kg / mol of polymer is obtained.
- poly (NAM-LY) polymer synthesized as described in paragraph a) and b) and the biological ligand (PEO-Maleimide Activated Biotin) are introduced with the solvent into a flask.
- the reaction mixture placed under argon is stirred, at a temperature of 30 ° C. and protected from light for 5 days.
- the resulting polymer, named poly (NAM-LY-B) is purified by dialysis.
- the purified poly (NAM-LY-B) is mixed with an Avidin-polystyrene latex (sold by ESTAPOR, reference 1080-06) in a PBS-Tween 1% o buffer containing albumin 0.1 gL "1. the reaction mixture is left for 4 h at 37 0 C. After centrifugation (4000 rpm) and six washes with buffer, the latex was analyzed by fluorescence.
- the fluorescence emission at 530nm is compared with the fluorescence of a solution of the poly (NAM-LY) fluorescent polymer (under the same operating conditions) This comparison makes it possible to determine the proportion of polymer chains carrying a biotin at their end, namely 15%.
- the fluorescence emission of the polymer does not vary, contrary to what is observed for the RPE which sees its emission decrease by 30%.
- the emission of poly (NAM-LY) decreased by 14% while that of the RPE decreased by 56%.
- Biotin-Fluoresceine conjugate (BF, sold by SIGMA, reference: B 8889 or MOLECULAR PROBES, reference: B-1370) is used as it is.
- Biotin-Lucifer Yellow conjugate (B-LY) is synthesized by reaction of EZ-link NHS-PEO 4 -Biotin (sold by PIERCE, reference 21330) and Lucifer Yellow Cadaverine (NHS lmol: 1.1 mol LY), in the same operating conditions as those of the coupling reaction of the fluorophore on the poly (NAM-st-NAS) described previously in paragraph b).
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Abstract
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FR0552031A FR2887892B1 (fr) | 2005-07-04 | 2005-07-04 | Polymeres fluorescents en solution aqueuse et procede de preparation de polymeres fluorescents solubles en solution aqueuse |
PCT/FR2006/001544 WO2007003781A1 (fr) | 2005-07-04 | 2006-06-30 | Polymeres fluorescents solubles en solution aqueuse et procede de preparation de plymeres fluorescents solubles en solution aqueuse |
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EP1899434A1 true EP1899434A1 (fr) | 2008-03-19 |
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EP06778734A Withdrawn EP1899434A1 (fr) | 2005-07-04 | 2006-06-30 | Polymeres fluorescents solubles en solution aqueuse et procede de preparation de polymeres fluorescents solubles en solution aqueuse |
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US (1) | US8133411B2 (fr) |
EP (1) | EP1899434A1 (fr) |
FR (1) | FR2887892B1 (fr) |
WO (1) | WO2007003781A1 (fr) |
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TWI395737B (zh) | 2006-11-08 | 2013-05-11 | Dow Agrosciences Llc | 作為殺蟲劑之雜芳基(取代的)烷基n-取代的磺醯亞胺 |
FR2927902B1 (fr) * | 2008-02-26 | 2011-11-11 | Oreal | Polymere ethylenique statistique comprenant un groupe reactif et un groupe ionisable, composition cosmetique et procede de traitement cosmetique. |
US20090221424A1 (en) | 2008-03-03 | 2009-09-03 | Dow Agrosciences Llc | Pesticides |
US9242010B2 (en) * | 2008-05-16 | 2016-01-26 | Research Foundation Of The City University Of New York | Living copolymer-protein/peptide hybrids for biomedical applications |
FR2950629B1 (fr) | 2009-09-25 | 2013-12-06 | Commissariat Energie Atomique | Composes fluorescents, polymerisables, de la 7-hydroxycoumarine, leur preparation, polymeres fluorescents de ceux-ci et capteurs chimiques les comprenant. |
WO2015134000A1 (fr) * | 2014-03-04 | 2015-09-11 | Empire Technology Development Llc | Composés conducteurs à base d'acrylamide, et leurs procédés de préparation et leurs utilisations |
US12105092B2 (en) | 2019-05-15 | 2024-10-01 | George Mason University | Molecularly imprinted particles |
US20230227657A1 (en) * | 2020-04-15 | 2023-07-20 | Queensland University Of Technology | Fluorescent macromolecule and uses thereof |
CN118406174B (zh) * | 2024-07-04 | 2024-08-30 | 上海美迪西生物医药股份有限公司 | 含甘露糖聚合物、联用raft聚合和点击化学制备含甘露糖聚合物的方法及其应用 |
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US4166105A (en) * | 1973-07-30 | 1979-08-28 | Block Engineering, Inc. | Dye tagged reagent |
EP0449505B1 (fr) * | 1990-03-26 | 1995-05-24 | Nalco Chemical Company | Polymères fluorescents et leurs composés de départ |
DE4114482A1 (de) * | 1991-05-03 | 1992-11-05 | Bayer Ag | Polymere farbstoffe, verfahren zu deren herstellung und deren verwendung |
US5750357A (en) * | 1994-05-18 | 1998-05-12 | Microquest Diagnostics, Inc. | Method of rapid analyte detection |
WO1998001478A1 (fr) | 1996-07-10 | 1998-01-15 | E.I. Du Pont De Nemours And Company | Polymerisation presentant des caracteristiques vivantes |
US5772894A (en) * | 1996-07-17 | 1998-06-30 | Nalco Chemical Company | Derivatized rhodamine dye and its copolymers |
WO2001007430A1 (fr) * | 1999-07-22 | 2001-02-01 | Nalco Chemical Compant | Polymeres fluorescents solubles dans l'eau |
US6280635B1 (en) * | 2000-05-01 | 2001-08-28 | Nalco Chemical Company | Autocycle control of cooling water systems |
FR2848556B1 (fr) | 2002-12-13 | 2006-06-16 | Bio Merieux | Procede de polymerisation radicalaire controlee |
-
2005
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2006
- 2006-06-30 US US11/921,873 patent/US8133411B2/en not_active Expired - Fee Related
- 2006-06-30 WO PCT/FR2006/001544 patent/WO2007003781A1/fr not_active Application Discontinuation
- 2006-06-30 EP EP06778734A patent/EP1899434A1/fr not_active Withdrawn
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US8133411B2 (en) | 2012-03-13 |
WO2007003781A1 (fr) | 2007-01-11 |
US20080290321A1 (en) | 2008-11-27 |
FR2887892B1 (fr) | 2007-09-07 |
FR2887892A1 (fr) | 2007-01-05 |
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