EP1732963A2 - Hochmolekulargewichtpolymer auf basis von n,n-dimethylacrylamid - Google Patents

Hochmolekulargewichtpolymer auf basis von n,n-dimethylacrylamid

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Publication number
EP1732963A2
EP1732963A2 EP05739585A EP05739585A EP1732963A2 EP 1732963 A2 EP1732963 A2 EP 1732963A2 EP 05739585 A EP05739585 A EP 05739585A EP 05739585 A EP05739585 A EP 05739585A EP 1732963 A2 EP1732963 A2 EP 1732963A2
Authority
EP
European Patent Office
Prior art keywords
polymer
solid support
molecules
chosen
polymers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05739585A
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English (en)
French (fr)
Inventor
Jan Sudor
Valessa Barbier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique CEA
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Publication date
Application filed by Commissariat a lEnergie Atomique CEA filed Critical Commissariat a lEnergie Atomique CEA
Publication of EP1732963A2 publication Critical patent/EP1732963A2/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
    • C08F265/10—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of amides or imides

Definitions

  • the present invention relates to high molecular weight statistical polymers based on N, N-dialkylacrylamide and monomers functionalized laterally by molecules of interest.
  • the present invention relates to N, N-DIMETHYLACRYLAMIDE AND MONOMERS FUNCTIONALIZED BY PROBE MOLECULES AND USES THEREOF. , when used for the preparation of solid supports comprising at least one surface on which said polymers are adsorbed.
  • the present invention also relates to the solid supports thus modified and to their process for obtaining, as well as to their various uses, in particular for the immobilization of molecules of interest or the carrying out of chemical, biochemical or biological reactions.
  • the biological molecules are immobilized on the support via a bond covalent so that the molecules do not detach from the support during the use of the chips, especially during the washing steps.
  • this immobilization is generally carried out in two distinct stages: - a first stage of functionalization of the supports which consists in a chemical modification of their surface by grafting of synthetic molecules
  • Another approach consists in immobilizing ON via the copolymerization of ON functionalized by acrylamide with a crosslinked polyacrylamide (Rehman FN, et al., Nucleic Acid Research, 1999, 27 (2), 649-655).
  • the gels can be prepared by the techniques of conventional polymerization and well known to those skilled in the art such as radical polymerization.
  • the surfaces thus prepared have a high density of probes stably attached.
  • the surfaces of these supports on the other hand, have the drawback of being permanently modified and therefore cannot be easily regenerated. It has also already been proposed, in particular by Southern E. et al, Nat.
  • 5,695,936 describes a method of detecting a nucleotide sequence of interest using a nucleotide probe labeled with a tracer.
  • a reagent consisting of a copolymer based on N-vinylpyrrolidone or maleic anhydride, having a molecular mass of between 5000 and 400,000 g / mol and comprising lateral substituents of oligonucleotide nature.
  • the immobilization of this copolymer on the surface of the support is carried out in a fairly complicated manner via a complex between the solid support, a probe labeled with a tracer, a reagent and a target nucleic acid.
  • US Patent 5,453,461 also describes biologically active polymers of formula P- (A) q in which P represents a linear, branched or crosslinked polymer formed for example from acrylic monomers, A represents a biologically active section and can for example be biotin or an ON comprising from 1 to 80 nucleotide units and q is an integer equal to 1 or 2. Depending on the structure of these polymers, A is always positioned at the end of the polymer chain and not sideways.
  • 5,723,344 describes the preparation of copolymers formed from an N-vinylpyrrolidone monomer and a second monomer comprising a reactive function allowing the attachment by covalent bond of a biological ligand capable of forming a complex with a target molecule such as for example an antigen / antibody complex, polynucleotide / polynucleotide, polynucleotide / nucleic acid, antibody / hapten, hormone / receptor, as well as their capacity to adsorb on the surface of solid supports.
  • Such copolymers have a molecular weight generally between 1000 and 500,000, preferably between 10,000 and 250,000 and contain 25 to 70% of units derived from N-vinylpyrrolidone.
  • the supports thus prepared can be used for the immobilization of target molecules in solution, in particular in methods for detecting and assaying nucleotide sequences.
  • the layers of polymers adsorbed on such supports nevertheless have the disadvantage of being very unstable, due to the relatively small molecular size of these copolymers and the high percentage of lateral substituents (30 to 75%).
  • American patent US Pat. No. 6,692,914 also describes sensors made up of a substrate, the surface of which comprises a plurality of chains of segmented polymers (block polymers) in the form of a brush comprising a water-soluble or water-dispersible segment and at least one chosen probe. among biological molecules, linked to said segment.
  • trypsin can be attached to the surface of reactors and undergoes, in this case, very little autolysis compared to trypsin in solution.
  • the fixation of the trypsin is carried out on quartz or silica microbeads which are then inserted into microcapillaries which serve as reactors for trypsin.
  • the fixation of trypsin is carried out by means of a Schiff base reduction reaction between a residual primary amino group of trypsin and an aldehyde group carried by the surface (see in particular Muilin C, in “Methods in Enzymology", Colowick, SP, Caplan NO Eds., Académie Press, New York, 1987, Vol 136).
  • Such methods are however long and laborious and are not entirely satisfactory.
  • after immobilization of the trypsin on the surface of the supports there remain parts of the surface which have not been modified and which it is necessary to saturate in order to minimize the non-specific reactions of adsorption of molecules. in solution (peptides, proteins for example).
  • the present invention firstly relates to a water-soluble statistical polymer (not segmented) comprising probe molecules as lateral substituents, characterized by the fact: - that it results from the copolymerization of a monomer A of
  • the water-soluble statistical polymers in accordance with the present invention and as defined above are capable of physically adsorbing stably to the surface of solid supports, and thus make it possible to lead to solid supports comprising at least one surface functionalized by probe molecules such as for example nucleic acid or protein chips on which it is then possible to immobilize target molecules of interest while minimizing the adsorption of other molecules in solution.
  • probe molecules such as for example nucleic acid or protein chips
  • the polymers in accordance with the invention do not lead to the formation of a brush of polymers adsorbed by one of their ends on the surface of the support.
  • the term “small organic molecule” is understood to mean organic molecules preferably having a molecular weight less than or equal to 1000 g / mole.
  • said polymer has a molecular mass greater than or equal to 1.10 g / mole. The inventors have indeed found that the stability of surfaces functionalized by such polymers was even greater when the polymers used had such a molecular weight.
  • N, N- [dialkyl (C] -C 4 ) acrylamide] constituting the copolymers in accordance with the invention, mention may be made of methyl, ethyl, w-propyl and zz-butyl groups, the methyl group being very particularly preferred.
  • monomer A the
  • N, N-dimethylacrylamide is therefore very particularly preferred.
  • monomers B mention may in particular be made of vinyl monomers and acrylamides such as acrylic and methacrylic acids, N-aminoalkyl acrylamides, N-aminoalkyl methacrylamides, aminoalkyl acrylates and aminoalkyl methacrylates, in which the alkyl group may represent for example the ethyl, propyl, butyl, pentyl, or hexyl group.
  • the probe molecules present in the polymer as lateral substituents are preferably chosen from active biological molecules having a limited lifespan, such as enzymes.
  • the degree of incorporation of the probe molecules is between 1 and 20% by number relative to the total number of monomeric units of monomer A of N, N- [dialkyl (Cj -
  • the polymers in accordance with the invention and as described above can be prepared according to conventional copolymerization techniques well known to those skilled in the art, such as, for example, radical copolymerization, by reacting the monomers A as defined above. above with monomers Bl or B2 as defined above in a suitable solvent such as water, in the presence of a polymerization activator such as for example a red / ox couple such as the ammonium persulfate / sodium metabisulfite couple.
  • a polymerization activator such as for example a red / ox couple such as the ammonium persulfate / sodium metabisulfite couple.
  • the preparation of the monomers B2 can also be carried out according to conventional methods and well known to those skilled in the art, for example by reacting, under anhydrous conditions in a suitable organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide ( DMF) or dimethylsulfoxide (DMSO), a probe molecule containing a carboxylic group (biotin for example) with the vinyl group of a copolymerized monomer such as for example N-aminopropyl methacrylamide, in the presence of a coupling agent such as for example the dicyclohexyl carbodiimide; the reaction of a carboxyl group and of an ine group being in fact well known for a long time in the literature, in particular in the article by Khorana HG et al, Chem.
  • a suitable organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide ( DMF) or dimethylsulfoxide (DM
  • trypsin modified with a vinyl group can be prepared according to the method described by Plate NA et al, Polymer Science USSR, 1989, 31, 216-219.
  • the polymers according to the invention can be characterized by classical analytical techniques such as refractive index diffusion exclusion chromatography and detection by light scattering or by viscosimetry in order to collect information on the size of the polymers or even by magnetic resonance techniques nuclear (NMR) to gather information on their structure.
  • NMR magnetic resonance techniques nuclear
  • the present invention also relates to the use of at least one water-soluble statistical polymer as described above for the preparation of a solid support comprising at least one surface functionalized by probe molecules, and in particular for the preparation of chips proteins or nucleic acids and in particular DNA.
  • a process for the preparation of a solid support comprising at least one surface functionalized with a water-soluble statistical polymer in accordance with the invention characterized in that it comprises the following steps: '' a solid support comprising at least one surface to be functionalized, with a solution in a compatible solvent of at least one water-soluble random polymer in accordance with the invention and as defined above, - incubation of said surface with said polymer solution for a time sufficient for the adsorption of the polymer on the surface of the solid support, - rinsing of the support solid using a polymer-free solvent to obtain a solid support comprising at least one surface functionalized by an adsorbed layer of polymers.
  • the solid supports which can be functionalized according to this method are preferably chosen from the supports comprising at least one surface of the silica type and its derivatives such as glass and quartz, or any other material covered with silica, glass or quartz.
  • the term “compatible solvent” is understood to mean any solvent allowing the dissolution of the polymer, not causing any alteration of the probe molecules which it comprises as lateral substituent and for which the surface of the solid support to be functionalized will have an affinity lower than that which it will have for the polymers confused with the invention.
  • the polymers in solution may spontaneously adsorb on the surface of the support.
  • the first factor is the level of interaction between the surface of the solid support and the monomeric units constituting the polymer, that is to say the amount of energy necessary for the adsorption of the polymer (forces of attraction).
  • the second factor is the reduction in the conformational states of the polymer on the surface of the solid support, that is to say the reduction in chain entropy. This is due to the impenetrability of the surface for the monomeric units of the polymer and corresponds to the energy which tends to repel the polymer from the surface of the solid support (repulsive forces). The thickness of the polymer layer adsorbed on the surface of the solid support will be greater the greater the repulsive forces.
  • the solidity of the adsorption of the polymer layer on the surface of the solid support will therefore depend on the ratio between the forces of attraction and the repulsive forces.
  • the duration of the incubation of the solid support with the polymers in solution is preferably between 1 and 60 minutes approximately, and even more preferably between 5 and 40 minutes approximately.
  • the quantity of polymers in solution is between approximately 0.001% and 5% by weight relative to the volume of the polymer solution and even more preferably between approximately 0.1% and 2% ( weight / volume).
  • the solvents used to rinse the support after adsorption of the polymers and before their use are preferably chosen from the solvents described above and used to make the polymer solution.
  • the solvent used to rinse the support is identical to that used to make the polymer solution.
  • the present invention also relates to the solid supports obtained by implementing the preparation process in accordance with the invention, said supports being characterized by the fact that they comprise at least one surface functionalized by an adsorbed layer of water-soluble carrier polymers of probe molecules as lateral substituents as defined above.
  • Such supports can in particular be in the form of studs, channels, capillaries, reactors or reaction chambers such as, for example, the devices usually used to carry out enzymatic reactions (enzymatic digestion).
  • the adsorption of the polymers on the surface of the solid support is carried out via the monomers A of N, N- [dialkyl (C] -C 4 ) a_crylamide].
  • These solid supports are in particular protein chips and in particular enzyme, peptide or polypeptide chips, acid chips nucleic acid and in particular DNA or RNA, oligosaccharide or polysaccharide chips.
  • the thickness of the polymer layer is generally between 1 and 100 nm.
  • the thickness of the polymer layers can be measured for example by elipsometry or using more sophisticated techniques using evanescent waves (Allain C. and ⁇ /., Phys. Rev. Leti, 1982, 49, 1694) or the neutron scattering (Barnett K. et al, "The effects of Polymers on Dispersion Stability", Tadros,
  • biochemical refers to reactions, processes and protocols which involve at least one substrate and its enzyme.
  • biochemical reaction can be used within the framework of the present invention to designate the methods of amplification of nucleic acids such as polymerase chain reactions (PCR), the determination of a genotype such as microsequencing, or alternatively nucleic acid sequencing.
  • PCR polymerase chain reactions
  • biochemical also includes other types of enzyme-catalyzed reactions such as digestion of proteins by proteases, cleavage of DNA by nucleases, phosphorylation of molecules by kinases, isomerization of molecules by isomerases, the conversion of dopamine to norepinephrine by dopamine hydrolase, etc.
  • chemical is used to denote reactions, methods and processes in which there is at least one step involving a reaction which is not catalyzed by an enzyme.
  • the term “chemical” can be used to designate syntheses of organic or inorganic molecules, degradation reactions in which one of the steps is not catalyzed by an enzyme as well as the chemical reactions catalyzed by the ultra violet radiation.
  • the term “biological” is used to designate reactions of which at least one of the stages involves a living organism such as a cell, a culture of cells, a cluster of cells. adherent, a mono- or multicellular organism, parts of tissues or organs.
  • the term “biological” used in the context of the present invention therefore includes mono- or pluricellular eukaryotic organisms, as well as prokaryotic organisms such as bacteria and viruses.
  • the subject of the invention is also a method for immobilizing target molecules and for screening complementary target molecules in solution or for carrying out biochemical, chemical or biological reactions on a solid support, characterized in that it comprises at least the following steps: a) bringing a solid support into contact with at least one surface functionalized by an adsorbed layer of water-soluble random polymers carrying probe molecules as lateral substituents with a liquid sample capable of containing target molecules, during sufficient time to carry out said immobilization or said reaction, b) desorption of the polymer layer from the surface of the support by rinsing the support with an alkaline solution or a solvent, c) optionally, repeating steps a) and b) above.
  • the alkaline solutions which can be used to carry out the stage of regeneration of the support can be chosen from solutions of sodium hydroxide, potassium or ammonium.
  • the solvents which can be used to carry out the stage of regeneration of the support are preferably chosen from solvents for which the adsorbed polymers have more affinity than for the surface on which they have previously been adsorbed.
  • step b) of regeneration of the support is carried out using a sodium hydroxide solution.
  • the invention also comprises other arrangements which will emerge from the description which follows, which refers to an example of the preparation of a copolymer of N, N-dimethylacrylamide and acrylic acid comprising trypsin as a probe molecule, to an example of the preparation of a glass capillary comprising a surface functionalized by such a polymer and to the use of such a capillary to carry out a creatine digestion reaction, to an example of preparation of a copolymer of N, N-dimethylacrylamide and biotin functionalized by a vinyl group, to an example of preparation of a solid support comprising a surface modified by such a copolymer and its use for the immobilization of target molecules biotinylated by through streptavidin. It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.
  • the mixture is then stirred for 20 minutes under strong bubbling with nitrogen in order to remove the dissolved oxygen.
  • the reaction mixture is then brought to a temperature of 32 ° C. using a water bath. 1% by mole of ammonium peroxydi sulphate and 0.1% by mole of sodium metabisulphite relative to the amount of monomers are added in order to initiate the copolymerization reaction.
  • the reaction is carried out for 1 hour and 30 minutes, the mixture becoming highly viscous after approximately 45 minutes.
  • the reaction medium is then diluted to 500 ml with MilliQ water, acidified to a pH of 3 with 3 N hydrochloric acid, then ultrafiltered on membranes of 1,000,000 g / mol and finally lyophilized.
  • the product of the reaction was characterized by proton NMR and acid-base assay to determine the rate of incorporation of acrylic acid.
  • a copolymer of P (DMA-s-AA) is obtained in which for an initial fraction of
  • the grafting protocol used is based on the EDC / NHS couple (ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride /
  • the reaction medium is stirred at room temperature for 15 minutes then filtered on a Microcon® membrane having a threshold of 100,000 g / mole cut off to remove unreacted NHS and EDC.
  • the reaction mixture is then centrifuged at 10,000 rpm for 1 hour and 15 minutes.
  • the reaction mixture is then heated at a temperature of 4 ° C followed by addition of 2.4 mg (1., 04.10 "7 moles) of a trypsin solution at 2 mg / ml in 0.1 M MES, 0 , 5 M NaCl, pH 6.
  • the mixture is then maintained at a temperature of
  • reaction mixture is diluted to 300 ml by addition of MilliQ water then ultrafiltered on membranes having a cutoff threshold of 100,000 Da in order to remove the unreacted trypsin, the secondary reaction product (NHS) and to desalt the medium.
  • the reaction mixture is then concentrated to a volume of 50 to 100 ml and then lyophilized.
  • EXAMPLE 2 Preparation of a glass capillary Functionalized AR COPOLYMER CARRIER TRYPSIN AS MOLECULE PROBE
  • the trypsin copolymer as prepared above in Example 1 is suspended in NH 4 HCO 3 buffer 25 mM ( pH 8) filtered through a 0.22 ⁇ m filter, at a rate of 3% (w / y) of trypsinized copolymer / ml of buffer.
  • a capillary 20 cm long and 75 ⁇ m in diameter (Polymicro®) is treated by passing the following solutions: NaOH 3 N; 25 mM NH 4 HCO 3 buffer; 0.2 M HCl; Buffer and finally the solution of P (D As-AA) trypsin.
  • EXAMPLE 3 PREPARATION OF A COPOLYMER OF NN-DIMETHYLACRYLA1MIDE AND BIOTIN FUNCTIONALIZED BY A VINYL GROUP
  • This example illustrates the synthesis of a copolymer of N, N-dimethylacrylamide and biotin functionalized with a vinyl group.
  • the functionalization of biotin is carried out by reaction of a carboxylic group of biotin with N-aminopropyl methacrylamide containing a vinyl group.
  • the reaction is carried out under anhydrous condition in an organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (D SO), using a coupling agent such as dicycloh exyl carbodiimide.
  • an organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (D SO)
  • a coupling agent such as dicycloh exyl carbodiimide.
  • the copolymerization of biotin with the monomer of N, N-dimethylacrylamide is carried out under the same conditions as those described above in Example 1 by reacting 2.7 g (0.0272 moles) and 1.2 g ( 0.0042 moles) of biotin modified by a vinyl group.
  • a copolymer is obtained which is purified by ultrafiltration using a threshold cut-off filter of 100,000 Da and then lyophilized.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
EP05739585A 2004-03-15 2005-03-10 Hochmolekulargewichtpolymer auf basis von n,n-dimethylacrylamid Withdrawn EP1732963A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0402630A FR2867479B1 (fr) 2004-03-15 2004-03-15 Polymere de haut poids moleculaire a base de n,n- dimethylacrylamide et de monomeres fonctionnalises par des molecules sondes et ses utilisations
PCT/FR2005/000573 WO2005100424A2 (fr) 2004-03-15 2005-03-10 Polymere de haut poids moleculaire a base de n,n-dimethylacrylamide

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EP1732963A2 true EP1732963A2 (de) 2006-12-20

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US (1) US20070178465A1 (de)
EP (1) EP1732963A2 (de)
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WO (1) WO2005100424A2 (de)

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WO2011140590A1 (en) * 2010-05-10 2011-11-17 Bio-Layer Pty Ltd Binding systems
US11529610B2 (en) 2012-09-17 2022-12-20 W.R. Grace & Co.-Conn. Functionalized particulate support material and methods of making and using the same
RU2015114330A (ru) 2012-09-17 2016-11-10 У.Р. Грейс Энд Ко.-Конн. Хроматографические среды и устройства
SG11201605712SA (en) 2014-01-16 2016-08-30 Grace W R & Co Affinity chromatography media and chromatography devices
ES2929099T3 (es) 2014-05-02 2022-11-24 Grace W R & Co Material de soporte funcionalizado y métodos de fabricación y uso de material de soporte funcionalizado
BR112017026193B1 (pt) 2015-06-05 2021-09-14 W.R. Grace & Co-Conn Adsorventes, método de produção dos adsorventes e uso dos adsorventes
CN112816688B (zh) * 2021-01-04 2022-05-13 深圳市柏明胜医疗器械有限公司 一种酶标板及其制备方法和应用

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CA1206457A (en) * 1981-10-07 1986-06-24 Alan Rosevear Synthesis of compounds
AU5785400A (en) * 1999-07-02 2001-01-22 Symyx Technologies, Inc. Polymer brushes for immobilizing molecules to a surface or substrate, where the polymers have water-soluble or water-dispersible segments and probes bonded thereto
FR2811083B1 (fr) * 2000-06-30 2002-11-22 Inst Curie Milieu liquide non-thermosensible pour l'analyse d'especes au sein d'un canal
WO2004011513A1 (en) * 2002-07-29 2004-02-05 Applera Corporation Graft copolymers, their preparation and use in capillary electrophoresis

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WO2005100424A2 (fr) 2005-10-27
WO2005100424A3 (fr) 2006-01-26
US20070178465A1 (en) 2007-08-02
FR2867479B1 (fr) 2007-11-02
FR2867479A1 (fr) 2005-09-16

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