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WO2005110593A2 - Procede de synthese de polymeres - Google Patents

Procede de synthese de polymeres Download PDF

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Publication number
WO2005110593A2
WO2005110593A2 PCT/EP2005/005359 EP2005005359W WO2005110593A2 WO 2005110593 A2 WO2005110593 A2 WO 2005110593A2 EP 2005005359 W EP2005005359 W EP 2005005359W WO 2005110593 A2 WO2005110593 A2 WO 2005110593A2
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Prior art keywords
polymer
process according
solvent
solution
synthesis
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WO2005110593A3 (fr
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Alain Laurent
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Apibio SAS
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Apibio SAS
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00378—Piezoelectric or ink jet dispensers
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    • B01J2219/00497—Features relating to the solid phase supports
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00675—In-situ synthesis on the substrate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00709—Type of synthesis
    • B01J2219/00713—Electrochemical synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00731—Saccharides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/12—Libraries containing saccharides or polysaccharides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to a process for the stepwise synthesis of polymers on a solid surface of a support and the use of the process for the manufacture of biochips. Further, the present invention relates to an automated polymer synthesis system for synthesizing on a solid support a polymer chain by sequentially adding polymer building blocks to specified loci on the surface of a substrate.
  • the functional groups (usually terminal OH groups) of the building blocks are temporarily protected by intermediate protecting groups which are removed when treated with appropriate reagents.
  • These protecting groups are usually either acid labile groups like for example DMT (dimethoxytrityl) and its derivatives or photolabile groups like for example NPPOC (2-nitrophenyl-propyloxycarbonyl).
  • Biopolymers like oligonucleotides or polypeptides, are very useful for the manufacture of so-called biochips or genechips, wherein the polymers are attached to an array on a usually planar substrate.
  • the polymers are applied on the substrate either by direct in-situ or after an ex-situ synthesis. In-situ synthesis on the substrate is often preferred because it provides the facile build-up of different or identical polymer chains directly on the respective substrate without tedious intermediate steps after ex-situ synthesis of the polymers before attaching the polymer chain(s) on the substrate.
  • acetonitrile is used especially in oligonudeotide synthesis because of its high polarity enabling the solubilisation of the respective reaction partners.
  • a typical reaction in oligonudeotide synthesis is the coupling reaction between a phosphoramidite and a hydroxy group, for example an alcoholic hydroxy group of the 5'OH or 3'OH of the deoxyribose unit. This reaction requires a polar and an aprotic solvent like acetonitrile.
  • acetonitrile has several disadvantages, like for example its low boiling point (81°C), its incompatibility with many materials used in commercially available ink jet head materials like glues, plastics etc. Due to its low boiling point, acetonitrile droplets evaporate quasi immediately after being ejected from the print head.
  • US Patent Nr. 6,028,129 proposes ethylene and propylene carbonate derivatives for use in the automated synthesis of two-dimensional arrays of oligonucleotides. These compounds are used in order to overcome the well known drawbacks encountered by the use of acetonitrile.
  • US Patent 6,419,883 proposes to use micro droplets of a solution comprising a solvent having a boiling point of 150°C or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/cm/sec.
  • Solvents disclosed in this patent are N-methyl- 2-pyrrolidone, propylene carbonate or ⁇ -butyrolactone.
  • the proposed solvents in the prior art are not always compatible with ink-jet print heads having a plurality of nozzles, especially when the dimensions of the nozzles are small and/or generate microdroplets which do not have a uniform size. Therefore, it is impossible to maintain an accurate reaction control and a reaction protocol especially with respect to the concentration of the polymer building blocks. Therefore it has been the problem underlying the invention to provide a solvent which is universally applicable in a process for the stepwise synthesis of polymers independent from the specific set-up like nature of ink-jet print head etc. for carrying out the process.
  • the use of a mixture of two different solvents enhances the overall yield of the reaction, especially for the single coupling steps.
  • the use of a second solvent further allows for the fine-tuning of the viscosity, the surface tension and of the polarity and the aprotic properties of the solution compared to the use of one single solvent. Further, the second solvent increases the solubility of the phosphorous compound, preferably a phosphoramidite, and/or the activator in the reaction mixture.
  • the first solvent has a viscosity in the range of 8-30, preferably in the range of 8-20 mPa • s. Less than 7 m Pa s.
  • the solvent can not be used with an ink-jet print head having the nozzle diameters as small as indicated below. More than 30 m Pa ⁇ s results in a too viscous fluid which cannot be used within usual ink-jet print heads. Further, the reactivity on the spot where the solution droplet is applied would decrease due to the increased viscosity of the first solvent. Surprisingly it was found that the selected range of viscosity enables the use of the first solvent in a process according to the invention without significant evaporation of the solvent. Therefore an encrusting of the nozzles etc.
  • the first solvent has a surface tension of more than 26- 32 dyne/cm. This enhanced surface tension in combination with the selected viscosity according to the invention of the solvent avoids efficiently the "blooming" of a droplet of the solution applied on a surface.
  • Specific preferred examples of the first solvent according to the invention comprise but are not limited to methyloleate, 1-phenyltetradecane, 1-cyclopentylpentadecane, 1- cyclohexyltridecane, hexylnaphthalene, dinonylether, dibutylsebacate, 1- phenylpentadecane, 1-cyclopentylhexadecane, hexachlorocyclopentadiene, bis- cyanoethylether, 1-cyclohexyltetradecane, benzyl benzoate, N-butylstearate, diethylphthalate, 3-methylsulfolane, 2-pyrrolidone, 1-decylnaphthalene, dimethylphthalate, dibutylphthalate, diisooctylphthalate, dioctylphthalate, diisodecylphthalate.
  • the first solvent comprises or is a chemical compound which contains a derivatized carboxyl group.
  • polycarboxylic acid derivatives i.e. compounds comprising two or more derivatized carboxylic functions which display the above mentioned physical properties are a very suitable and preferred solvent especially in the field of the synthesis of biopolymers.
  • the term "derivatized carboxyl acid or function” means that the acid proton (or in other words the acid OH moiety) of the COOH groups has been substituted by a non-protic moiety. Examples of such derivatives include but are not limited to alkyl, aryl, aralkyl esters and the like.
  • the carboxylic functions are normally attached to a backbone which may be for example an aromatic, heteroaromatic, alkyl, alkenyl or alkinyl backbone and the like.
  • the most preferred chemical compounds have the formula (R 2 OOC) n - B - (COO ⁇ ) m wherein n and m are the same or different and represent an even number > 1
  • Ri, R 2 are the same or different and represent a linear or branched Ci to C ⁇ 0 alkyl, alkenyl or alkinyl group
  • B is a linear or branched Ci to C 10 alkyl, alkenyl, alkinyl group, a substituted or unsubstituted aromatic, heteroaromatic, alicyclic or heterocyclic ring.
  • Preferred examples are advantageously selected from the group consisting of alkyl diesters of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, maleic acid, fumaric acid, muconic acid, acetylendicarboxylic acid phthalic acid, isophthalic acid, terephthaiic acid.
  • alkyl esters of the polycarboxylic most preferably of dicarboxylic acids, whereby the alkyl group is preferably a branched or linear Ci to C ⁇ 0 alkyl, preferably a Ci to C 6 alkyl, namely methyl, ethyl, propyl, butyl, pentyl.
  • the amount of the second solvent in the solution is lower than the amount of the first solvent. If the second solvent would be present in excess, then the nature of this second solvent would predominate.
  • the amount of the second solvent is therefore in the range of 1- 30 vol. %, preferably 5-30 vol. %, most preferably 10-30 vol. %, with respect to the total volume of the solution.
  • the second solvent has a viscosity of less than 6 mPa ⁇ s.
  • Preferred second solvents are for example glymes like triglyme, diglyme, adiponitrile, acetonitrile and the like.
  • Table 1 shows the influence of the second solvent on the coupling yield of for each reaction step in the stepwise syntheses of oligonucleotides
  • the second solvent used alone does not give a reaction at all, but the solvent mixture consisting of 80 vol % dibutyl sebacate and 20 vol % triglyme gives high reaction yields.
  • substrate comprises any suitable substrate like glass, silica, silicon materials, but also polymer materials like polyethylene, polypropylene, polystyrene and the like which may be functionalized or not and the respective composite materials.
  • the substrate is provided with an array of metallic electrodes.
  • an electrically conducting polymer is attached to the metallic electrodes and whereby the electrically conducting polymer is the same or different for each electrode of the array.
  • the electrically conducting polymer is synthesized in situ on the metallic electrode
  • the synthesis of the electrically conducting polymer is in a most preferred embodiment an electrochemical synthesis.
  • the polymeric building blocks are therefore in addition to biomolecular building blocks like nucleotides, oligonucleotides, amino acids, oligopeptides, carbohydrates also preferably electropolymerizable monomers or macromers (oligomers), polymers etc. It is further preferred, that biomolecules as mentioned above are already chemically linked to electropolymerizable monomers or oligomers or even polymers before being applied on the surface.
  • ECPs electrically conductive polymers
  • methods of electrochemical polymerization generally used for the preparation of ECPs such as polymerization at set current (chronopotentiometry) or at set potential (chronoamperometry) are also applicable to the method according to the invention.
  • the quality of the deposit of the electrically conducting polymer may be controlled by the choice of experimental conditions: the concentration of the monomer e. g. pyrrole, thf and the like, the oligonucleotide-monomer/monomer ratio, the nature of the solvent, the electrochemical method used (cyclic voltammetry, chronoamperometry or chronopotentiometry).
  • the copolymer obtained may accordingly have different qualities of porosity and of accessibility depending on the desired subsequent use, and if the amount of bound oligonudeotide may be modified.
  • the electrode effectively makes it possible to monitor the progress of the polymerization reaction (for example the thickness of the polymer formed) or the progress of subsequent reactions carried out on the copolymer.
  • the process in accordance with the invention in one or other of its variants, it additionally comprises the elongation of an oligonudeotide chain in several successive steps, each of these steps consisting of the binding of one or more nucleotides, or oligonucleotides.
  • Elongation of the oligonudeotide is carried out at the surface of the support by assembling the protected monomers, starting with at least one nucleotide or oligonudeotide bound to the surface of the electrically conductive polymer.
  • the standard methods for the chemical synthesis of nucleic acids may be used in the implementation of this embodiment.
  • the electrically conducting polymer has a chemical functionality allowing for the attachment of a polymer building block.
  • chemical functionality denotes a chemical reactive group (masked or unmasked) like for example an amino, acetyl, carboxyl, hydroxyl group etc.
  • the polymer building blocks are selected from the group comprising nucleosides, nucleotides, oligonucleotides, amino acids, oligopeptides, carbohydrates, dimers and trimers thereof.
  • This selection facilitates the manufacture of so-called bio- or genechips considerably.
  • a redox couple like organometallic complexes as metallocenes like ferrocene, nickelocene, ruthenocene etc. is introduced into the polymer before, during or after electrochemical synthesis of the polymer. The introduction of a redox couple enhances the electrical conducting properties of the electrically conducting polymer.
  • a biochip comprising a substrate, an electrode, attached to said electrode an electrically conducting polymer and bonded to said conducting polymer a biomolecule selected from nucleic acid and their derivatives, peptides, proteins and carbohydrates, obtainable by a process according to the invention.
  • an automated polymer synthesis system for synthesizing on a solid support a polymer chain by sequentially adding polymer polymer building blocks comprising the following elements: a) an ink jet print head comprising a plurality of nozzles for jetting a microdroplet on a selected locus of the support b) a treatment cell for treating the support on which a microdroplet has been deposited with an agent c) transport means for moving the support adjacent to the print head and/or to the treatment unit d) a vessel comprising a solution containing a polymer building block and wherein the diameter of the nozzles is in the range of 30-70 microns.
  • the diameter of the nozzles is advantageously in the range of 30-70 microns in order to generate small microdroplets with an uniform size and volume.
  • the volume of a microdroplet is preferably in the range of 60-100 pL to control the amount of polymer building block to be applied.
  • each nozzle is individually addressable thereby allowing for the construction of a plurality of different polymer chains each or a predefined number thereof containing different building blocks, before starting the reaction.
  • the nozzle spacing is in the range of 0.020-0.005 ⁇ m, more preferably in the range of 0.015-0.010 ⁇ m. This close arrangement makes it easy to apply the microdroplets on complex patterns on the surface of the biochip.
  • the inkjet head comprises a tank in connection with each nozzle. This simplifies the system set-up because in prior art each nozzle had to be connected with a reservoir thus requiring more sophisticated controlling means and setups.
  • Polymer building block denotes a chemical moiety which is comprised within the final polymer.
  • the chemical moiety may therefore comprise functional groups before incorporation in the final polymer.
  • suitable polymeric building blocks according to the invention are substituted or non-substituted phosphoramidites, mono-, oligo - and polynucleotides, amino acids, peptides, sugars (furanoses, riboses, etc.), biotin, avidin, streptavidin, antibodies and the like.
  • a microdroplet is a separate and discrete unit preferably having a volume of about 100 to 200 pL, most preferably between 5 pL and 70 pL.
  • solution as used herein comprises the solvent perse and the solute or several solutes.
  • the microdroplets when reacted to the surface form so-called spots or microdots.
  • the arrays of polymers obtained according to the present invention are arranged in these microdots or spots which are separate and discrete units.
  • the diameter of each microdot can be greater than 1,000 ⁇ m but ranges typically from about 5 ⁇ m - 800 ⁇ m, preferably from about 10 ⁇ m - about 500 ⁇ m and most preferred from 20-200 ⁇ m.
  • the distance between the individual microdots is typically from about 1 ⁇ m - about 500 ⁇ m, preferably from about 20 ⁇ m - about 400 ⁇ m. Generally, the distance between the microdots should preferably be in the range of the respective site of the microdoplets to as to avoid a using of neighbouring spots.
  • the physical separation of the microspots is obtained by the reaction of the remaining functional groups with a non-removable protecting group preferably comprising phosphorous. These areas provide then an unreactive protective surface which is chemically inert.
  • biomolecule or “biopolymer” as used herein means any biological molecule in the form of a polymer, such as oligonucleotides, amino acids, peptides, proteins, carbohydrates, antibodies, etc.
  • nucleoside as used herein comprises both deoxyribonudeosides and ribonudeosides.
  • oligonudeotide refers to an oligonudeotide which has deoxyribonucleotide or ribonucleotide units.
  • Suitable nucleotides useful for the synthesis of oligonucleotides according to the present invention are those nucleotides that contain activated phosphorous containing groups such as phosphotriester, H-phosphonate and phosphoramidite groups.
  • activator usually means a catalyst which in the case of oligonudeotide synthesis is a catalyst which fosters the reaction between the 3' phosphoramidite group of a nucleoside and the hydroxyl groups of the next nucleoside or nucleotide. This may be 5- methylthiotetrazole, tetrazole, or 5-ethylthiotetrazole, DCI or pyridiniumchloride.
  • alkyl refers to any saturated straight chain, branched or cyclic hydrocarbon group of 1 to 10 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n- butyl, etc. The term alkyl also includes the cycloalkyl groups such as cyclopentyl, cyclohexyl, cycloheptyl, etc.
  • lower alkyl denotes an alkyl group of 1 to 4 carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.
  • heterocyclic refers to any five-membered or six-membered monocyclic structures or to an eight-membered to eleven-membered bicydic structure which is either saturated or unsaturated.
  • the heterocyclics comprise at least one heteroatom selected from the group consisting of nitrogen, oxygen, sulfur, phosphorous, arsenic and the like.
  • nitrogen heteroatoms and “sulfur heteroatoms” as well as “phosphorous heteroatoms” include any oxidized form of nitrogen, sulfur and phosphorous as well as a quartemized form of any basic nitrogen.
  • heterocyclic compounds include piperidinyl, morpholinyl and pyrrolidinyl.
  • oligonudeotide designates polydeoxynucleotides (containing 2- deoxy-D-ribose), polyribonucleotides (containing D-ribose) to any other type of polynudeotide which is an N-glycoside of a purine or pyrimidine base and to other polymers containing non-nucleotidic backbones providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • protecting group a species is designated which prevents a segment of a molecule from undergoing a specific chemical reaction but which is removable from the molecule following completion of this reaction. This is in contrast to a “capping group” which permanently binds to a segment or a functional group of a molecule or to the functional groups at the surface of the substrate to prevent any further chemical transformation of that segment.
  • the method of the present invention may also be used to synthesize peptides by standard solid phase peptide synthesis methodologies.
  • a linker containing an activated carboxyl group is keyed to amino groups which can link to the activated surface of a support according to the invention.
  • Any of the usual "temporary" protecting groups routinely used in polypeptide synthetic chemistry are suitable for use in the present invention. Non-limiting examples among these are, for example, BOC (t-butoxycarbonyl) and FMOC (N ⁇ 9 -fluorenylmethyloxycarbonl) groups.
  • suitable amino protecting groups include but are not limited to 2-(4-biphenyl)propyl[2]oxycarbonyl (Bpoc), 1-(1- adamantyl)-l-methylethoxy-carbonyl (Adpoc) and the like.
  • Representative activators or so-called in-situ coupling reagent suitable for use in the present invention include but are not limited to N,N'-dicyclohexylcarbodiimide (DCC) and the like.
  • Preferred is their use in conjunction with the use of further accelerators or additives such as 1- hydroxybenzotriazole (HOBt), benzotriazol-1-yl-oxy-tris (dimethylamino)phosphonium hexafluorophosphate (BOP) and the like.
  • HOBt 1- hydroxybenzotriazole
  • BOP benzotriazol-1-yl-oxy-tris
  • BOP dimethylamino)phosphonium hexafluorophosphate
  • the method of the present invention is advantageously employed to synthesize deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymer species (so-called oligonucleotides) by any of the several known chemistries for solid-phase DNA or RNA synthesis including phosphite triester, phophoramidite synthesis and H-phosponate synthesis.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • oligonucleotides any of the several known chemistries for solid-phase DNA or RNA synthesis including phosphite triester, phophoramidite synthesis and H-phosponate synthesis.
  • the method of the present invention is useful for practising any iterative nucleic acid synthetic technique.
  • oligonucleotides are synthesized by the phosphoramidite method.
  • the reactive sites on the surface of the substrate are sometimes functionalized with an additional spacer according to methods known in the art.
  • the introduction of the spacer can take place before starting the generation of the polymer chain, or, for example after step a) of the process according to the invention.
  • the spacer groups may also be applied to preselected portions of the reaction support only.
  • monomeric nucleotide or nucleoside polymer building blocks also called synthons
  • synthons have temporary protecting groups at appropriate nucleobase or 2'-O positions.
  • Solid phase nucleic acid synthetic techniques employ so-called temporary and permanent protecting groups in analogous fashion to solid phase peptide synthesis.
  • Base labile protecting groups are used to protect the exocyclic amino groups of the heterocyclic nucleobases during the synthesis. This type of protection is usually achieved by acylation with acylating agents such as benzoylchloride and isobutyrylchloride.
  • Acid labile protecting groups are used to protect the nucleotide 5' hydroxyl during synthesis.
  • Representative hydroxyl protecting groups are known to persons skilled in the art. These include but are not limited to dimethoxytrityl, monomethoxytrityl, trityl and 9-phenyl-xanthene (pixyl) groups. Dimethoxytrityl (DMT) protecting groups are widely used to the great acid lability which affords efficient removal even by very dilute acids.
  • the first step in the iterative chain elongation cycle according to the phosphoramidite technique is the removal of the 5'-O-protecting group (deprotection) of the initial monomer by immersing the reaction support in a solution of the deprotecting agent. This is followed by the addition of a rinsing reagent.
  • Suitable reagents for deprotection include Lewis acids such as ZnBr 2 , AICI 3 , BF 3 and TiCI 4 in various solvents such as dichloromethane nitromethane, tetrahydrofuran and mixed solvents such as nitromethane and lower alkyl alcohols such as methanol or ethanol and mixtures thereof.
  • Protic acids, alone or in combinations, such as acetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid and toluenesulfonic acid may also be used.
  • Chains are lengthened by addition and reaction of activated 5'-O-protected monomeric synthons.
  • a 5'-DMTr-deoxynucleoside-3'-0-(N,N- diisopropylamino)- ⁇ -cyanoethylphospite is deposited onto the reaction support.
  • Phosphoramidites of numerous nucleosides are commercially available.
  • a mild organic catalyst or activator, typically tetrazole, ethylthiotetrazole or methylthiotetrazole is deposited onto the reaction support with the phosphoramidite.
  • the coupling reaction is followed by the addition of a rinsing solvent, typically anhydrous acetonitrile.
  • a capping reagent is added for example by immersing the substrate in a solution of the capping reagent onto the preselected portions of the reaction support to cap free hydroxyl species remaining due to incomplete reaction of phosphite monomers (capping 1).
  • surface capping or capping 1
  • capping 2 The capping reagent for the "capping 2" which is typically a solution of an acid anhydride, also functions to reverse any inadvertent phosphitylation of guanine O-6 positions.
  • Oxidation of the resulting phosphite triester to the corresponding phosphate triester may be accomplished by adding an oxidant known in the art to be suitable such as a solution of alkaline iodine in water.
  • oligonucleotides may either take place in 3'-5' or in 5'-3' direction.
  • the method of the present invention may be employed in the synthesis of oligonucleotides having the naturally occurring nucleobase adenine (A), thymine (T), guanine (G), cytosine (C) and uracil (U) as well as non-naturally occurring nucleobases.
  • Non-naturally occurring nucleobases are molecular moieties which are known in the art to mimic the function of naturally occurring nucleobases in their biological role as components of nucleic acids.
  • Oligonudeotide species having a wide variety of modifications to nucleobases, sugars or inter-sugar linkages can be prepared in accordance with the method of the invention which are generally applicable to the synthesis of any oligomers synthesizable by solid phase techniques as, for example, also to polycarbohydrates.
  • the method of the present invention may be employed in the synthesis of S- phosphorodithioates, phosphorothioates, etc.
  • the polymers produced according to the method of the invention may be composed of more than one type of monomeric subunit (for example, amino acids, peptide nucleic acids, nucleotides, sugars (carbohydrates), etc.) and may possess more than one type of inter-subunit linkage.
  • monomeric subunit for example, amino acids, peptide nucleic acids, nucleotides, sugars (carbohydrates), etc.
  • Illustrative polymers produced according to the method of the invention include peptides, peptoids (N-alkylated glycines), ⁇ -polyesters, polythioamides, N-hydroxy amino acids, ⁇ -esters, polysulfonamides, N-alkylates polysulfonamides, polyureas, peptide nucleic acids, nucleotides, polysaccharides, polycarbonates, oligonucleotides, oligonucleosides and the like and chimeric molecules that contain one or more of these polymers joined together as a single macro molecule.
  • Libraries of monomeric species can also be prepared by the method of the invention. These include benzodiazepine libraries and other such analog libraries including but not limited to antihypertensive agents, antiulcer drugs, antifungal agents, antibiotics, antiinflammatories, etc.

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Abstract

La présente invention concerne un procédé de synthèse progressive de polymères, et notamment de biopolymères, sur une surface de support solide. Ce procédé consiste à utiliser une surface de support solide possédant un site de liaison, à appliquer une première micro-gouttelette d'une solution renfermant un premier élément constitutif de polymère sur le site de liaison en vue de fixer le premier élément constitutif de polymère au site de liaison, et à appliquer, sur le site de liaison modifié de l'étape précédente, une seconde micro-gouttelette d'une solution renfermant le même élément constitutif de polymère ou un élément différent, cette solution contenant un solvant présentant une viscosité supérieure à 7 mPa s. La présente invention concerne également une biopuce pouvant être obtenue au moyen d'un procédé, ainsi qu'un système automatisé de synthèse de polymères permettant de synthétiser, sur un support solide, une chaîne polymère par addition successive d'éléments constitutifs de polymère, et comprenant une tête d'impression à jet d'encre, une cellule de traitement, une unité de transport et un récipient contenant une solution renfermant un élément constitutif de polymère.
PCT/EP2005/005359 2004-05-17 2005-05-17 Procede de synthese de polymeres Ceased WO2005110593A2 (fr)

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US8361396B2 (en) 2010-02-22 2013-01-29 Oligoco, Inc. Automated polymer-synthesis system

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US8361396B2 (en) 2010-02-22 2013-01-29 Oligoco, Inc. Automated polymer-synthesis system

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