DE102004024364A1 - Process for the preparation of polymers - Google Patents

Abstract

The present invention relates to a process for the stepwise synthesis of polymers, in particular biopolymers, on a solid support surface, comprising the steps of providing a solid support surface with a binding site, applying a first microdroplet of a solution comprising a first polymer building block to the attachment site for attachment of the first Polymer building blocks on the binding site and applying the modified binding site of the previous step a second microdroplet of a solution comprising the same or a different polymer building block, wherein the solution contains a solvent having a viscosity of more than 7 mPaÈs. The present invention further relates to a biochip obtained by a method according to the invention and in another aspect to an automated polymer synthesis system for synthesizing a polymer chain on a solid support by sequential addition of polymer building blocks comprising an ink jet printhead, a treatment cell, transport means and a container comprising a solution and a polymer building block.

Description

The The present invention relates to a process for the stepwise synthesis of polymers on a solid support surface and the use of a such process for the production of biochips. Furthermore the present invention is a system for automated polymer synthesis for the synthesis of a polymer of a polymer chain on a solid carrier by sequential addition of polymer building block to specific locations on the surface a substrate.

nowadays the need for systems responsible for the synthesis of polymers decreases are suitable, in particular in the field of production of oligonucleotides or polypeptide polymers. Various synthetic methodologies were for developed this purpose. The two most important synthetic methodologies i) the stepwise photochemical synthesis and ii) the classical one chemical synthesis of such polymers. Both methodologies include the conversion of at least one functional group of building blocks of the desired Polymers by successively converting these building blocks to step by step Education of the desired Polymer.

The functional groups (usually terminal OH groups) the Building blocks are temporarily protected by intermediate protecting groups, the after treatment with suitable reagents. These Protecting groups are common either acid labile Groups such as DMT (dimethoxytrityl) and its derivatives or photolabile groups such as NPPOC (2-nitrophenyl-propyloxycarbonyl).

It Many efforts have been made to protect these groups vary. For example, U.S. Patent No. 6,222,030 describes the use of carbonate-protected Hydroxyl groups in a 3'-5 'oligonucleotide.

The Polymers such as oligonucleotides or polypeptides are for production so-called biochips or gene chips, the polymers on an array of one usually planar substrate are attached. The polymers are on the substrate either by direct in situ or after an ex situ synthesis applied. In situ synthesis on the substrate is often preferred as it is the easy construction of different or identical polymer chains directly on the corresponding substrate without elaborate intermediate steps after the ex situ synthesis of the polymers prior to attachment of the polymer chains on the substrate requires.

however have most of the known synthetic methodologies for the photochemical In situ synthesis, serious disadvantages due to a required sophisticated construction to precisely define the reaction site, where the polymer chain has to be built up. This includes i.a. the Use of a plurality of masks or a plurality of micromirrors for the exact selection of the reaction site.

As well has the chemical synthetic methodology using the classic "wet chemistry" serious disadvantages with respect to the solvents used in these reactions, which must meet a number of requirements, for example suitable for inkjet printheads are.

• a) ausreichende Löslichkeit der Mischung Base/Aktivator
• b) Unterstützen der Kopplungsreaktion zwischen beispielsweise einem Phosphoramiditderivat und dem Träger/Nukleosideinheit (Lösungsmitteleffekt, polar und aprotisch zur gleichen Zeit)
• c) einen hohen Siedepunkt zur Vermeidung einer Verflüchtigung des Tröpfchens auf der Oberfläche
• d) sie dürfen nicht zu teuer sein und müssen eine geringe Toxizität aufweisen
• e) sie sollen wasserfrei sein
• f) sie sollen chemisch kompatibel mit der Mischung Base/Aktivator sein
• g) und sollen durch einen Tintenstrahldruckkopf aufspritzbar sein, insbesondere im Hinblick auf die Dimension der Düsen des Druckkopfes.

These requirements are for example

• a) sufficient solubility of the mixture base / activator
• b) Assisting the coupling reaction between, for example, a phosphoramidite derivative and the carrier / nucleoside unit (solvent effect, polar and aprotic at the same time)
• c) a high boiling point to avoid volatilization of the droplet on the surface
• d) they must not be too expensive and have low toxicity
• e) they should be anhydrous
• f) they should be chemically compatible with the mixture base / activator
• g) and are to be sprayed by an ink jet print head, in particular with regard to the dimension of the nozzles of the print head.

typically, acetonitrile is due in particular in the oligonucleotide synthesis its high polarity used what the solution the corresponding reaction partner allows. Furthermore, one is typical reaction in the synthesis of oligonucleotides 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 such as acetonitrile.

however For example, acetonitrile has several disadvantages, such as its low boiling point (81 ° C), its incompatibility with many materials used in commercially available inkjet printheads etc. be used as adhesives, plastics. Because of its low Boiling point, the acetonitrile drops evaporate almost immediately after sprayed it out of the printhead were.

therefore It was an objective in the art to use acetonitrile with other solvents which better meet the above-mentioned requirements.

The U.S. Patent No. 6,028,129 Ethylene and propylene carbonate derivatives for use in automated Synthesis of two-dimensional oligonucleotide arrays. These connections are used to overcome the well-known disadvantages caused by to overcome the use of acetonitrile.

The Revelation of US Pat. No. 6,177,558 also relates to Replacement of acetonitrile especially with regard to the problem the evaporation. Accordingly, includes the reagent solution the on the surface sprayed on, for example on a biochip, further reactants, in at least solvent are contained with a relatively high boiling point, which preferably a polar, aprotic solvent which has a boiling point of at least about 140 ° C and is selected from the group consisting of dinitriles, glymes, diglyme and theirs Derivatives.

Farther beats U.S. Patent 6,419,883 discloses the use of microdroplets of a solution ago, which is a solvent which has a boiling point of 150 ° C or higher, a surface tension of 30 dyne / cm or higher and a viscosity of 0.015 g / cm / sec. The solvents, disclosed in this patent are N-methyl-2-pyrolidone, Propylene carbonate or γ-butyrolactone.

however are the proposed solvents from the prior art is not always with ink jet print heads with a variety of nozzles compatible, especially if the dimensions of the nozzles are small are and microdroplets can generate which do not have a uniform size. Therefore, it is impossible to maintain a precise reaction control and a reaction protocol in particular with regard to the concentration of the polymer building blocks.

Therefore was the underlying problem of the invention, the provision a solvent, generally in a stepwise synthesis process of polymers is applicable regardless of specific systems, such as the type of ink jet print head, etc. for performing the Process.

• a) zur Verfügungstellens einer festen Trägeroberfläche, die eine chemische funktionalisierte Bindungsstelle aufweist
• b) Auftragen eines ersten Mikrotröpfchens einer Lösung umfassend einen ersten Polymerbaustein auf dieser Bindungsstelle zur Befestigung des ersten Polymerbausteins an die Bindungsstelle
• c) des Auftragens auf die modifizierte Bindungsstelle aus Schritt b) eines zweiten Mikrotröpfchens einer Lösung umfassend den gleichen oder einen verschiedenen Polymerbaustein

wobei die Lösung ein Lösungsmittel mit einer Viskosität von mehr 7 mPa·Sekunde enthält.The problem underlying the invention is therefore solved by a process for the stepwise synthesis of polymers on a solid support surface, comprising the steps of:

• a) providing a solid support surface having a chemical functionalized binding site
• b) applying a first microdroplet of a solution comprising a first polymer building block on this binding site for attaching the first polymer building block to the binding site
• c) applying to the modified binding site from step b) a second microdroplet of a solution comprising the same or a different polymer building block

wherein the solution contains a solvent having a viscosity of more than 7 mPa.sec.

In a preferred embodiment has the solvent a viscosity in the range of 8 to 30, preferably in the range of 8 to 20 mPa · second. Less than 7 mPa · second are not preferred because in this case the solvent is not with a Inkjet printhead can be used, the nozzle diameter which are as small as indicated below. More than 30 mPa · second results in too viscous liquid that is not usual Inkjet printheads can be used. Furthermore, the reactivity on the Place where the solvent droplet applied will decrease due to the increasing viscosity of the solvent. It was surprisingly found that the selected Range of viscosity the use of the solvent in a method according to the invention, without the solvent evaporated to a greater extent. Therefore, the crusting of the nozzles, etc. the device used for spraying the solution containing the polymeric building blocks contains (also referred to as "synthons") or the so-called "blooming" of the droplet, once it's on the surface of the substrate is successfully avoided.

The Formation of crusts around the nozzles due to the evaporation of the solvent not only decreases the concentration of the reagent in the droplet and It also influences the result of the coupling reaction and the formation of unwanted By-products on the surface. Furthermore crusting results in chemical degradation of the material, that forms the crust that even contaminate the subsequent droplets can. Most of the solvents used in the prior art require a regular cleaning of the Device, in particular the nozzles of the inkjet printhead. The present invention also allows considerably extended Cleaning intervals, i. the cleaning will be two or even three times less often than in the prior art.

Farther it is preferred that the solvent a surface tension of more than 26 to 32 dyne / cm. This increased surface tension in combination with the selected one viscosity according to the invention of the solvent efficiently avoids the "blooming" of a droplet the solution, the on the surface was applied.

specific preferred examples of a solvent according to the invention include, but are not limited to on methyl oleate, 1-phenyl tetradecane, 1-cyclopentyl pentadecane, 1-cyclohexyl tridecane, Hexylnaphthol, dinonyl ether, dibutyl sebacate, 1-phenylpentadecane, 1-cyclopentylhexadecane, hexachlorocyclopentadiene, biscyanoethyl ether, 1-cyclohexyltetradecane, benzyl benzoate, N-butyl stearate, diethyl phthalate, 3-methyl, 2-pyrrolidone, 1-decylnaphthol, dimethyl phthalate, dibutyl phthalate, Diisooctyl phosphate, dioctyl phthalate, diisodecyl phthalate.

These Connections provide the required polarity, which in a first Aspect necessary is to solve most polymeric building blocks and in a second aspect to support the coupling reaction, which tend to be polar and aprotic solvent due to the reaction intermediates in the coupling reactions to prefer.

In a particularly advantageous embodiment of the invention the solvent or is a chemical compound that is a derivatized carboxyl group contains. It has surprisingly been found in particular polycarboxylic acid derivatives, i.e. Compounds the two or more derivatized carboxylic functions and which include the aforementioned physical properties have, are particularly suitable and a preferred solvent especially in the field of synthesis of biopolymers. The term "derivatized carboxylic acid or function "means that the acid proton (or in other words the acidic OH unit) the COOH groups substituted by a non-protic moiety has been. Examples of such derivatives include, but are not limited to Alkyl, aryl, aralkyl esters and the like.

The carboxylic functions are usually attached to a scaffold, for example, an aromatic, heteroaromatic, alkyl, Alkenyl or alkynyl skeleton or the like.

The most preferred chemical compounds have the formula (R ₂ OOC) _n -B - (COOR ₁ ) _m in which n and m are the same or different and represent an even number ≥ 1,
R ¹ , R ² are the same or different and represent a linear or branched C ₁ -C ₁₀ alkyl, alkenyl or alkynyl group, B is a linear or branched C ₁ -C ₁₀ alkyl, alkenyl, alkynyl, a substituted or unsubstituted aromatic, heteroaromatic, alicyclic or heterocyclic ring.

preferred Examples are preferably selected from the group consisting of alkyl diesters of oxalic, malonic, succinic, glutaric, adipic, suberic, azelaic, sebacic, maleic, fumaric, muconic, acetylenedicarboxylic, phthalic, isophthalic, terephthalic.

Most preferred are the alkyl esters of the polycarboxylic, most preferably the dicarboxylic acids wherein the alkyl group is preferably a branched linear C ₁ -C ₁₀ alkyl, preferably a C ₁ -C ₆ alkyl group, especially methyl, ethyl, propyl, butyl, pentyl is.

In another preferred embodiment not only one solvent is used, but the solution contains a second solvent (the first solvent is the carboxylic acid derivative). The use of a second solvent he heightens the overall yield of the reaction, especially for the individual NEN coupling steps. The use of a second solvent further allows fine tuning of the viscosity, surface tension, and polarity and aprotic properties of the solution. Furthermore, the second solvent increases the solubility of the phosphorus compound, preferably a phosphoramidite and / or the activator in the reaction mixture.

In In that sense it is for the success of the invention important that the amount of the second solvent in the solution is lower than the amount of the first solvent. If the second solvent in excess would be present would the Type of this second solvent prevalence. Preferably, therefore, the amount of the second solvent in the range of 1 to 30% by volume, preferably 5 to 30% by volume, on most preferably from 10 to 30% by volume with respect to the total volume the solution. Furthermore, it is preferable that the second solvent has a viscosity of less as 6 mPa · second having. Preferred solvents For example, glymes such as triglyme, diglyme, adiponitrile, acetonitrile and the same.

table Figure 1 shows in a flow of the second solvent on the coupling yield for each Reaction step in the stepwise synthesis of oligonucleotides.

If the second solvent is used alone (triglyme), no reaction takes place, however the solution consisting of a mixture of 80% by volume dibutyl sebacate and 20% Vol .-% triglyme yields high reaction yields.

The relative viscosity μ _rel = μ _{second solvent} / μ _{first solvent of} the solution is preferably lower than 0.95. It has surprisingly been found that the value of the relative viscosity of less than 0.95 leads to an increase in the reaction rate in the range of a factor of 2 to 10, preferably 3 to 8 and an increase in the yield (up to 15 to 20%). higher yield) of each of the consecutive coupling steps.

Of the The term "substrate" includes any suitable one Substrate such as glass, silica, silicone materials, but as well Polymer materials such as polyethylene, polypropylene, polystyrene and the like, which may or may not be functionalized and the corresponding ones Composite materials.

In a particularly preferred embodiment The invention relates to the substrate with an array of metallic electrodes Mistake. Before the implementation of step a) is an electrically conductive polymer to the metallic Electrodes applied, wherein the electrically conductive polymer the same or different is for every electrode of the array.

It is particularly preferred that the electrically conductive polymer in situ is synthesized on the metallic electrode. The synthesis of the electrically conductive polymer is in a particularly preferred embodiment an electrochemical synthesis. In one embodiment of the invention polymeric building blocks therefore also in addition to biomolecular building blocks such as nucleotides, oligonucleotides, amino acids, oligopeptides, Carbohydrates also preferably electropolymerizable monomers or macromers (oligomers), polymers, etc. It is further preferred that the above mentioned biomolecules already chemically to the electropolymerisierbaren monomers or oligomers or even polymers are bound before being applied to the surface become. The methods of electrochemical polymerization, usually for the Production of ECPs (electrically conductive polymers) can be used like a polymerization on the set current (chronopotentiometry) or at a set potential (chronoamperometry) are also in the process according to the invention applicable.

The quality the deposition of the electrically conductive polymer can by the Choice of experimental conditions to be controlled: the concentration of the monomer, for example pyrrole, THF and the like, of the ratio of oligonucleotide monomer / monomers, the type of solvent, the electrochemical method used (cyclic voltammetry, Chronoamperometry or chronopotentiometry). The obtained copolymer can therefore different qualities the porosity and accessibility dependent on of desired have subsequent use and if the amount of bound Oligonucleotide can be modified.

By measuring the while In response to the reaction current, it makes the electrode effective possible, to follow the progress of the polymerization reaction (for example, via the Thickness of the polymer formed) or the progress of the following Reaction carried out on the copolymer.

According to one preferred embodiment the method according to the invention in one or other of its variants, it still includes the renewal an oligonucleotide chain via several successive steps, each of these steps being taken from the Binding of one or more nucleotides or oligonucleotides.

The renewal of the oligonucleotide is deposited on the surface of the support by assembling the protected Monomers performed, starting with at least one nucleotide or oligonucleotide of the surface of the electrically conductive Polymers is bound. The standard methods for chemical synthesis of nucleic acids can to carry out this embodiment be used.

Around a good adhesion of further polymer building blocks on the electrically conductive polymer to enable it is preferred that the electrically conductive polymer is a chemical functionality has, which allows the attachment of a polymer block. The term "chemical Functionality "refers to a chemical reactive group (masked or unmasked) such as a Amino, acetyl, carboxyl, hydroxyl, etc.

Preferably 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 production of so-called bio or gene chips considerably. In a further preferred embodiment The invention provides a redox couple such as organometallic complexes such as ferrocene, nickelocene, ruthenocene in the polymer before, during or incorporated after the electrochemical synthesis of the polymer. The introduction a redox couple enlarges the electrically conductive properties of the electrically conductive polymer.

The The problem underlying the invention is further by a biochip solved, which comprises a substrate, an electrode, wherein the electrode electrically conductive polymer is attached and to the conductive polymer a biomolecule is bound, the selected is from nucleic acids and their derivatives, peptides, proteins and carbohydrates, obtainable by a method according to the invention.

• a) einen Tintenstrahldruckkopf umfassend eine Mehrzahl von Düsen zum Aufsprühen eines Mikrotröpfchens auf einen ausgewählten Platz auf dem Träger
• b) eine Behandlungseinheit zur Behandlung des Trägers auf dem ein Mikrotröpfchen abgelagert wurde mit einem Reagens
• c) Transportmittel zu Bewegung des Trägers benachbart zum Druckkopf und/oder zur Behandlungseinheit
• d) ein Gefäß umfassend eine Lösung enthaltend einen Polymerbaustein

und wobei die Lösung enthaltend einen Polymerbaustein ein Lösungsmittel umfasst, das eine Viskosität von mehr als 7 mPa·Sekunde aufweist.In yet another aspect, the problem of the invention is solved by an automated polymer synthesis system for synthesizing a polymer chain on a solid support by sequential addition of polymer building blocks, comprising the following elements:

• a) an ink jet printhead comprising a plurality of nozzles for spraying a microdroplet to a selected location on the support
• b) a treatment unit for treating the carrier on which a microdroplet has been deposited with a reagent
• c) transport means for movement of the carrier adjacent to the print head and / or the treatment unit
• d) a vessel comprising a solution containing a polymer building block

and wherein the solution comprising a polymer building block comprises a solvent having a viscosity of more than 7 mPa · s.

In a preferred embodiment is every single nozzle individually controllable, what with the construction of a plurality of different polymer chains or a pre-defined number containing different building blocks allowed before the reaction started becomes.

Preferably is the distance of the nozzles in the range of 0.02 to 0.005 μm, more preferably in the range of 0.015 to 0.1 microns. This close arrangement facilitates it's the microdroplets in complex structures on the surface of the biochip.

Of the Diameter of the nozzles is preferably in the range of 30 to 70 microns, with small microdroplets a uniform size and volume to create. The volume of a microdroplet is preferably in the Range of 60 to 100 picoliters to control the amount of applied Polymer block.

In another preferred embodiment, the ink jet head includes a tank in communication with each nozzle. This simplifies the construction of the system, because in the prior art each nozzle with a Reservoir had to be connected, and thus required more complicated means of control.

definitions and abbreviations

Polymer: The term polymer building block (alternatively also called Synton) denotes a chemical entity within the final polymer is included. The chemical entity can therefore be functional groups prior to incorporation into the final polymer. Non-limiting examples suitable polymeric building blocks according to the invention are substituted and unsubstituted phosphoramidites, mono-, oligo and polynucleotides, Amino acids, Peptides, sugars (furanoses, riboses, etc.), biotin, avidin, streptavidin, antibodies and like.

Microdroplets: a microdroplet is a separate and discrete unit, preferably with one volume of about 100 to 200 picoliters, most preferably between 5 and 70 picoliters.

It It is understood that the term "solution" as used here becomes, the solvent designated per se and the solved Product or more dissolved Products.

The microdroplets form when on the surface react, so-called spots or microdots. The arrays of the obtained Polymers according to the present invention Invention are arranged in these microdots or spots, the separate and discrete units. The diameter of each microdot can be bigger than 1000 μm but is typically in the range of 5 microns to 8 microns, preferably of 10 μm until about 500 μm and most preferably from 20 to 200 microns.

Of the Distance between the individual microdots is typically about 1 μm to about 500 μm, preferably of 20 μm until about 400 μm. In general, the distance between the microdots should preferably be be in the area of the appropriate spot of the microdroplets to use to avoid from neighboring spots.

The physical separation of the microspots is determined by the reaction of the remaining functional groups with a non-removable protecting group, preferably comprising phosphorus. These areas form one non-reactive protective surface, which is chemically inert.

Of the Term "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. The term "nucleoside" as used herein includes both deoxyribonucleosides and ribonucleosides. Of the The term "oligonucleotide" refers to an oligonucleotide, the deoxyribonucleotide or ribonucleotide units having.

suitable Nucleotides suitable for the synthesis of oligonucleotides according to the present invention Invention are those nucleotides containing active phosphorus Groups such as phosphorus triester, H-phosphonate and phosphoramidite groups.

Of the The term "activator" usually means a catalyst which in the case of oligonucleotide synthesis a Catalyst is the reaction between the 3'-phosphoramidite group of a nucleoside and the hydroxyl group of the next Nucleosides or nucleotides favored. This may be 5-methylthiotetrazole, tetrazole or 5-ethylthiotetrazole, DCI or pyridinium chloride.

Of the Term "alkyl" as used herein is, refers to any saturated straight chain, branched or cyclic hydrocarbon group from 1 to 1 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. The term alkyl also includes cycloalkyl groups such as cyclopentyl, Cyclohexyl, cycloheptyl, etc.

Of the Term "lower alkyl" refers to a Alkyl group of 1 to 4 carbon atoms and includes methyl, Ethyl, n -propyl, isopropyl, n -butyl, isobutyl, t -butyl.

The term "heterocyclic" refers to any five-membered or six-membered monocyclic structure or an eight-membered to eleven-membered bicyclic structure which is either unsaturated or saturated. The heterocycles comprise at least one heteroatom selected from the group consisting of nitrogen, oxygen, sulfur, phosphorus, arsenic and the like. The terms "nitrogen heteroatoms" and "sulfur heteroatoms" as well as "phosphorus heteroatoms" include any oxidized form of nitrogen, sulfur and phosphorus as well as the quaternized form of any basic nitrogen. Examples of "heterocyclic compounds" include piperidinyl, morpholinyl and pyrrolidinyl.

Of the Term "oligonucleotide" as used herein is called polydeoxynucleotides (containing 2-deoxy-D-ribose), Polyribonucleotides (containing D-ribose) or any other type of one Polynucleotide, which is an n-glycoside of a purine of pyridimine base or other polymers containing non-nucleotide scaffolds, provided that the polymeric nucleobases contain a configuration, which allows the base pairing and base stacking, as is the case with DNA and RNA is.

With the term "protecting group" as used herein is called a species that prevents a segment of a molecule a specific chemical reaction is received, but that of the molecule after completion this reaction can be removed. This is in contrast to a "capping group" that is constantly on a segment or a functional group of a molecule binds or to functional groups on the surface of the substrate for prevention from any further transformation of this segment.

The Method of the present invention may also be used be synthesized peptides by standard solid-phase peptide synthesis methodologies.

Typically, solid phase peptide synthesis is performed in C to N direction. Therefore, linker groups are required such that cleavage at the end of the synthetic protocol produces a C-acid or amide. In preferred embodiments, a linker containing an active of the carboxyl group is attached to amino groups, as can bind to the activated surface of a carrier according to the invention. Any of the common "temporary" protecting groups commonly used in polypeptide synthesis chemistry are suitable for use with the present invention. Non-limiting examples of these are, for example, BOC (t-butoxycarbonyl) and FMOC (N ^α9 -fluorenylmethoxycarbonyl) groups.

Other suitable amino protecting groups include, but are not limited to 2- (4-biphenyl) propyl [2] oxycarbonyl (BPOC), 1-1-1-adamantyl-1-methylethoxycarbonyl (ADPOC) and the same. Representative Activators or so-called in situ coupling reagents suitable but do not include for use in the present invention limited on N, N'-dicyclohexylcarbodiimide (DCC) and the like. Their use is in conjunction with the Use of other accelerators or additives such as 1-hydroxybenzotriazole (HoBt), benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) and the like are preferable.

The inventive method is preferably used to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to produce polymer species (so-called oligonucleotides), but comprising any of the known chemistries for solid phase DNA or RNA synthesis Phosphite triester, phosphoramidite synthesis and H-phosphonate synthesis can also be used.

in principle For example, the process of the present invention is useful for carrying out each iterative nucleic acid synthesis technique. In a preferred embodiment The invention provides oligonucleotides by the phosphoramidite method produced. For the phosphoramidite synthesis according to the invention sometimes the reactive sites on the surface of the substrate become with another spacer molecule functionalized according to known from the prior art compounds. The introduction the spacer can take place before the beginning of the production of the polymer chain or, for example, after step a) of the method according to the invention. The spacer groups can also to previously selected ones Make the reaction carrier be applied.

typically, have monomeric nucleotide and Nukleosidpolymerbausteine (or as well Called syntons) temporary Protecting groups at corresponding nucleobases or 2'-O-positions.

Solid phase nucleic acid synthesis techniques use the so-called temporary and permanent protecting groups in a manner analogous to solid phase peptide synthesis. Base-labile protecting groups are used to protect exocyclic amino groups of heterocyclic nucleobases during synthesis. This type of protection is usually achieved by acylation with acylating reagents such as benzyl chloride and isobutyryl chloride. Acid labile protecting groups are used to protect the nucleotide 5'-hydroxyl during synthesis. Representative hydroxyl protecting groups are known to one of ordinary skill in the art. These include but are not limited to dimethoxytrityl, monomethoxytrityl, trityl and 9-phe nylxanthene (pixyl) groups. Dimethoxytrityl (DNP) protecting groups are widely used because of the high acid lability which enables efficient removal even by very dilute acids.

The first step of the iterative chain extension cycle according to the phosphoramide technique is the removal of the 5'-O-protecting group (deprotection) of the original monomer by immersing the reaction carrier in a solution of the deprotecting agent. This is followed by the addition of a rinse. Suitable reagents for deprotection include Lewis acids such as ZnBa ₂ , AlCl ₃ , BF ₃ and TiCl ₄ in various solvents such as dichloromethane, nitromethane, tetrahydrofuran and mixed solvents such as nitromethane and lower alkyl alcohols such as methanol, ethanol or mixtures thereof. Protic acids, alone or in combination, such as acetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid and toluenesulfonic acid can also be used. The chains are extended by addition and reaction of activated 5'-O-protected monomeric syntons. In the phosphoramidite technique, a 5'-DMTA-deoxynucleoside-3'-O- (N, N-diisopropylamino) -β-cyanoethyl phosphite is applied to the reaction support. Phosphoramidites of numerous nucleosides are commercially available.

One mild organic catalyst or activator, typically tetrazole, Ethylthiotetrazole of Metylthiotetrazole is on the reaction medium with applied to the phosphoramidite. The coupling reaction is through the addition of a rinse solvent, typically followed by dry acetonitrile.

To the rinse a capping agent is added, for example by immersion of the substrate in a solution of the capping agent on the preselected positions of the reaction carrier for Capping of free hydroxyl species due to an incomplete reaction the phosphite monomers remained (capping 1). In the context of the present Invention is found to be a fundamental difference between the capping of unreacted reactive groups on the surface of the solid support exists (also referred to as "surface capping" or capping 1) and the nucleotide / nucleoside itself (capping 2). Both reactions are referred to as "capping", however they should not be confused with each other. The capping agent for the "second caps," that typically a solution an acid anhydride also acts to reverse the unwanted phosphitylation of Guanin O-6 positions.

The Oxidation of the resulting Phosphittriesters to the corresponding Phosphate triesters can be made by adding one from the prior art known oxidizing agent, which is suitable to be achieved, such as about a solution of alkaline iodine in water.

a One of ordinary skill in the art will appreciate that the synthesis of the oligonucleotides either in 3'-5 'or 5'-3' direction can.

The inventive method can be used in the synthesis of oligonucleotides, the naturally occurring nucleobases Avenin (A), Thymine (T), Guanine (G), Cytosine (C) and Uracil (U) have, as well not natural occurring nucleobases. Non-naturally occurring nucleobases are molecular entities known in the art are that they have the function of naturally occurring nucleobases imitate the components of nucleic acids in their biological role.

oligonucleotides Species have a big one Number of modifications for Nucleobases, sugars or intersugar linkages and may be prepared according to the methods of the invention, generally for synthesis any oligomers synthesizable by solid-phase techniques are, for example, also on polycarbohydrates.

For example can the method according to the present Invention in the synthesis of S-phosphorodithioates, phosphorothioates, etc. are also used.

The produced according to the invention Polymers can composed of more than one kind of a monomeric subunit be (for example, amino acids, Peptide nucleic acids, Nucleotides, sugars (carbohydrates) etc.) and can be more than one type of Between subunit link exhibit. Illustrative polymers made according to the methods of the invention include peptides, peptoids (n-alkylated glycines), Alpha polyesters, polythioamides, n-hydroxyamino acids, beta esters, polysulfonamides, n-alkylate polysulfonamides, polyureas, peptide nucleic acids, nucleotides, Polysaccharides, polycarbonates, oligonucleotides, oligonucleosides and the like and chimera molecules the one or more of these polymers together as a single macromolecule exhibit.

libraries monomeric species can also be prepared by the process of the invention. These include benzodiazepine libraries and others analog libraries, but limited to anti-hypertensives, Anti-ulcer agents, Fungicides, antibiotics, anti-inflammatories, Etc.

a One of ordinary skill in the art will appreciate that the scope of the invention not just the ones mentioned above Features includes, but also combinations thereof and that of Scope of the invention also with respect to each individual feature to the invention as described above comprises.

Claims (26)

A process for the stepwise synthesis of polymers on a solid support surface comprising the steps of: a) providing a solid support surface having a chemical functionalized binding site, b) applying a first microdroplet of a solution comprising a first polymer building block on said attachment site for attachment of the first polymer building block at the binding site c) of applying the modified binding site of step b) a second microdroplet of a solution comprising the same or a different polymer building block, characterized in that the solution contains a solvent having a viscosity of more than 7 mPa · s. Method according to claim 1, characterized in that that the solvent a viscosity from 8 to 30, preferably 8 to 20 mPa · second. Method according to claim 2, characterized in that that the solvent a surface tension of more than 26 to 32 dyne / cm. Method according to one of Claims 1, 2, 3, characterized that the solvent a carboxylic acid derivative is. Method according to claim 4, characterized in that that the carboxylic acid a derivative of a polycarboxylic acid is. Method according to claim 5, characterized in that that the polycarboxylic acid derivative selected is from the group consisting of the alkyl esters of sebacic acid and Phthalic acid. Method according to one of the preceding claims, characterized marked that the solution a second solvent contains. Method according to claim 7, characterized in that that the amount of the second solvent in the solution is lower than the amount of the first solvent. Method according to claim 8, characterized in that that the amount of the second solvent in the range of 1 to 30% by volume, preferably 5 to 30% by volume, on most preferably 10 to 30% by volume with respect to the total volume the solution is. Method according to claim 8 or 9, characterized that the second solvent a viscosity of less than 6 mPa · second having. A method according to claim 10, characterized in that the value of the relative viscosity μ _rel = μ second solvent / μ first solvent of the solution is lower than 0.95. Method according to one of the preceding claims, characterized characterized in that the substrate with an array of metallic electrodes is provided. Method according to claim 12, characterized in that that before performing of step a) an electrically conductive polymer on the metallic Electrodes is attached. Method according to claim 13, characterized in that that the electrically conductive Polymer same or different for each electrode of the array is. Method according to claim 14, characterized in that that the electrically conductive Polymer is synthesized in situ on the metallic electrode. Method according to claim 15, characterized in that that the synthesis of the electrically conductive polymer is an electrochemical Synthesis is. Method according to one of claims 14 to 16, characterized that the electrically conductive polymer a chemical functionality has, which allows the attachment of a polymer block. Method according to one of the preceding claims, characterized in that the polymer building blocks are selected from the group consisting from nucleosides, nucleotides, oligonucleotides, amino acids, oligopeptides, Carbohydrates, dimers and trimers thereof. Method according to claim 18, characterized that a redox couple in the polymer before, during or after the electrochemical mediated synthesis introduced becomes. Biochip comprising a substrate, an electrode, and attaches to the electrode an electrically conductive polymer and to the conductive one Polymer bound a biomolecule selected from a nucleic acid and their derivatives, peptides, proteins and carbohydrates obtainable by a method according to a of the preceding claims. An automated polymer synthesis system for synthesis a polymer chain on a solid support by sequential addition of polymer building blocks comprising the following elements: a) an ink jet print head comprising a plurality of nozzles for spray on a microdroplet on a selected one Location of the carrier, b) a treatment unit for treating the carrier on which a microdroplet is deposited was with an active ingredient, c) means of transport for movement of the carrier adjacent to the print head and / or the treatment unit, d) a container comprising a solution containing a polymer building block characterized in that the solution containing a polymer building block a solvent, with a viscosity of more as 7 mPa · second includes. System according to claim 21, characterized that every nozzle is individually controllable. System according to claim 22, characterized in that that the distance of the nozzles in the range of 0.020 to 0.005 μm, preferably in the range of 0.015 to 0.010 microns. System according to claim 23, characterized that the diameter of the nozzles in the range of 30 to 70 microns. System according to one of Claims 21 to 24, characterized the printhead includes a tank in communication with each nozzle. System according to claim 24 or 25, characterized that the volume of a microdroplet in the Range of 60 to 100 picoliters.