DE19543232A1 - Production of matrix-bound miniaturised combinatorial polymer and oligomer library - Google Patents

Abstract

Production of a matrix-bound miniaturised combinatorial polymer and oligomer library of nucleic acids, peptides, sugars and other chemical compounds, which are prepared by multistep synthesis, uses a chemically inert stamp. The active surface of the stamp is micro-structured in a manner determined by the selected synthesis algorithm to allow selective access to defined loci on a substrate. The substrate is an insert solid support on whose surface a monolayer of a linker with a terminal protected functional group is covalently bound. The process comprises: (a) dipping the stamp in a solution containing a reagent capable of deprotecting the terminal functional group, (b) contacting the stamp with the substrate to deprotect the functional groups at the contact sites, (c) washing the substrate with an inert solvent, (d) coating the substrate with a first activated chemical building block so that chain extension takes place on the deprotected functional groups, and (e) repeating steps (a)-(d) using stamps whose microstructure patterns are adapted to access predetermined loci on the substrate according to the synthesis algorithm.

Description

Molecular interactions between polymers and ligands play an important role in biological processes. Small changes in the physico-chemical properties of polymers and / or ligands can cause significant changes in the interaction between the polymer and ligand. Ideally, all sequence variations of the polymer should be available to investigate the binding properties of a ligand (library). The interaction with the ligands should be investigated in parallel. This suggests placing all polymer variations on one surface so that they can interact with ligands in one step. This would result in identical incubation conditions for all molecules and a direct comparison would be possible. If specific interactions are known, the influence of other molecules (such as active substances) on specific interactions can be determined by joint incubation. These investigations presuppose the presence of a technology that allows small amounts of polymers to be applied specifically to defined locations on a solid support. Since the length of the polymer L is included as a power for a given number of monomers M, the following applies to the total number G of all variations of a polymer: G = M ^L. This makes it clear that miniaturization of such a matrix is also necessary. This is also desirable given the price of the reagents.

There are various efforts to create polymer libraries. A successful Employees of the companies Affymax and Affymetrix succeeded in realizing this project (Combinatorial Chemistry - applications of light directed chemical synthesis; Jacobs, Jeffrey W .; Fodor, Stephen P.A .; Trends in Biotechnology, 1994, 12 (1), 19-26). Here is a novel synthetic chemistry with light-sensitive protective groups used. By means of suitable Masks, as are common in photolithography, are defined on molecules Areas of a surface activated and so gradually polymers with defined Positions built. So it has already succeeded in combining all variations Build oligonucleotidoctamers, which means that 65536 molecular species were synthesized (Light generated oligonucleotide arrays for rapid DNA sequence analysis; Pease, Ann Cviani; Solas, Dennis; Sullivan, Edvard J .; Cronin, Maureen, T .; Holm Christopher P .; Fodor, Stephen P.A .; Proc. Natl. Acad. Sci. U.S.A. (1994), 91 (11), 5022-6). It is also by means of the same process and light-activated amino groups succeeded in creating peptide libraries (light directed combinatorial peptide synthesis, Gruber, S.M .; Yu-Pang, P .; Fodor, Stephen P.A .; Proc. At the. Pep. Symp., 12th (1992), Meeting Date 1991, 489-91).

An alternative method for the preparation of oligonucleotide libraries was by Southern and Maskos (Parallel synthesis and analysis of large numbers of related chemical compounds: applications to oligonucleotides; Southern, Edwin M .; Maskos, Uwe; J. Biotechnology (1994), 35 (2-3), 217-27) and successfully applied. Here, the conventional chemistry known from solid phase synthesis  applied. Capillaries were formed on a support and these were filled with reagents. Depending on the loading of the capillaries, it was possible to add a specific nucleotide locally.

Both methods have decisive disadvantages. Affymax's procedure requires a special protecting group chemistry that is not commonly used and consequently is expensive and immature. This leads to limitations on the length of the synthesizing polymers. Furthermore, photolithographic synthesis techniques used, which are standard in microstructure technology today. This makes working in the A clean room is required to achieve high spatial resolutions. Nevertheless there are physical limits; even under ideal conditions are resolutions not to be expected from <200 nm (λ / 2). By using a photolithographic Equipment in each synthesis step increases the synthesis effort. Thus, with long polymers are not synthesized in this process; the edges are only in Framework of the photolithographic technique sharp.

The method of Southern and Maskos offers the possibility of use conventional synthesis reagents, but is uneconomical here, the capillaries must be filled to the full length with the reagent. Also when using the described Synthesis algorithm the synthesis of all variations of a polymer length impossible. The The method can be miniaturized to the same extent as that described above. However the microstructuring of the carrier itself is required.

Thus, there is a need for an alternative method of making one miniaturized polymer and oligomer bank, in which the disadvantages of the known Procedures to be avoided. The task is therefore to provide a corresponding alternative procedure.

The invention relates to a method for producing a matrix-bound miniaturized combinatorial polymer and oligomer library of nucleic acids, Peptides, sugars and other chemical compounds by multi-step synthesis be prepared, and combinations thereof, using a chemically inert Stamp, whose active surface one by the chosen synthesis algorithm has predetermined microstructuring, which results in a selective response defined loci is made possible on a substrate, and a substrate in which it is is a solid inert carrier, on the surface of which a monolayer of a Linker is covalently bound to a terminal protected functional group, characterized in that

• a. said stamp in a solution containing a for the release of the terminal Protective reagent suitable for protected functional groups is immersed,
• b. the stamp wetted with the deprotection reagent with said substrate in Is brought in contact, whereby the terminal functional at the contact surfaces Groups are released
• c. after washing with an inert solvent, the substrate was activated with a first one chemical building block is overlaid, resulting in the released terminal functional groups a chain extension by said chemical building block he follows,
• d. the synthesis sequence of steps a-c is repeated any number of times until the according Synthesis algorithm is produced desired chain length, with each Synthesis step defined loci on said substrate by using stamps can be addressed with predetermined patterns.

The combinatorial polymer library is on a solid; inert to the reagents used in the synthesis steps (matrix), which itself does not have to be microstructured. Both flat surfaces ( Fig. 1, step 1.1) and micro-structured ones can be used. These conditions are met by each of the wafers used in microstructuring, e.g. B. silicon / silicon oxide, but also of appropriately polished quartz glass, for. B. Suprasil. The surface of the solid support is connected via a linker and, if necessary, additionally to the first chemical building block ( Fig. 1, steps 1.2 and 1.3). In the following, a solid support prepared in this way is referred to as a substrate.

A linker is a suitable chemical connection between the carrier surface and the first chemical building block. The linker molecule is freely selectable and adjusts itself prospective application. The linker is a polyfunctional, in particular bifunctional molecule that forms a covalent bond with separate functional groups forms with the carrier and with the first chemical building block. The substrate contains the Linker with a terminal protected functional group. When choosing the suitable linker for the production of an oligomer or polymer library is too note that the linkers are stable under the chosen synthesis conditions. Such Linkers are used in the case of oligonucleotide synthesis e.g. B. from 3-glycidoxypropyl trimethoxysilane or 3-aminopropyl-triethoxysilane (Chemically Bonded Stationary Phases for Aqueous High Performance Exclusion Chromatography; Engelhardt, H. and Mathes, D .; Journal of Chromatography, 142 (1977) 311-320).

The linker can also already contain the first chemical building block or is linked to the first chemical building block in a first synthesis step. A chemical building block is basically a monomer protected by its functional groups and suitable for the construction of oligomers and polymers (e.g. CE nucleotide phosphoramidites, FMoc amino acids, etc.). This building block is activated by deprotection (release) of the functional group intended for chain extension and can thus interact with suitable groups (e.g. a second chemical building block). A further polymerization (chain extension) step is thus opened when the functional group of the first building block necessary for this is deprotected and can thus react with a previously activated functional group of a second chemical building block. This is done in the method according to the invention with the aid of a topologically defined (microstructured) stamp which is inert to the reagents used in the synthesis steps, so that deprotection is only carried out in a positionally correct manner on the defined (predetermined) regions of the substrate ( FIG. 1, step 1.4). Such stamping techniques are known per se (Patterning self assembled monolayers: Applications in Materials Science; Amit Kumar, Hans A. Biebuyck, George M. Whitesides; Langmuir, 1994 (10), 1498-1511). After deprotection, there is a deprotected area on the surface of the substrate ( Fig. 1, step 1.5) that reacts with an activated chemical building block ( Fig. 1, step 1.6).

In principle, this method enables the synthesis of all polymers and oligomers, as long as they are stable under the chosen conditions (e.g. nucleic acids, peptides, Polysaccharides and other chemical compounds; through multi-step synthesis are produced, e.g. B. polyterpenes, etc.). For the synthesis Production of oligonucleotides, polynucleotides, peptides, polysaccharides, etc. suitable monomers are used as chemical building blocks. The synthesis also continues  can be carried out by copolymers from such chemical building blocks; so z. B. with glycopeptides provided with a nucleic acid tag. The maximum length of the synthesized chains depends on the step efficiency (yield) of the chosen ones Synthetic chemistry.

The arrangement of the polymers on the carrier surface results from the chosen one Synthesis algorithm, and this is basically freely selectable. The polymers have one length determined by the stamp algorithm and are clear on the surface localizable. The methods for the synthesis of the polymers mentioned are state of the art.

The procedure for producing the polymer library is as follows in detail:
The surface of the carrier is cleaned and completely provided with a suitable linker. This linker bears a removable protective group on the terminal functional group, or in an additional step the entire surface is occupied by a first chemical building block with a terminal protected functional group; in both cases the surface is protected and can be selectively activated locally. The stamps are made of a chemically inert material that adsorbs organic solvents, e.g. B. poly-di-methyl-siloxane (PDMS), whereby a sufficient amount of reagents for deprotection of specific terminal functional groups are applied in the correct position on the substrate and so the coupling reactions take place at these positions on the substrate.

Stamps whose active surface has a pattern predetermined by the selected synthesis algorithm are produced as follows. A negative of the stamps is microstructured and cleaned in a predetermined manner in the photoresist layer of coated wafers ( Fig. 2, step 2.1). The microstructuring enables the desired synthesis algorithm to be realized. This also makes it possible to selectively address defined areas on a surface. Di-methyl-siloxane mixed with a suitable hardener is added to the negative and covered with a glass plate for better handling and protection against contamination. The hardened PDMS is covalently bound to the cover slip ( Fig. 2, step 2.2), but can be easily removed from the silicon and the remaining photoresist on the stamp negative ( Fig. 2, step 2.3). This process is repeated accordingly until all stamp patterns necessary for the synthesis have been produced. The number of microstructured stamps required for the production of a specific polymer library results from the chosen synthesis algorithm.

To activate a specific area on the substrate; the specifically microstructured stamp provided for this purpose is coated with the deprotection reagent ( Fig. 2, step 2.4) and brought into contact with the substrate, ie pressed onto the substrate. The areas exposed to the stamp (contact points) on the substrate (carrier with linker and terminally protected functional group or correspondingly protected first chemical building block, which thus becomes part of the linker) are thereby deprotected ( Fig. 2, steps 2.5 and 1.5). After removing the stamp and washing thoroughly, the surface of the substrate becomes the first chemical building block; whose functional group intended for the reaction was previously activated. This extends the substrate by one monomer at the activated sites. The surface is then completely protected again ( Fig. 1, step 1.6). A washing step with an inert solvent is interposed between all synthesis steps. The steps mentioned are repeated in the same order until the desired chain length is reached.

The number of locations on the surface ("loci") that have to be addressed separately is equal to the number of desired variations of a polymer chain. If all variations of a polymer of length L are to be produced and the number M of chemical building blocks is used, then the number of separate loci Z results in:
Z = M ^L. However, the number of stamps S required for this is only: S = M × L ( Fig. 3). The dimensions of the polymer library produced in this way can be kept extremely small, since even negatives produced by electron lithography can be copied precisely. The limit for the miniaturization of the polymer library is thus identical to the limit for microsystem technology (Microcontact Printing of Self-Assembled Monolayers: A flexible New Technique for Microfabrication; James L. Wilbur, Amit Kumar, Enoch Kirn; George M. Whitesides; Journal of the American Chemical Society, 1992 (114), 9188-89). The maximum length of the polymers depends only on the synthetic chemistry chosen; the most promising according to the literature or own experience can be used. The process is economical because the surface can be kept small with a large variance of the polymers.

It is possible to capture the protective groups that are split off during deprotection. Your Quantification is a direct measure of step efficiency. It is also conceivable that terminal protective group on the chain and thus a marker for To have quantity determination available. Another possibility is the Terminal labeling of the synthesis products with a fluorescent group.

The fields of application of such libraries which can be produced according to the invention are extremely diverse. Examples of such fields of application are molecular Screening for interactions between different molecular species (see above) and the Sequencing of nucleic acids by hybridization.

Embodiment Preparation of an oligonucleotide library with all variations a trimer with the four naturally occurring bases

The total number of oligonucleotides to be synthesized is 64. Each of the 64 Oligonucleotides are three bases long.

The linker is manufactured as follows: a quartz glass is cleaned in an ultrasonic bath and in a 25% solution of 3-glycidoxypropyltrimethoxysilane in xylene containing one catalytic amount of N-ethyldiisopropylamine immersed; the vessel becomes tight completed and kept at 80 ° C for 8 h. Then it is in xylene and ethylene glycol washed. The glass is placed in ethylene glycol and the pH of the solution is determined Addition of sulfuric acid set to 5; the reaction lasts 8 h at 80 ° C. After At the end of the reaction, the glass is washed thoroughly with methanol and diethyl ether and dried in a drying oven (Chemically Bonded Stationary Phases for Aqueous High Performance Exclusion Chromatography, Engelhardt, H. and Mathes, D., Journal of Chromatography, 142 (1977) 311-320). So there is one on the glass Monolayer of hydroxyethyloxyethyloxypropyl siloxane, which readily with tetrazolyl Nucleotide phosphoramidites (activated chemical building block) reacts. All of the following Manufacturing steps are carried out at room temperature and under dry protective gas  carried out because the reagents used are sensitive to moisture (e.g. in a Laboratory tent (Atmosbag, which is filled with argon). Any FOD (almost oligonucleotide deprotection) nucleotide phosphoramidite (e.g. FOD-dG-CE- Phosphoramidite), whose 5′OH group is protected with dimethoxytrityl (DMT), becomes equal parts dissolved in acetonitrile and tetrazole, so that a 0.1 M solution of a activated nucleotide phosphoramidite is present. This solution is based on the previous one Given acetonitrile washed glass for 30 minutes. After washing with acetonitrile the phosphite using a 0.02 M iodine solution (in tetrahydrofuran; pyridine and water) Oxidized to phosphate and then washed thoroughly with acetonitrile. So that's one Di-methoxy-trityl-protected surface on the glass in front.

The stamps are produced and applied according to the algorithm shown in Figs. 2 and 3. Each synthesis cycle contains a stamp step and consists of a uniform procedure: 1.) washing with dichloromethane (0.5 min), 2.) deprotection of the 5′-hydroxyl group (detritylation, 5 min) with 2% trichloroacetic acid (TCA) in dichloromethane; done with the stamp, 3.) repeated washing with acetonitrile (2.5 min each), 4.) condensation (addition of tetrazolyl nucleotide phosphoramidite, 5 min), coupling only takes place at previously deprotected points; 5.) Washing with acetonitrile (2.5 min), 6.) Oxidation with 0.02 M iodine in tetrahydrofuran, pyridine and water (5 min), 7.) Washing with acetonitrile (2 min). This completes a synthesis cycle.

The solutions and reagents used are standard in the FOD phosphoramidite Nucleotide synthesis (Models 392-394 DNA / RNA Synthesizers, Users Manual, ABI, 1992). The washing solutions are continuously rinsed over the surface, the reagents remain on the surface for the specified time.

After the first four stamping steps there are four oligonucleotides with length 1 synthesized. After four further stamping steps, 16 oligonucleotides are 2 in length synthesized, and after a further four stamping steps there are 64 oligonucleotides of length 3 synthesized. The terminal protecting groups are split off with 2% TCA, existing protective groups of the exocyclic amino groups of the bases (Dimethylformamide group for adenosine and guanosine and isobutyryl group for Cytidine, thymidine has no exocyclic amino group) and on the phosphate (β-cyanoethyl group) are split off with concentrated ammonia solution (1 h at 55 ° C or 8 h at room temperature). The linker used is stable in all steps, so that after complete deprotection the oligonucleotides remain on the support. In order to is the oligonucleotide trimer library made. The yield is through Absorption measurements of the cleaved DMT groups are determined and are almost quantitatively.

Claims (3)

1. A method for producing a matrix-bound miniaturized combinatorial polymer and oligomer library of nucleic acids, peptides, sugars and other chemical compounds which are produced by multi-step synthesis; and combinations thereof, using a chemically inert stamp, the active surface of which has a microstructuring predetermined by the chosen synthesis algorithm, which enables a selective response of defined loci on a substrate, and a substrate which is a solid inert carrier, on the surface of which a monolayer of a linker with a terminal protected functional group is covalently bound, characterized in that

• a. said stamp is immersed in a solution containing a deprotection reagent suitable for the release of the terminal protected functional groups,
• b. the stamp wetted with the deprotection reagent is brought into contact with the said substrate, as a result of which the terminal functional groups are released at the contact areas,
• c. after washing with an inert solvent, the substrate is overlaid with a first activated chemical building block, as a result of which the chemical building block extends the released terminal functional groups,
• d. the synthesis sequence of steps ac is repeated any number of times until the chain length desired according to the synthesis algorithm is produced, with defined loci on said substrate being addressed in each synthesis step by using stamps with predetermined patterns.

2. The method according to claim 1, characterized in that the chemical building block protected on its functional groups, for the construction of oligomers and polymers suitable monomer. 3. The method according to claim 1, characterized in that as a chemical building block for the production of oligonucleotides, polynucleotides, peptides, polysaccharides or appropriate copolymer suitable monomer is used.