DE102007062154A1 - Process for the preparation and use of stochastically arranged arrays of test substances - Google Patents

Abstract

The present invention relates to a method for the efficient production and use of stochastically arranged arrays of test substances. The production and testing of large series of substances, typically drug candidates, involves a great deal of preparation. So far, the test substances are dissolved separately in a biocompatible medium and dosed into individual, specially addressed sample chambers. In addition, due to the small quantities and the geometry of the sample chambers, an unfavorable surface-volume ratio, especially for cell biological tests, results. In the method according to the invention, the test substances equipped with molecular building blocks specially tailored to the process are bound to carrier resin beads and immobilized in a randomly distributed arrangement on a carrier plate. From this template, the test substances can be transferred precisely to one or more assay plates on the surface of the substance tests are performed. The workload is independent of the number of test substances. The assignment of the individual substance spots to the originals on the carrier plate is realized by intelligent pattern recognition methods. The method is particularly suitable for extensive series of tests on the medical-biological activity of substances, but also for the investigation of cell biological samples and for antibody tests.

Description

The The present invention relates to a method for efficient production and application of stochastically arranged arrays of test substances.

The Method is characterized in that an array random Distributed substances by a randomly distributed Array of polymeric carrier resins, while maintaining the spatial arrangement mirror image transferred to a test surface becomes. The method is also characterized in that on this Identify the number of steps required to prepare the arrays for the preferably medical-biological and diagnostic Tests are independent of the number of substances that are on their biological activity should be tested.

The Protocol to demonstrate the chemical structure of the assay as biologically active substances were identified to the requirements adapted to the stochastically arranged arrays. The assignment of the individual Substance spots in the test array to the carrier resin particles and thus the identification of the test compounds by means of intelligent pattern recognition process.

State of the art

Since its invention in the early 90s . , the microarrays, especially the biochips, have greatly simplified the processes of medical-biological research. The test substances, usually nucleic acids, proteins or peptides, which interact with medically-biologically relevant molecules, cells or microorganisms, are applied in a highly dense arrangement in specifically addressable compartments of a test area. This arrangement allows the parallel examination of a sample with a large number of different reactants.

however is the production of arrays arranged spatially resolved Test substances, in particular the systematic production and arrangement a very large number of drug candidates, with the Current technologies are still very time and material consuming.

The currently most widely used techniques for the production of microarrays are in addition to the synthesis of the test substances directly on the test surfaces ( US 5,591,646 ), the fixation of the test substances by photolithographic methods ( US 5,412,087 . US 5,489,678 . WO 03/089900 ) and the application of the test substances by printing techniques ( US 6,079,283 . US 6,083,762 . US 6,094,966 ). An overview of the current state of science in this area is provided by the work of Ng / Ilag , Jain and Müller / Röder ,

The Methods of combinatorial solid phase synthesis allow by appropriate reaction the synthesis of millions of compounds within a few days. These compounds can be very high expenditure on equipment and time released from the synthesis beads and then tested in individual containers.

This usually happens by pipetting of stock solutions of Test substances in individual sample chambers of test plates. Therefore stand z. B. automated xyz pipetting devices, the Address compartments in which the assay is performed should be available.

to rational implementation of a large number of Medical-biological and diagnostic tests are increasing miniaturized method used. So stand for the biological testing of substances z. B. Microtiter plates with 1536 Sample chambers available. Due to the small volumes, which are pipetted into these sample chambers and due to the unfavorable Ratio of surface to volume, in particular the performance of cell biological tests in these microtiter plates only limited possible.

Studies have also been carried out in which the synthesized compounds remain on the synthesis beads and directly with the target molecules (other small molecules, proteins, DNA or other biopolymers). However, there is a risk here that the test results are influenced by the carrier resins.

to Reduction of the effort can collections of having different Substances loaded carrier resin beads examined together become. Set the obtained test results for the substance mixtures Are then difficult to interpret and from limited benefit for drug development.

An alternative approach to spatially resolved addressing is to perform the assays in liquid suspensions. In this case, the test substances are bound to carrier particles coded by color, shape, size or chemical-physical parameters. The coding is carried out in most variants by one or more fluorescent dyes ( US 2005/0106711 . US 2005/0106712 ), which are incorporated in the carrier particles. In another variant, the molecules bound to the test substances are marked by fluorescent dyes, in a further variant by a combination of both ( US 7,011,945 ). However, the number of possible codes and thus the number of simultaneously to be detected test substances is limited. The identification of the test substances is then carried out by methods of flow cytometry ( US 5,981,180 ) or the detection on optical fiber bundles ( US 6,023,540 . US 6,266,459 ). A more recent approach is to identify the test substances due to nanocrystals entrapped in the carrier particles ,

All of these technologies are instrumentally very complex. Therefore, some methods for the production and application of stochastic arrays have been developed. Here, the test substances are also bound to carrier particles and from the liquid phase by special gelatin coatings ( WO 2005/016516 ), electromagnetic fields ( US 2007/0231825 ), chemical bonding or mechanical methods ( US 2002/0051971 ) is immobilized in a random arrangement on a support surface. The identification of the test substances is carried out, similar to the above-mentioned methods, by analyzing the color coding of the carrier particles and / or the fluorescent markers of bound molecules, with the aid of CCD cameras ( US 2003/0143542 ) or spectrometers ( US 7,034,941 ) coupled microscopic method. Also applied are chemical methods or DNA sequencing ( WO 2007/044245 ). The identification is carried out directly on the support surface, optionally before and / or after the assays.

Except for nucleic acids ( WO 2007/044245 ) No methods for generating stochastic arrays of carrier particle-independent test substances are known. Likewise, the production of spatially resolved copies of stochastically arranged arrays of test substances has hitherto only been described for nucleic acids ( US 2003/0124594 ). Similar methods for proteins, peptides or other organic molecules are not yet known. Furthermore, we are not aware of any methods that identify the test substances by association with their original carrier particle and not directly on the assay surface.

Even though the approach of spectrally addressed carrier particles a significant improvement over the usual spatially resolved addressed microarray represents exists There is still a significant need for simplifying the production process and lowering the cost of medical biological microarrays.

task

The Invention is therefore the task of an efficient method for the spatially resolved production of arrays by combinatorial Provide solid phase synthesis prepared test substances, a transfer of the test substances from a carrier plate enabled on one or more test plates and in which the preparation and transfer time independent of the number of compounds to be tested. In addition, the inventive Procedures the implementation of medical-biological and diagnostic tests directly on the test surfaces, preferably at a test level, with a wide range of media and independent of a stock solution.

The Task therefore includes both the solution of the problem the enormous increase of the necessary pipetting steps with the Increase in the substances to be tested, as well as the solution the problem of the unfavorable relationship of Surface to volume, which in the miniaturization medical-biological Tests are done.

The object is achieved by a first-described method for the production and application of stochastically arranged arrays of test substances, as set forth in claim 1. Preferred embodiments of this method are claimed in claims 2 to 35. Claims 36 and 38 relate to the use of the method in the performance of drug screening, medical biological testing and in medical diagnostics. The wording of all claims is hereby incorporated by reference into the content of the description.

The inventive method consists in its essential Characteristics of the synthesis of bound to a carrier resin and in their properties specifically adapted to the process Test substances, an immobilized carrier resin bead monolayer on a carrier plate as a source of the test substances, a surface-modified test plate for recording of the stochastically arranged array of test substances, a routine to assign the individual substance spots to their source as well an analytical protocol for the detection of the chemical structure the test substances on the support plate.

To The teaching of the invention are those for the inventive Process required test compounds prepared so that they are bound to a carrier resin and in their properties are specifically adapted to the process. They contain one Orthogonal to the synthesis steps cleavable linker (Principle 1), a transmission principle (principle 2) and a biocompatible Spacer (principle 3), these principles also individually or in Any combination can occur. The principles 1 to 3 can be before, during, as well as after the synthesis, preferably during the solid phase synthesis, be introduced into the test compound molecules.

at the preferred preparation of the test substances with the known Methods of combinatorial solid phase synthesis become the starting molecules attached to a pretreated, functionalized carrier resin and then the target substances in successive Synthesis steps systematically constructed. Particularly preferred as starting molecule of the linker according to principle 1 used. With the help of this method and by appropriate reaction can create complex connection libraries within a few days getting produced.

To the completion of the syntheses can carrier resins be mixed from several syntheses. Thus, a stochastic lies distributed powder of preferably singulated synthesis beads, loaded with various test substances.

The mixed synthesis beads bearing the principles 1 to 3 Test compounds are placed on a preferably planar pad (Support plate) by a biotest compatible, non-toxic adhesive in random arrangement, preferably in a monolayer, fixed. This is preferably done by powder, Dip or centrifugal coating and subsequent combing. On the support plate creates a stochastically arranged Array of synthetic beads loaded with different test substances.

to Facilitating the deconvolution of positively tested compounds, preferably in mass spectroscopic analysis but also compartmentalizations are made on the carrier plate. For this purpose, specific mixtures or even pure beads, which presorted according to certain criteria due to the synthesis process were, in sample chambers, preferably the cavities of Filled microtiter plates. The synthesis beads are in the predetermined arrangement, preferably by immersion or centrifugal coating and subsequent combing, on the adhesive layer of the Support plate fixed in a monolayer. On the carrier plate arise from each other delimited fields defined by arrays Mixtures of synthetic beads loaded with test substances.

For transmission The test substances are the carrier plate and preferably planar test plate placed on top of each other. On the carrier resin beads form, ideally supported by a medium, preferably singular contact zones. That's the transmission supporting medium is preferably a solvent film, preferably from water or buffer solution.

With physical, biological / enzymatic or chemical methods become the test compounds on the linkers (principle 1) of the synthesis beads split off and then within the contact zones, mediated by Principle 2 and Principle 3, on the surface transferred to the test plate. Here, principle 2 ensures a specific and spatially resolved interaction of the test compounds with the test surface.

The surface contact between the carrier plate, carrier resin bead and test plate is determined by the material and the size of the synthesis beads, the medium used and the properties of the surfaces and the test environment, which are preferably matched to each other case-specifically. As interaction principle (principle 2), basic chemical, physical and biological, preferably covalent, ionic, electrostatic, adhesive or ligand-receptor interactions are used.

After separation from the carrier plate, a stochastic array of test substances (in mirror image to the carrier plate) is produced on the test plate. 1 ).

In this are the test compounds, due to the direct contact between the synthesis beads and the surfaces and under Use of principles 2 and 3, in largely singular, homogeneous substance spots. The connections are through the assay adapted biocompatible transfer principle (Principle 2) fixed in a uniform orientation on the substrate and by the biocompatible spacer adapted to the assay (Principle 3) spatially separated from each other and from the substrate.

by virtue of the loading of the multi-molecule synthesis beads a test substance can from a carrier plate by repeating the transfer process several identical Test plates are made as copies. Also, the transmitted Substance quantity through the duration and intensity of the transfer process controlled, which used for the production of concentration series can be.

The appropriate evidence to study the interaction of the test substances with specific target molecules, so-called targets, the preferably from native, modified cells or the diagnostic material be isolated and sorted or in target mixtures, soluble or bound on / in cells are directly on the test plate carried out.

The assignment of in the medical-biological or diagnostic tests positively / negatively identified spots to the corresponding test substances by means of intelligent methods of pattern recognition. For this purpose, the carrier plate is characterized by transferable with the method according to the invention markers. These markers are preferably carrier resin beads loaded with compounds containing fluorescent dyes and having the principles 1 to 3. The markers are applied to the carrier plate together with the synthesis bead mixture. Ideally, the labeled carrier resin beads are stochastically distributed over the entire surface of the carrier plate. On the basis of the superimposition of at least 3 points existing marker patterns on the carrier and the test plate is an exact determination of the coordinates of all spots on the plates and thus a one-to-one assignment of the individual substance spots on the test plates to the synthesis beads on the Support plate possible. Alternatively, markings or a scale can be placed directly on the plates. An example of the mirror-image transfer of the marker pattern from the carrier to the test plate and a possible coordinate system shows 1 ,

mathematical Algorithms for correcting transmission errors, such as distortion, Torsion and shrinkage effects, can help improve the accuracy of the assignment can be used.

With suitable analytical methods, preferably laser desorption and mass spectrometry, can the correspondingly effective test substances by Analysis of the on the positive / negative tested carrier resin beads remaining test compound molecules are identified.

The The invention also encompasses fields of application of the invention Process. It is mainly used in the implementation of biochemical and biological test series in which the effect of a large number of compounds, preferably peptides, proteins and organic molecules, in as much as one step to be examined comparatively. Especially in the investigation the biological activity of specific test substances, in the study of cell biological samples, in antibody tests and in medical-biological diagnostics are inventive Application fields.

In pharmacy is the inventive Method especially for the study of structure-activity relationships in the context of drug development, where the interaction of a large Number of test substances with isolated targets, such as cells and microorganisms, is looked at.

The Inventive process of the invention first described here spatially resolved transmission of stochastically arranged Arrays of test substances without pipetting steps and apparatus engineering Effort solves the problems of the prior art that in the usual methods of preparation of test environments for screening experiments and by the Miniaturization of medical-biological tests arise. That concerns both the problems of enormous increase in the required pipetting steps with the increase in the substances to be tested as well as the unfavorable one Ratio of surface to volume.

The Invention solves the above problems by being stochastic Arranged arrays of test substances by direct contact between Synthesis beads and preferably prepared planar surfaces and transferred. Arrays emerge singular, each consisting of molecules of a single compound existing Substance spots. The studies on the interaction of the test substances are performed directly on the test surface. In this way, eliminates all pipetting for Production of a test environment. This offers the advantage that the Number of steps for the transfer the test compounds and thus the time required for the Preparation independent of the number of test substances is. By the ideally monomolecular coating of the test plate it is possible to carry out the tests in a test level, which is another advantage of the invention Represents process.

To the preparation of a carrier plate can several identical test plates with very high precision getting produced. The time required for the transfer is manageable. The transfer process itself takes a period of about 20 s, regardless on the number of test substances and the size the carrier / test plates.

Depending on the size of the carrier resin beads, a loading of the test surfaces comparable to commercial bio chips is possible. For example, with a bead diameter of 20 μm, 250,000 beads per cm ^{2 could be} immobilized on the test plate.

The spatial assignment of after implementation present the medical-biological or diagnostic tests Spots on the carrier resin beads on the carrier plate is variable depending on the bead diameter less than 2 μm possible.

The Principle of the stochastic distribution of the test substances on the Test plates, the small amount of time and the opportunity the production of test plate copies now make it possible perform the tests with high redundancy. this includes with respect to random effects or environmental influences individual molecules or positions of the test plate very much robust tests another advantage of the invention Process.

moreover can by selecting the Abspaltungsbedingungen concentration series to be created.

The inventive method for the preparation of stochastically arranged arrays of test substances is thus very flexible, robust and feasible without technical effort.

For details, to particularly preferred embodiments of the invention Method and other features and advantages of the invention from the following description of exemplary embodiments in conjunction with the subclaims, wherein the above mentioned and to be explained below individual Features in each case and in any combination are claimed together. The embodiments are purely illustrative and limit the reach of Invention not a.

In The attached figures show:

1 Schematic representation of the corresponding spot patterns on a carrier plate (picture on the right) and the associated test plate (mirror image on the left) after carrying out a binding assay with fluorescent dyes.

2 Synthetic scheme for attaching the photolinker 4-bromomethyl-3-nitrobenzoic acid to a synthesis bead and subsequent methylamidation.

3 Synthesis scheme for the photochemical cleavage of the synthetic conjugate from the synthesis bead to 4-bromomethyl-3-nitrobenzoic acid.

4 Structural formula of Pam ₃ Cys-OH.

5 Structural formula of an immobilized conjugate containing all three principles.

6 Structural formula of an intermediate after attachment of the Pam ₃ Cys-OH and before binding of the biologically active test compounds via the β-alanines.

7 Synthetic Scheme for Connecting the Photolinker (Step 1).

8th Synthesis scheme for methylamidation of the photolinker (step 2).

9 Synthetic scheme for the attachment of Fmoc-amino acids (step 3).

10 Synthetic scheme for cleavage of the Fmoc protecting groups (step 4).

11 Synthesis scheme for photochemical cleavage of the synthesis conjugate (step 7).

12 ESI-MS of Pam ₃ Cys-SK ₃ K (βA-βA-Aca-Aca-biotin) -NCH. ₃

13 ESI-MS of a Pam ₃ Cys-SK ₃ K (βA-βA-dipeptide) -NCH ₃ collection from 400 individual compounds (aa _n = amino acid).

14 Extract of the ESI-MS of a Pam ₃ Cys-SK ₃ K (βA-βA-dipeptide) -NCH ₃ collection from 400 individual compounds with assignment of the peptide sequences.

15 Microscopic image of a coating produced by powder coating and combing with carrier resin beads (Ø 20 μm, image section 780 × 600 μm).

16 Microscopic image of a coating produced by dip coating and combing with carrier resin beads (Ø 20 μm, image section 2600 × 2000 μm).

17 Microscopic image of a coating produced by centrifugal coating and combing with carrier resin beads (Ø 20 μm, image section 2600 × 2000 μm).

18 Schematic representation of the spatial structure of a test compound coupled to the carrier resin bead.

19 Schematic representation of a loaded with a test compound and immobilized in the adhesive layer of the support plate carrier resin beads.

20 Schematic representation of the transfer process of test compounds from the surface of a carrier resin bead to the test surface.

21 Schematic representation of a resulting from the transfer of a carrier resin bead singular substance spot on the test plate.

22 Structural formula of Pam ₃ Cys-SK ₃ K (carboxyfluorescein) -NCH ₃ .

23 Structural formula of Pam ₃ Cys-SK ₃ K (Aca-tetramethylrhodamine) -NCH ₃ .

24 Microscopic images of the spot pattern of fluorescence-labeled beads on the support plate (top left), a first test plate (bottom left), a second test plate (bottom right) and showing the superposition of all three plates (top right) (image sections 520 × 400 microns). In the superimposed illustration rhodamine-labeled beads are light gray, fluorescein-labeled beads are white and unlabeled beads are shown in dark gray. In the images of the test plates, only the fluorescence of the rhodamine and the fluorescein-labeled beads are visible. In the recording of the support plate all beads are visible.

embodiments

Example A - Execution the principles

For the realization of the method according to the invention become additional in the synthesis of the test compounds functional groups incorporated into the molecular structure, that represent the principles described.

Principle 1 - orthogonally cleavable left

The basis of this principle is an orthogonally cleavable linker to the synthesis steps. The photolinker 4-bromomethyl-3-nitrobenzoic acid attached to the NH ₂ functionality of the carrier resin beads offers, after the substitution with methylamine, an amide group for the coupling and / or synthesis of the desired test substances.

Therefor became the classical solid-phase synthesis with Fmoc and Aloc-protecting group chemistry applied. The side chains were also provided with classical amino acid protecting groups like Boc and tBu protected.

To completed synthesis and the application of the bead monolayer on the Support plate became the synthesis conjugate by UV irradiation deliberately released. With the irradiation time, the wavelength and the intensity of the UV light used could Amount of synthetic conjugate released per synthesis bead to be influenced.

Of the used photolinker was stable during synthesis. Photolysis left a methylamide group on the synthetic one Conjugate that does not have the preferably medical-biological tests affected.

The synthetic schemes for attaching the photolinker to the carrier resin beads as well as the cleavage mechanism are in 2 and 3 specified.

Principle 2 - Transmission principle

basis this principle is a functional and functional transferring and anchoring Group in the synthesis conjugate always to the test environment and the Functionalization of the test surface is adjusted. Preferably you use basic chemical and physical interactions such as hydrophobic or electrostatic interactions, covalent or ionic bonds, photoaffinity or biological Ligand-receptor interactions.

In the example shown, the test substances were prepared by attachment of Pam ₃ Cys-OH ( 4 ) of the company EMC microcollections GmbH provided with a lipophilic functionality. The test plates were lipophilically coated with trimethoxypalmitoylsilane. The hydrophobized surfaces then had a contact angle of 110 to 115 ° with respect to water. Once in contact with the surface of the test plate, the Pam ₃ Cys conjugates anchored through hydrophobic interactions and aligned in a dense, ordered molecular layer. A space requirement of 0.8 nm ² per molecule was determined.

The mutual attractions were so strong that the Substance layer on the test plate in aqueous solutions was stable. Only with lipid-dissolving organic solvents, Like dichloromethane, this anchorage can be restored to solve.

Principle 3 - biocompatible spacer

Principle 3 has two functional properties as a spacer. On the one hand, it should spatially separate the lipophilic Pam ₃ Cys group (representative of all conjugates of Principle 2) from the biologically active part of the synthesis conjugate. Thus, the influence of the properties of the basic conjugate, such as the greatly reduced water solubility of the test substances or toxicity effects due to the principle 2, is minimized to the test results. On the other hand, it should support the mutual alignment of the total conjugates on the test surface. The biocompatible spacer used was the pentapeptide serine-lysine-lysine-lysine-lysine. Two molecules of β-alanine were attached as a lateral extension of the lysine coupled to the photolinker. Furthermore, the biologically active conjugates were synthesized using this functionality.

This resulted in the 5 represented total conjugate.

The biologically active conjugates could be prepared by conventional solid-phase synthesis getting produced. Due to the principles 1 to 3 behaved all the total conjugates in the transmission of the arrays of the carrier plate to the test plate, regardless of the individual test substance, similar. In addition, the worried positive charge of lysine for increased water solubility and the mutual ionic repulsion of the individual conjugates.

Example B - Solid Phase Synthesis a basic conjugate

The carrier resin TentaGel S-NH ₂ was charged with the photolinker 4-bromomethyl-3-nitrobenzoic acid (step 1) and substituted with methylamine (step 2).

Subsequently, the spacer was constructed by stepwise coupling of Fmoc-Lys (Aloc) -OH, 3 molecules Fmoc-Lys (Boc) -OH and Fmoc-Ser (tBu) -OH, each after cleavage of the Fmoc-protecting group (Steps 3 and 4). After further cleavage of the Fmoc protecting group, the Pam ₃ Cys-OH was attached as a lipophilic group. The resulting basic conjugate shows 6 ,

After cleavage of the Aloc protective group (step 5) of the first lysine, 2 molecules of Fmoc-β-alanine-OH and the biologically active test compound in the side chain were attached. The 5 shows the resulting after cleavage of the Boc and all other protecting groups (step 6) total conjugate.

The after separation from the carrier resin (step 7) resulting Compound showed hydrophobic properties with nevertheless good Water solubility of at least 1 mg / ml.

Of the Success of the individual synthesis steps was checked by means of HPLC-ESI-MS. For this purpose, in each case samples of the loaded with the intermediate product Removed carrier resin and split off all protective groups (Step 6). The synthesis conjugate became photochemically from the carrier resin separated (step 7) and the intermediate analyzed.

The individual synthesis steps are described in detail below. All stated amounts refer to a starting amount of 1 g of carrier resin.

Step 1 - Attachment of the Photolinker to the Carrier Resin ( 7 )

1 g of TentaGel S-NH ₂ resin (Rapp Polymere GmbH, loading capacity 0.25 mmol / g, diameter 20 μm) was washed 4 times with 5 ml of DMF each time.

4-Bromomethyl-3-nitrobenzoic acid (3 equiv, 0.195 g, 0.75 mmol) and HOBt · H ₂ O (3 equiv; 0.115 g, 0.75 mmol) were dissolved in 12.5 ml of DMF, DIC ( 3 equiv; 1.16 ml; 7.5 mmol) was added and the solution was stirred at room temperature for 30 min.

Subsequently The solution was added to the washed resin and the Shake mixture for at least 16 h at room temperature.

The Resin was filtered off, washed (6 × 5 ml DMF, DCM, MeOH) and dried under vacuum.

The Completeness of the reaction was checked with the Kaiser-ninhydrin test.

Step 2 - Methylamidation of the Photolinker ( 8th )

To To the dried resin was added 8 ml of dry DMSO and then Add 8M methylamine in EtOH (20 equiv; 0.575 mL, 4.6 mmol). The mixture was shaken for 3-3.5 h at room temperature.

The Resin was filtered off and washed (3 x 8 ml DMSO, 6 x 8 ml DMF, 6 x 8 ml DCM, 6 x 8 ml MeOH, 3 x 8 ml of DMF).

Of the Chloranil test was positive.

Step 3 - Attachment of Fmoc Amino Acids ( 9 )

3 Equiv. a Fmoc amino acid or other compound having a carboxyl group, such as. PAM ₃ Cys-OH, and HOBt · H ₂ O (3 equiv, 0.115 g, 0.75 mmol) were dissolved in 8 mL of DMF and DIC (3 equiv; 1.16 mL, 7.5 mmol). was added. In the case of Pam ₃ Cys-OH, a mixture of DCM and DMF (1: 1 v: v) was used to improve solubility.

The Solution was added to the resin and the mixture at least 45 min, for longer-chained or poorly soluble Amino acids at least 3 h, shaken.

The Resin was filtered off and washed with 8x8 ml of DMF.

Of the Chloranil test was negative.

Step 4 - Cleavage of the Fmoc protecting groups ( 10 )

A Solution of piperidine in DMF (8 ml, 30% v: v) became the Add the resin and shake the mixture for 20 minutes.

The Resin was filtered off, washed once with 8 ml of DMF and again for 15 min with a solution of piperidine in DMF (8 ml, 30% v: v) shaken.

The Resin was filtered off and washed with 6 x 8 ml of DMF.

The Completeness of the reaction was checked with the Kaiser-ninhydrin test. In the case of a negative test result, the cleavage was done with twice the incubation time repeated.

Step 5 - Cleave the Aloc-protecting groups

Pd (PPh ₃ ) ₄ (1.1 equiv.) Was dissolved in 10 ml DCM with 5% acetic acid and 2.5% morpholine.

The Solution was added to the resin washed 5 times with DCM and shake the mixture for at least 6 hours.

The Resin was filtered off and 5 times each with DCM with 5% acetic acid, washed pure DCM, DMF and DCM.

The Completeness of the reaction was checked with the Kaiser-ninhydrin test.

Step 6 - Splitting the Boc and page groups

8 ml of a solution of 95% TFA and 5% H ₂ O (v: v) was added to the resin and the mixture was shaken for 40-60 min.

The Resin was filtered off and washed with 8x8 ml of DMF.

The Completeness of the reaction was checked with the Kaiser-ninhydrin test.

Step 7 - Photochemical cleavage from the carrier resin ( 11 )

For checking 5 mg of resin were taken from the synthesis in each case and 80% in 1 ml. tert. Butyl alcohol / water added. The mixture was 10 min shaken and filtered off.

Subsequently was again 1 ml of 80% tert. Butyl alcohol / water added. Now was the mixture for 90 min with UV light of the wavelength Irradiated 365 nm and then filtered off.

The Irradiation was tert after further addition of 1 ml of 80% tert. Butyl alcohol / water repeated with an exposure time of 30 minutes and then the resin filtered off.

The Resin was mixed with 400 μl ethanol and after 3 minutes incubation time filtered off.

The Filtrates of all steps of UV irradiation were combined, snap frozen and lyophilized.

The colorless powder obtained was for further study used.

Example C - coupling of the basic conjugate with biotin

At that produced according to embodiment B. Basic conjugate was biotin, representative of various potentially bioactive compounds, attached and the reaction product analyzed analytically.

Carrying out the coupling reaction

For synthesis, 50 mg of the basic conjugate-loaded resin ( 6 ) used.

When Spacer side chain were 2 molecules of β-alanine and 2 molecules of ε-aminocaproic acid the Aloc-protected lysine and then the Biotin coupled. Afterwards all protecting groups were split off (Steps 3 to 6 in Embodiment B).

10 mg of the carrier resin loaded with the synthesis product used for the following photochemical cleavage. The cleavage procedure was different from step 7 in Example B repeated 4 times.

Analysis of the reaction product

A portion of the resulting 3.2 mg of the test compound Pam ₃ Cys-SK ₃ K (βA-βA-Aca-Aca-biotin) -NCH ₃ was dissolved in water and analyzed by HPLC-ESI-MS.

In the in 12 represented mass spectrum (calculated mass 2118 g / mol) represent the peaks 707.1 [MH ₃ ] ³⁺ 1,059.7 [MH ₂ ] ²⁺ 712.5 [MH ₃ ] ³⁺ Oxidation product with an oxidized sulfur atom 1,067.8 [MH ₂ ] ²⁺ Oxidation product with an oxidized sulfur atom 717.6 [MH ₃ ] ³⁺ Oxidation product with two oxidized sulfur atoms 1,075.5 [MH ₂ ] ²⁺ Oxidation product with two oxidized sulfur atoms

As can be seen from the mass spectrum, provides the target compound the main product.

Example D - Coupling of the basic conjugate with a chemical library

According to the split-and-mix principle, the basic conjugate ( 6 ) synthesized a dipeptide library after 2 molecules of β-alanine as spacer side chain. To this end, the 20 proteinogenic amino acids were used, resulting in 400 different conjugates.

8 mg of this library were released by photochemical cleavage and concentrated. The obtained mass spectra ( 13 and 14 ) clearly show that it is a library. The highest and lowest masses as well as the most common peaks were assigned to the corresponding amino acids.

Example E - Production a carrier plate with a bead monolayer

starting point for the spatially resolved transmission process The test substances is the fixation of the synthesis beads on one Carrier plate, ideally in a monolayer.

When Support plate was a plastic polymer used on a thin, only a few microns thick, UV-permeable and proven biologically inactive adhesive layer was applied.

Around To make a bead monolayer, you need 4 sub-steps, which are described below.

Specification of the beads

The carrier resin beads have a uniform size of 20 μm in diameter in the dry, unswollen state. One gram of dry material contains 240 million beads. The average loading of the carrier resin is 240 μmol per g. This corresponds to a charge capacity of about 1 pmol or 6 x 10 ^-11 molecules per bead.

All carrier resin beads have a uniform structure, size, shape, loading and functionalization. In the following, an NH ₂ -functionalization of the beads is used.

Separating the beads

Around from the bead powder, the carrier resin particles to a monolayer Being able to shape it on the carrier plate is what it is indispensable to separate these before, since in the synthesis occasionally Adhesions occur.

Therefor the beads were heated in 60 ° C tert. Butyl alcohol added, sonicated for 20 min and then Shaken for another 20 min, taking the temperature was always kept above 40 ° C.

Were the beads in the powder were not isolated enough, were shaken for 1 h in n-methyl-pyrrolidinone and then Washed 3 times with diethyl ether. Then they became again in 60 ° C warmer tert. Butyl alcohol added and the original procedure is repeated.

The Solution with the isolated beads was by liquid Nitrogen flash frozen and stored at -80 ° C and 0.5 mbar pressure lyophilized.

It remained a fine powder, which did not singulate less than 0.5 ‰ Contained beads.

Apply the beads to the support plate

For this sub-step can alternatively be three different ones Coating techniques are used.

at the powder coating became the carrier plate with the adhesive layer mounted down in the upper part of a coating chamber. One from the bottom into the coating chamber entering gas jet swirled the beads lying there on the ground and carried them to the carrier plate, where they stuck. Once the adhesive layer was covered, no further beads adhered to the support plate. It formed mainly a monolayer. For dip coating the beads were placed on the adhesive layer of the carrier plate scattered, or the support plate was immersed in the beads. On the adhesive film remained only beads adhere, the direct contact had with the carrier plate. A monolayer was created. excess Material was removed by wiping, brushing or blowing off with a Gas jet removed.

at the centrifugal coating was the support plate together with the beads in the coating chamber. The coating chamber was inserted into a centrifuge so that the beads through the Centrifugal force in the adhesive layer of the carrier plate were pressed. Thereafter, the coating chamber was turned over, so that by centrifugal forces arising during centrifugation again the excess beads from the backing plate were removed. Distance and speed were each chosen so that the beads are pressed firmly into the adhesive layer or were not removed from this again. Back remained a monolayer.

Immobilization of the beads on the carrier plate

microscopic Images of the coated carrier plates still showed some bead clusters and overlays. Furthermore the beads were not fixed enough. That's why the next step is combing.

To a metal ridge turned over at an acute angle to the pulling direction the surface of the bead-coated carrier plate drawn. Here, the top of the ridge was so lead that the underlying beads pressed into the adhesive layer were. By pulling the beads arranged themselves to a tight bearings, defects in the layer were closed.

Removal of unfixed and / or protruding Beads

in the second step of combing, the ridge became dull in one Angle to the direction of pull on the carrier surface stated. As a result, supernatant beads, not solid sticking fragments and bead clusters raised and out the coating removed.

Finally the carrier surface was blown off with compressed air, to remove debris and dust.

It created a monolayer, which almost no protruding Beads or clusters contained more.

Comparison of coating techniques

With all three coating techniques, a bead monolayer could be achieved. Powder coating and dip coating show nearly ideal layers after combing ( 15 and 16 ). The centrifugal coating also gives a monolayer ( 17 ). However, this is not as densely packed as the layers resulting from the other two techniques.

The Size of the backing plate can be at all principle, three techniques can be chosen arbitrarily.

Example F - Transmission a stochastically arranged array of test substances on the test drive

The screening compounds used here by way of example were anchored via the photochemically cleavable linker 4-bromomethyl-3-nitrobenzoic acid to the NH ₂ -functionalized carrier resin beads. As a bioinert spacer, the pentapeptide serine-lysine-lysine-lysine-lysine with a β-alanine-β-alanine side chain and as the lipohilic group Pam ₃ Cys-OH was used ( 18 ). The test plate was a slide with hydrophobed by Trimethoxypalmitoylsilan glass surface.

The coated side of the carrier plate prepared according to Embodiment E, which carried about 4 million beads on an area of 25 × 74 mm ( 19 ), was moistened with water. After a short residence time of a few minutes, the test plate with the lipophilized side was placed on the coating of the carrier plate. At each bead, a singular contact zone formed between the carrier and the test plate.

The photolinker was split by irradiation of UV light (365 nm). The test compounds separated from the carrier resin diffused through the moist swelling layer of the beads ( 20 ). As soon as they reached the surface of the test plate, the lipophilic groups interacted with the hydrophobic surface. The molecules of the liberated test compounds, supported by the spacer contained in their structure, aligned on the test surface to form a dense, adherent monolayer.

After removal of the carrier plate, an isolated substance spot containing exclusively molecules of a test compound remained per synthesis bead ( 21 ). The substance spots were arranged in mirror image to the bead array on the carrier plate.

Under the selected reaction conditions (irradiation and residence times) the spin-off and transfer process was not complete. There remained therefore still enough synthetic conjugate the test compounds on the beads. Thus could a carrier plate by repeating the process of making several identical ones Test plates (copies) can be used.

Example G - Evaluation of the transfer

Transfer of fluorophores

For the internal control of the transfer and the assignment of spot patterns on the basic conjugate ( 6 ) Fluorophores anchored according to the methods described in Example C at the site of biotin. Carboxyfluorescein ( 22 ) and tetramethylrhodamine ( 23 ) used.

The were fluorescent beads, as in the embodiment E, applied to a carrier plate. From that were three according to the methods of embodiment F produced almost identical test plates.

Around the assignment of the spot pattern of the layer to the bead pattern of the To facilitate carrier plate, was no dense monolayer applied.

The one-to-one allocation of the individual spots was possible within a variation width of a maximum of 1 μm. Also torsion effects could be corrected. Only deformations of the test plates led to slight deviations ( 24 ).

Influence of binding reactions on the test surface

to Check the stability of the spot pattern on the test plates during the medical-biological tests was another Trial performed. This was a stochastic arranged array of unloaded, with carboxyfluorescein and loaded with biotin carrier resin beads by the methods of embodiment E on a support plate applied. Of these, three identical test plates were prepared by the methods made of embodiment F.

On The test surfaces were given a binding reaction with a Fluorescence-labeled streptavidin performed. The spot pattern all four plates were then compared. To Correction of the torsion and distortion effects was an assignment Spots with a maximum variation of 2 μm possible.

Consequently could be shown that performed after the transfer of the array Binding reaction does not affect the stability of the Had spot pattern on the test plate. Single proven transmission errors are due to the redundant information from the stochastic Array unproblematic.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (38)

Process for the production and use of stochastically arranged arrays of test substances, characterized in that an array of randomly distributed substances is transferred from a carrier plate to a test surface. Method according to claim 1, characterized in that that the transfer is spatially resolved while retaining the spatial arrangement takes place. Method according to claim 1, characterized in that that the spatially resolved transmission in dependence of the diameter of the carrier particles with a deviation less than 2 μm is possible. Method according to claim 1, characterized in that that with a carrier plate one or more identical Test plates can be produced. Method according to claim 1, characterized in that that he  5.1. the test substances with specific to the process equipped with coordinated features, 5.2. the test substances binds to a carrier resin, 5.3. the loaded carrier resin beads on a carrier plate, 5.4. the test substances from the carrier plate to one or more test plates, 5.5. performs the substance tests directly on the test surfaces, 5.6. the substance spots on the test plates the carrier resin beads on the carrier plate assigns and 5.7. the test substances on the loaded carrier resin beads accurately to the corresponding Position of the carrier plate identified. Method according to claim 5, characterized in that that in step 5.1. prepared test compounds in addition to the active, preferably the biologically active, part of the principles 5.1.1. an orthogonal to the synthesis steps cleavable linker, 5.1.2. a transmission principle and 5.1.3. a biocompatible Spacers individually or in any combination. Method according to Claim 6, characterized that the orthogonal to the synthesis steps cleavable linker cleaved by physical, biological / enzymatic or chemical methods can be. Method according to Claim 6, characterized that the orthogonal to the synthesis steps cleavable linker Photolinker, preferably 4-bromomethyl-3-nitrobenzoic acid is. Method according to Claim 6, characterized that the transfer principle represents functional groups, the adhesive or electrostatic interactions, ionic or covalent bonds or receptor-ligand interactions with the Surface of the test plate can go. A method according to claim 6, characterized in that the transfer principle is realized by a hydrophobic group, preferably Pam ₃ Cys-OH. Method according to Claim 6, characterized that the biocompatible spacer the active, preferably the biological active, part of the test substance molecules of the transfer principle and spatially separates the test surface. Method according to Claim 6, characterized that the biocompatible spacer by the spatial separation the influence of the test results by transmission principle and / or test surface prevented. Method according to Claim 6, characterized that the biocompatible spacer is a linear molecular chain, preferably a peptidic compound, more preferably a pentapeptide with short side chains. Method according to Claim 6, characterized that the principles 5.1.1. to 5.1.3. before, during and / or after the synthesis, preferably the solid phase synthesis, the test substances be introduced. Method according to claim 5, characterized in that that in step 5.2. realized connection to the Carrier resin before, during and / or after the synthesis, prefers solid phase synthesis, the test substance molecules he follows. Method according to claim 15, characterized in that that the test substance molecules via the orthogonal to the synthesis steps cleavable linker to the carrier resin are bound. Method according to claim 5, characterized in that that in process step 5.3. a dense monolayer of stochastic distributed, loaded with different test substances carrier resin beads is applied to the carrier plate. Method according to claim 17, characterized in that that a mixture of carrier resin beads of different Synthesis is applied. Method according to claim 17, characterized in that that mixtures of carrier resin beads from different syntheses or also carrier resin beads from a synthesis in spatially applied to separate compartments of the carrier plate become. Method according to claim 17, characterized in that that the monolayer is through 5.3.1. Separating the carrier resin beads, 5.3.2. Powder, dip or centrifugal coating, 5.3.3. then Combing and 5.3.4. Dusting is generated. Method according to claim 17, characterized in that that on the carrier plate a biocompatible, non-toxic Adhesive layer is applied. Method according to claim 5, characterized in that that in step 5.4. realized transmission the test substances 5.4.1. Cleavage of the orthogonal linker, 5.4.2. Diffusion of the test substance molecules in the source layer the carrier resin beads, 5.4.3. Interaction of the transmission principle with the test surface, 5.4.4. Alignment of the test substance molecules on the test surface and 5.4.5. Binding the transmission principle takes place at the test surface. Method according to claim 22, characterized in that that the surface of the test plate, matching the respective transmission principle modified, preferably hydrophobed. Method according to claim 22, characterized in that that the transfer process by a the carrier resin beads enveloping film of a medium, preferably water or buffer solution, is taught. Method according to claim 22, characterized in that that the transmission of the test substances in singular Contact zones between the test plate and the carrier resin beads he follows. Method according to claim 22, characterized in that that per carrier resin bead a homogeneous substance spot containing only one test substance on which test plate is generated. Method according to claim 22, characterized in that that method step 5.4. to generate several identical ones Test plates can be repeated. Method according to claim 5, characterized in that that the process step 5.5., the investigations of the activity and / or mode of action of the test substances, directly on the surface the test plate is performed. Method according to claim 5, characterized in that that in step 5.6. Realized assignment of substance spots done by intelligent pattern recognition methods. Method according to claim 29, characterized that the carrier plate and the test plate with markings, which are suitable for defining a coordinate system provided are. Method according to claim 29, characterized that on the support plate from a stochastic pattern markers transferable by the method according to claim 1, preferably made of carrier resin beads containing fluorescence dye molecules, containing the principles 5.1.1. to 5.1.3., are loaded. Method according to claim 29, characterized that based on one through the markers and / or marker patterns defined coordinate system each test substance spot unique Coordinates can be assigned. Method according to claim 29, characterized that due to the coordinates determined a one-to-one assignment each test substance spot to the original carrier resin bead is possible. Method according to claim 29, characterized that transmission errors such as distortion, torsion and Shrinkage effects corrected by special mathematical algorithms can be. Method according to claim 5, characterized in that that the process step 5.7, the detection of the chemical structure selected test substances, directly on the carrier plate takes place at the associated carrier resin beads. Application of the method according to claim 1, characterized characterized that it is for extensive testing for the investigation of interactions of the test substances with specific ones Can use target molecules. Application of the method according to claim 1, characterized characterized that it is for extensive testing to study the biological activity of test substances, preferred for drug screening and antibody testing can use. Application of the method according to claim 1, characterized characterized that it is for the medical-biological Can use diagnostics.