WO2009080766A2 - Procédé de fabrication et application de réseaux ordonnés aléatoires de composés chimiques - Google Patents

Procédé de fabrication et application de réseaux ordonnés aléatoires de composés chimiques Download PDF

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WO2009080766A2
WO2009080766A2 PCT/EP2008/068056 EP2008068056W WO2009080766A2 WO 2009080766 A2 WO2009080766 A2 WO 2009080766A2 EP 2008068056 W EP2008068056 W EP 2008068056W WO 2009080766 A2 WO2009080766 A2 WO 2009080766A2
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chemical
beads
bead
plate
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WO2009080766A3 (fr
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Manfred Auer
Hubert Gstach
Guenter Roth
Karl-Heinz Wiesmueller
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Novartis Ag
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B20/00—Methods specially adapted for identifying library members
    • C40B20/02—Identifying library members by their fixed physical location on a support or substrate
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00459—Beads
    • B01J2219/00466—Beads in a slurry
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
    • B01J2219/00648—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of solid beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides

Definitions

  • the following invention describes a method for efficient generation and application of random ordered arrays of chemical compounds.
  • an array of random ordered chemical substances is generated by a transfer (printing step) from an array of random ordered array of particles (master).
  • the particles are polymer beads which are bearing these chemical substances.
  • the "printed" array is mirror symmetrical to the array bearing the beads. The spatial allocation between the master and the print could be therefore made and each structure on the print could be allocated to its "progenitor" particle on the master.
  • the methods significance is also that the generation of the print, is independent from the number of transferred chemical substances. This is due to the parallel transfer of all compounds within one transfer step (instead of sequential transfer of chemical compounds in other methods).
  • the printed arrays are mainly being used for biomedical, diagnostic or biochemical assays for biological activity.
  • the main claim of this patent is not this pattern recognition.
  • the main claim is the parallel transfer of all chemical compounds for the purpose to generate an array, time independently of the number of chemical compounds.
  • microarrays (Ekins, R.P.: Multi-analyte immunoassay. J.Pharm.Biomed.Anal. 1989, 7:155-168. Fodor, S.P.; Read, J. L; Pirrung, M. C; Stryer, L; Lu 1 AT. und Solas, D.: Light- directed, spatially addressable parallel chemical synthesis. Science 1991, 251 :767-773) have been derived as logical miniaturisation and parallelisation step from already used assays and arrays.
  • Today microarrays, also named biochips, are used to simplify and parallelise biomedical, biochemical, pharmaceutical or biological tests, processes and assays.
  • the used chemical compounds are manifold like nucleic acids, proteins, peptides, small chemical compounds or sugars, which are interacting either with other molecules, biochemical compounds, cells, microorganisms or whole organs or species. Mainly the activity of a chemical compound is detected as molecular binding event on microarrays. Microarrays consist of a multitude of different spots with each spot bearing one single chemical compound. This high density order of spots allows the parallel detection of activity with a small amount of sample. To prepare such arrays each compound has to be deposited on a predefined position, such that in case of activity from the position of the active spot one could conclude which chemical compound is active.
  • the methods of the combinatorial solid phase synthesis opened the possibility to synthesis millions of different chemical compounds within several days.
  • the basic idea of this method is to synthesise all compounds on microparticles (beads). According to the spit-and-mix synthesis method after the synthesis each bead is bearing exactly one chemical compound. Actual this beads were separated from each other an the chemical compounds are cleaved of the bead with each bead in another vial, thus ensuring that each vial contains only one compound in solution (stock solution). This means a high technological and time consuming effort.
  • the chemical compounds are either tested for activity direct within the vial or for performance of a multitude of experiments instead of only one the chemical compounds are transferred onto a microarray or a microtiter plate, and tested there.
  • the transfer of the chemical compounds is made by pipetting the stock solutions of the chemical compounds onto a mircoarray of into a well of a microtiter plate.
  • pipetting robots with automatic xyz-pipetting devices are used to transfer the millions of compounds. But still it is a technique which is time consuming and quite a lot of effort.
  • microarrays The production of microarrays is mainly limited due to the time of transfer of the chemical compounds. But the printing process takes a minimum limit of time to take up the chemical compound solution and transfer it to the microarrays. To reduce this transfer time multiparallel print heads had been developed with up to 384 printing heads. But still the printing time is increasing with the number of chemical compounds which have to be transferred.
  • the assays could be made directly on bead.
  • the chemical compounds therefore are still bound to the synthesis beads and the assay with small target molecules (other small molecules, proteins, DNA and other bio molecules) were performed by direct mixing of beads with these target molecules.
  • Some of these sequencing techniques use a non-bead based method and generate a random distribution of the DNA on the surface.
  • This invention therefore comprises to solve the need for an efficient method for the production of arrays directly made from chemical compounds synthesised on beads by combinatorial solid phase synthesis. It solves the complete task from generation of the random ordered bead array (bead plate) with chemical compounds, their transfer onto a microarray (assay plate) via a print process and the performance of a biological assay.
  • the printing step which is generating the assay plate as print from the bead plate could be repeated many times until the beads on the bead plate are depleted from chemical compounds.
  • the invention combines the advantages of a bead based array for the generation of an array, but is than transferring the chemical compounds onto a surface, so that the advantages of a standard mircoarray could be applied whilst performing a biological assay.
  • the method of the invention comprises the following combination of features to allow the generation of random ordered bead arrays, the transfer from each bead its chemical compound onto a microarrays (one bead generates one dot) and the performance of a biochemical assay. Therefore it comprises the following points
  • the whole process is shown in figure 0.
  • the method comprises chemical compounds that are bound to the beads.
  • the chemical compounds are synthesised to have the following principles.
  • the preferred synthesis of the chemical compounds may be made with classical combinatorial solid phase synthesis the starting molecules are bound onto pre-treated, functionalised beads and sequentially build up to the final chemical compounds.
  • a residue preferably comprises already principle 1.
  • beads from different libraries could be mixed to generate a larger library of chemical compounds. Also beads bearing other compounds could be used as markers for bioassays or to allocate them within the bead matrix on the bead plate. Within this mix of beads all chemical compounds are randomly distributed.
  • the beads with the chemical compounds were immobilised onto a bead plate (preferably on a planar plate).
  • the immobilisation procedure allows the formation of a bead monolayer. This could be done (but not only) by a biocompatible non-toxic adhesive layer on the surface of the bead plate. Whilst the transfer process the beads are immobilised in a random ordered manner and form like such a bead array.
  • the procedure to immobilize the beads on the bead plate may include powder coating, dip coating or centrifugal coating.
  • combing removes simply of the multilayer beads by moving an edge close to the surface of the bead plate.
  • a compartmentation of the bead plate could be performed.
  • a specific mixture or sublibrary could be filled in cavities, which than are used for coating of the bead plate.
  • a microtiter plate could be used as cavity for storage of this sublibraries and for the coating.
  • the transfer again could be made like on the non- compartmented bead plate, but will lead to a compartmented bead plate. This allows sub- compartments and therefore subarrays.
  • the transfer process from bead plate to assay surface can be performed easily by the skilled person.
  • a planar bead plate is brought in close physical contact to the assay plate, which is assisted or mediated with a medium which is ensuring or enforcing a single contact zone from each bead to the assay plate.
  • the compounds are deposited on the surface of the assay plate and is generating a "dot" which is coated with the chemical compound from its progenitor bead.
  • water or cell media is used as transfer media.
  • the contact zone between the bead of the bead plate and the assay plate depends on the materials of the beads, the surface of the assay plate, the size of the bead, the amount and chemical properties of the transfer medium and applied pressure. All this variables have to be optimized to ensure a homogenous transfer from each chemical compound of each bead to each dot on the assay plate. Principle 2 is optimized to allow this transfer.
  • the anchoring of the chemical compounds onto the assay surface could be made by any physico-chemical interaction like covalent, ionic, electrostatic, magnetic, adhesive, adsorptive or ligand-receptor- interactions.
  • the assay plate generated as print of the bead array is mirror symmetrical in respect to the bead array (figure 1).
  • Each bead is generating one dot on the surface of the assay plate. Due to the immobilisation of the beads on the bead array the pattern of beads is printed in a one-to-one allocation onto the assay plate.
  • Principle 1 allows the release of the compounds if the print could be performed.
  • Principle 2 allows the transfer and anchors the compound in a manner so that the compounds of a bead are only generating a dot on the assay plate directly underneath the bead (figure 0).
  • the chemical compounds form a dense monolayer in this dot due to the structure of principle 2.
  • Principle 3 allows now a screening, due to the fact that the biocompatible spacer is optimized to prevent non-specific interactions.
  • the transfer process could be repeated until all chemical compounds are transferred and the beads are empty.
  • the amount of transferred chemical compounds could be controlled by contact time between bead plate and assay plate and by releasing time according to principle 1. This could be used to perform dilutions and will lead to arrays with different compound densities and concentration rows.
  • a positive or negative identified dot could be allocated to its progenitor bead on the bead plate.
  • the allocation is made due to a doping of the beads with beads that are generating a colour on the assay surface, which allows to allocate this pattern to the pattern of the bead plate. This could be made due to pattern recognition and is based on the theorems pronounced by Riemann 1849 about manifolds in geometry.
  • the coloured beads contain coloured chemical compounds bearing principle 1 to 3 like all other beads, but with coloured or fluorescent residues.
  • the coloured beads are mixed to all other beads so that each transfer will generate a fluorescent pattern on each assay plate (like the dark dots in figure 1).
  • According to the allocation of 3 dots to their progenitor beads all beads and dots could be allocated to each other by using mathematical algorithms like, torsion, shrinking and rotation.
  • the according progenitor bead could be allocated.
  • the allocated bead than could be analysed for its chemical compound preferably by MALDI or mass spectroscopy. Than the analysed chemical compound could be determined as positive and therefore active compound.
  • the invention covers all applications to this method of transfer.
  • the main applications are used whilst biochemical or biological probing, in which large amounts of chemical compounds are tested for binding against peptides, proteins or other molecules.
  • the chemical compounds could be any chemical molecules, but are preferably peptides.
  • the invention is predestined as method in the field of pharmacy for the screening of structure- effect-relationship for drug screening, where the interaction of one single molecule is tested against a large number of binding partners.
  • This binding partners are the chemical compounds which could be transferred by the method comprised by the patent.
  • the present invention therefore solves for the first time the spatial resolved transfer of a random ordered bead array to a random ordered array of chemical compounds without any pipetting steps and a low technical effort. It also solve the problem of increasing time amount with increasing number of chemical compounds that have to be transferred. It solves as well the problem of screening of bead arrays and of manufacturing of chemical arrays by combining both advantages without the disadvantages.
  • the beads are used to form fast a random ordered bead array, than the printing step allows to generate a normal array and this could be used for normal screening.
  • An one-to-one allocation allows than to identify the active dot and the active bead. So a chemical analysis of the chemical compound from the bead could be made according to the positive dot on the microarray, without any pipetting steps for the transfer of the compounds or the array generation.
  • the invention interconnects the bead arrays to the surface arrays by a simple printing step which transfers the chemical compounds from each bead to a according single dot on the microarray.
  • Each dot consists of the chemical compounds according to its progenitor bead.
  • the assays could be performed later on the assay plate as on any commercially available microarray.
  • This printing step solves the problem that the time amount doesn't increase if the number of chemical compounds is increased. In all other methods a dramatic increase of time consumption is observed. Therefore the time consumption of the transfer step to generate the microarray is independent of the number of transferred chemical compounds.
  • the transfer process could generate in an ideal way a monomolecular layer of the chemical compounds and therefore a performance of an assay would lead to a significant signal yield, which could also be counted as advantage of the invention.
  • the preparation of the bead plate could be seen as master, which could be copied multiple times onto an assay plate. Therefore multiple copies of each master could be made.
  • This multiple copying is another advantage of the invention.
  • the transfer process itself is instantaneously and could be done within 20 seconds and as said is completely independent from the number of transferred chemical compounds, used beads or size of assay or bead plate.
  • the spatial resolution of dots after performance of a biochemical assay was about 10% of the bead diameter which means 2 microns or less.
  • This invention comprises therefore the fast production of a variety of arrays which isn't possible with all the other methods. No other method allows this easy control.
  • Figure O Schematic view of the transfer process a) formation of a bead monolayer on the master plate b) physical contact between the master and the assay plate c) release of chemical compounds from bead (principle 1 ) d) transfer from bead and anchoring on the assay plate surfaces of the molecules (principle 2) e) removal of master plate and repeated printing f) performance of biochemical assay with less background due to spacing between anchoring and screening part of the chemical compounds (principle 3) g) allocation of positive dots to their progenitor beads
  • Figure 1 Schematic view of the random pattern on the bead plate (right) and the corresponding mirror symmetrical pattern on the assay plate after performance of an biological assay. Positive dots are shown in black.
  • Figure 6 Chemical structure of a intermediate after coupling of Pam 3 Cys-OH and before further coupling of biological active substances after spacing with ⁇ -Alanine.
  • FIG. 7 Synthesis scheme for coupling of photo cleavable linker (step 1 ).
  • FIG. 8 Synthesis scheme for methylamidation of photo cleavable linker (step 2).
  • Figure 14 Enlargement of Figure 13 with assignment of some compounds of the peptide collection
  • Figure 15 Microscopy picture of bead array generated by powder coating and combing with synthesis beads (020 ⁇ m, image size 780x600 ⁇ m).
  • Figure 16 Microscopy picture of bead array generated by dip coating and combing with synthesis beads (020 ⁇ m, image size 2600x2000 ⁇ m).
  • Figure 17 Microscopy picture of bead array generated by centrifugal coating and combing with synthesis beads (020 ⁇ m, image size 780 x600 ⁇ m).
  • FIG 19 Scheme about immobilised bead on the bead plate.
  • the immobilisation is shown on the example of a adhesive thin film.
  • FIG. 20 Scheme of the transfer step of chemical compounds.
  • the transfer medium comprises a contact zone which determines the dot which is generated after the transfer.
  • the release of the compounds is made in this example by radiation.
  • FIG. 22 Chemical structure of Pam 3 Cys-SK 3 K(Carboxyfluorescein)-NCH 3 .
  • Figure 23 Chemical structure of Pam3Cys-SK 3 K(Aca-Tetramethylrhodamin)-NCH 3 .
  • Figure 24 Microscopic picture of the generated dot pattern of fluorescent beads (upper left picture), the first copy as array (lower left) and a second copy as array (lower right).
  • Figure 25 Microscope images of sub-compartments within the bead arrays of the master plate.
  • the master plate was manufactured with centrifugal coating.
  • FIG. 26 Master plate with 65 sub-compartments on the storage plate. Each compartment contains another chemical sub library and doping with different amounts of fluorophores, wielding in different colours.
  • FIG 27 ESI-MS spectra of a biotinylated compound Figure 28 Master plate with massive doping of rhodamine (red) or fluorescein (green) bearing beads. Cross contaminations after printing processes lead to yellow colour.
  • the arrows show missing beads on the master plate.
  • the circles mark special positions of beads which are allocated to the assay plate shown in figure 29. Especially the larger bead (lower right circle) generates a larger dot in figure 29.
  • Figure 29 Assay plate generated from master plate in figure 28. Clearly visible that there is no cross contamination between the dots and beads (white arrows) could stick to the surface, but could be removed by washing steps. A larger dot was generated (lower right circle) by a larger bead (see figure 28)
  • Figure 30 Performance of a bioassay by binding of streptavidine (green) onto the dots of an assay plate. Before the incubation (left) of streptavidine only the pattern of the red dots (doping with red labelled beads on the master plate) is visible and a contamination (green). After the incubation (right) the dots which bound streptavidine are clearly visible.
  • Figure 31 Left side is before, right after binding assay. All assay plates bear a red and a green pattern as 100% control and for establishment of an internal coordinate system to find back the same position within the assay plate. From top to bottom different ratios of red and green labelled streptavidine has been used, (green to red ratio: top 3:1 ; 1 :1 ; 1 :2 and bottom 1 :4).
  • Figure 32 Example for an allocation.
  • the bead array on the master plate (upper left, transmission light micrography) was printed onto an assay plate (upper right, fluorescence microscopic image).
  • An overlay of these two pictures showed a perfect fit (lower left).
  • a fluorescence image for green beads of the master plate showed that the beads which are illuminate are over green dots on the assay plate and in any case not on any red dot (lower right).
  • Figure 33 Another example of allocation.
  • the master plate (upper left) was printed several times. Due to different process parameters the generated arrays on the assay plates show differences. But in total the pattern of the red dots could be allocated to each other easily by eye. In cases of larger images a software is needed to allocate to each dot its progenitor bead. But this image is only a example to show that the allocation works fine.
  • Figure 34 Regaining of a bead by laser microdissection. First (upper left) a bead is marked and than cut out by a laser. A final laser impulse is shooting the bead out of the area of the plate (upper right) into a eppendorf cup (lower left). The regained bead was a fluorescent one. Its fluorescence was directly proven in the cup (lower right).
  • Application examples
  • the applied dose determines the amount of cleaved and therefore released chemical compound.
  • the photo cleavable linker is stable during the whole synthesis process. Due to the UV light the covalent binding breaks as such that the released chemical compound bears only an additional methyl group. Due to the difference in size of whole molecule and methyl group it could be suggested that it wont interfere with biomedical assays.
  • the synthesis scheme is shown in figure 2, the cleavage reaction in figure 3.
  • the transfer parts have to be adapted onto the used transfer media and the anchor part has to be adapted onto the surface the compound should be anchored.
  • a hydrophilic transfer media water
  • the anchoring is made by a lipophilic (hydrophobic biomembrane like surface). Therefore the used compound was Pam 3 Cys-OH ( Figure 4) of the company EMC microcollections GmbH was elongated with serine followed by 4 lysines. This molecule provides a lipophilic part (the fatty acids) which will anchor the molecule on the hydrophobic surface and a hydrophilic part (the lysines, K 4 ) which allow the compound be transferred from the bead onto the surface via the hydrophilic solution.
  • the hydrophilicity of the liquid and the hydrophobicity of the surface determine the contact angle of the liquid and therefore the size of the contact area.
  • the assay plate was functionalised with trimethoxypalmitoylsilan to gain a lipophilic surface for the Pam3 anchor of the chemical compound. Contact angles have been between 100° and 115° against pure water
  • the Pam3Cys-compound anchors instantaneously onto this hydrophobic surface by hydrophilic interactions and build up a molecular monolayer. A density of 0.8 nm ⁇ 2 per molecule could be observed. The adhesion of this molecules to this surface was high enough that assays could be performed in hydrous solutions. Only with a mixture of organic solvents together with heat and mechanical shear forces the compound could be removed.
  • the whole example conjugate is shown in Figure 5.
  • the biological active part of the chemical compound could be synthesised via traditional solid phase synthesis. All chemical compounds are identical according to principle 1 to 3. Therefore they reacted in the same way for the transfer steps. Mainly not influenced by the different biological active parts of the molecules. This decouples the transfer from the screening compound in terms of transferability.
  • TentaGel S-NH 2 was used as synthesis beads.
  • the Fmoc protection group was cleaved of after each coupling step respectively. (Step 3 and 4).
  • Step 7 After photo cleavage (Step 7) and separation from the beads the resulting chemical compound showed hydrophobic attributes but was soluble in water with > 1 mg/ml.
  • Step 1 Coupling of photo cleavable linker ( Figure 2 and 7)
  • the washed resin was incubated with this solution and shook for 16 h at 20 0 C.
  • the resin was filtered, washed (each 6 x 5 ml DMF, DCM, MeOH) and dried under vacuum.
  • the mixture was shaken at 20 0 C for 3-3,5 h.
  • the resin was filtered and washed (3 x 8 ml DMSO, 6 x 8 ml DMF, 6 x 8 ml DCM, 6 x 8 ml
  • the final mixture was added to the washed resin and incubated under shaking for at least 45 min. In cases of larger or low soluble molecules or fatty acids the incubation time could be elongated for several hours.
  • the resin was filtered and washed with 8 x 8 ml DMF.
  • the resin was filtered and washed with 6 x 8 ml DMF.
  • the resin was filtered and washed with 8 x 8 ml DMF.
  • the resin was resolved in 1 ml 80% tert. Butylalcohol /water again. This mixture than is radiated for 90 min with UV-light at a wavelength of 365 nm.
  • the resin was than incubated with 400 ⁇ l ethanol, incubated for 3 min and than filtrated again.
  • the obtained colourless powder was used for further analysis.
  • the synthesised basic chemical compound was elongated with biotin.
  • Biotin was used representative for any potential biochemical active compound and also representative for any chemical residue that could be coupled onto the basic chemical compound.
  • One main part of the invention is to generate a primary array of beads. To remain the spatial order of this random ordered array the beads on the master plate have to be immobilised. A monolayer would be preferred finally.
  • master plate a plastic polymer on which an adhesive thin film has been coated was used. The thin film is thinner than the radius of the beads.
  • the adhesive layer is UV- permeable and biological inert (in means that it doesn't produce nonspecific binding in a biochemical assay).
  • an immobilised bead monolayer the following 4 steps have been performed.
  • the synthesis beads bearing the chemical compounds show in a dry and non-swollen state a uniform size and shape of a 20 micron diameter sphere.
  • 1 gram of this resin consists of roughly 240 million beads with a loading capacity of 1 picomol molecules of each chemical compound. This means that each bead could carry 6-10 '11 molecules. All beads are uniform in means of shape, size, chemical functionalisation, loading capacity and surface coating.
  • a NH 2 -functionalisation was used in this case.
  • each bead has to be a single bead and does not stick together with others. This is a prerequisite condition for the monolayer formation.
  • the beads For the singularisation the beads have been solved in 60 0 C warm water free tert. Butylalcohol and were sonicated for 20 min in an ultrasonic bath. Thereafter the suspension was shaken for another 20 min, at a temperature above 4O 0 C. In case that no homogenous suspension could be generated the beads were filtered and resuspended in n-Methyl-pyrrolidon and shaken for 1 h, washed than 3 times in diethyl ether. Than again treated with the 60 0 C warm tert. Butylalcohol.
  • the bead solution was shock frosted in liquid nitrogen and than lyophilised at -80 0 C and 0,5 mbar until a fine powder was gained, with an amount of less than 0,5 %o non-singled beads.
  • Powder coating is a widely used technique and allows coating of the master plate very easy.
  • the master plate is put in the upper part of the coating device and the powder is blown up from the bottom of the device. Beads which are fast enough to penetrate the adhesive layer penetrate it and will stick into it. Whilst beads which are to slow will only touch it and fall back into the containment to the other beads and will get blown up again.
  • Dip coating is another widely used technique. It could be used to coat the master plate but it is less efficient like the powder coating and dorms more multilayers than monolayers. In case of dip coating the master plate is simply dipped into an excess of beads and shaken. The movement together with the dipping ensured that the beads would stick onto the master plate. Due to electrostatic charging multilayers could also be generated. In that case the multilayers have been removed with a brush.
  • Centrifugal coating is a coating principle developed for this invention. It is not know to the author if there had been a similar development elsewhere. Even if this coating principle is not of importance for the invention, it allows making sub compartments within the bead layer on the master plate, preferably for each compartment a sub library from the synthesis is used. The working principle is shown in figure 30.
  • the singularised beads were put into a compartment and the compartment is sealed with the master plate.
  • the adhesive thin film will seal the compartment.
  • the beads are centrifuged into the adhesive layer.
  • the compartment is taken out and put back into the centrifuge in a reversed position.
  • the beads are forced back into the compartment. Only the beads which are stick into the adhesive layer will stay on the master plate.
  • the penetration depth of the beads into the adhesive layer could be controlled by the revolutions per minute of the centrifuge and the centrifugal time.
  • Combing 1 Final immobilisation of the beads on the master plate
  • Microscopy images showed that not all beads have been brought into a nice order in means of penetration depth into the adhesive thin film or multilayers.
  • a technique called combing was used.
  • To bring all beads into the same penetration depth a metal edge was carried in a defined distance over the master plate surface. The angle (in direction of the edge movement) between the edge and the master plate was less than 30°, so that the beads have been pressed into the adhesive layer. This step was closing non filled structures with multilayer beads and ensured that all beads stuck into the adhesive.
  • the generated master plate was a bead monolayer array with nearly no multilayer beads.
  • Example F Transfer of the chemical compounds from the beads of the master plate into a array onto the assay plate
  • a chemical structure was designed and synthesised bearing the photo cleavable linker 4-Bromomethyl-3-nitrobenzoic acid which was coupled to the amino group of the synthesis beads (principle 1).
  • the pentapeptide Serin- Lysin-Lysin-Lysin-Lysin with a side chain elongation of ⁇ -Alanin- ⁇ -Alanin was made (principle 3).
  • the liphophilic group Pam 3 Cys-OH was used (principle 2).
  • the pentapeptide Serin-Lysin-Lysin-Lysin-Lysin-Lysin also enhanced the solubility of the molecule in water and counts therefore also to principle 2.
  • As assay plate a microscope slide was used after silanisation with trimethoxypalmitoylsilan performing a hydrophobic surface.
  • Bead arrays generated like described in example E were used as master plates with a size of a microscope slide.
  • the coated area was 24 x 70 mm with roughly 4,000,000 beads per master plate.
  • the beads were wetted with water. An excess of water was removed with a gas blow.
  • Than the master plate was brought in a close physical contact with the hydrophobic assay plate.
  • the hydrophobic coating of the assay plate ensures that a small singular contact area is generated.
  • Each bead is generating exactly one contact area in the shape of a circular dot.
  • an array of dots bearing the chemical compounds remain on the assay plate.
  • the pattern of the dots could be allocated to the bead pattern due to a doping with beads bearing a fluorescent labelled compound.
  • the beads Due to the fact that the beads have a chemical loading capacity in the range of a picomol and a dot only consumes 200 to 300 attomol there is enough material to generate (mathematically) more than 1000 copies from each master plate. Several copies (up to 20) have been made manually. By changes in the illumination time for the cleavage the amount of released chemical compounds could be determined. In a efficient printing step only the amount of molecules which could be anchored on a dot will be released.
  • Biotin is a small molecule which could easily coupled to a chemical compound (figure 27), whilst streptavidine is used widely in many biochemical assays and staining steps. Therefore a master plate has been generated with different beads (non-biotinylated, biotinylated and red fluorescent).
  • the red fluorescent beads generate a pattern of red fluorescent dots on the surface of the assay plate (figure 30 left). This pattern allows the recovery of this position after performance of assay. Before the assay only the red fluorescence is visible (figure 30 left). After the assay the red pattern has slightly changed due to washing effects but could be still allocated to the original pattern. But now a green fluorescence occurred (figure 30 right) as a significant sign for binding of green labelled streptavidine. Thus it could be proven that it is possible to perform a biochemical binding assay on the assay plate surfaces.
  • a ratiometric assay is a commonly used technique. Within this test two different samples were used. One sample is marked with a green, the other with a red fluorophore and than mixed. The mixture is poured onto a microarray and both samples than compete with each other for binding on the array. In cases that both samples bind equally strong to the spot 50% is green and 50% is red, which is resulting in a yellow colour. In cases that one sample binds stronger the colour will shift to that of the strongest binder.
  • Such an assay could be simulated by using a known pair of molecular binders. In this case as chemical compound a biotin was used. Streptavidin is binding biotin. Therefore a amount of streptavidine was labelled green and another red. A mixture of both streptavidine derivatives was made in different ratios.
  • Assay plates have been generated containing a biontinylated compound and a doping with red and green labelled compounds. Before the binding only this pattern of red and green dots is visible (figure 31 left). After the incubation with the streptavidine mixture the before non- visible dots are now fluorescent, according to the ratio of the streptavidines to each other, in a different colour (figure 31 right). Thus is proving that this assay could be used as ratiometric assay as well.
  • the master plate which is allocated to a positive dot on the assay plate. This could be made with laser microdissection. Like from a cell tissue the polymer of the master plate could be cut out with the laser and the bead is catapulted out into a compartment like an eppendorf cup. The sequence of marking, cutting and the regained bead in the cup is shown in figure 34 The bead could be used like any other for analysis.

Abstract

Cette invention concerne un procédé de fabrication et d'application efficaces de réseaux ordonnés aléatoires de composés chimiques. Le procédé implique la génération d'un réseau ordonné aléatoire de billes, chacune portant exactement un composé chimique. Les composés chimiques des billes du réseau de billes (matrice) sont transférés sur une autre surface (plaque de dosage) générant une copie chimique du réseau de billes sur cette surface. La plaque de dosage peut être utilisée comme n'importe quelle micromatrice.
PCT/EP2008/068056 2007-12-20 2008-12-19 Procédé de fabrication et application de réseaux ordonnés aléatoires de composés chimiques WO2009080766A2 (fr)

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