CA2347923A1 - Solid phase synthesis with the solid phase consisting of two different substrates - Google Patents
Solid phase synthesis with the solid phase consisting of two different substrates Download PDFInfo
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- CA2347923A1 CA2347923A1 CA002347923A CA2347923A CA2347923A1 CA 2347923 A1 CA2347923 A1 CA 2347923A1 CA 002347923 A CA002347923 A CA 002347923A CA 2347923 A CA2347923 A CA 2347923A CA 2347923 A1 CA2347923 A1 CA 2347923A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
-
- 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
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
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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
- 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
- B01J2219/00279—Features relating to reactor vessels
- B01J2219/00281—Individual reactor vessels
- B01J2219/00283—Reactor vessels with top opening
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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
- 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
- B01J2219/00279—Features relating to reactor vessels
- B01J2219/00306—Reactor vessels in a multiple arrangement
- B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
- B01J2219/00315—Microtiter plates
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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
- 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
- B01J2219/00351—Means for dispensing and evacuation of reagents
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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
- 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
- B01J2219/00351—Means for dispensing and evacuation of reagents
- B01J2219/00373—Hollow needles
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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
- 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
- B01J2219/0047—Pins
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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
- 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
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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
- 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
- B01J2219/00599—Solution-phase processes
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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
- 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
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00702—Processes involving means for analysing and characterising the products
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
- C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
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- Genetics & Genomics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
- Ceramic Capacitors (AREA)
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- Inorganic Compounds Of Heavy Metals (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention relates to the synthesis of substances on a solid phase consisting of two different substrates, to a suitable reaction system and to a method comprising solid-phase synthesis and an analytical process during whi ch the chemical structure and the biological properties of the synthesised substance are analysed. The inventive solid-phase synthesis of substances in a reaction vessel containing a liquid phase is characterised in that the solid phase for the synthesis comprises a substrate that is reversibly added in th e liquid phase and the wall of the reaction vessel in its entirety or in part.
Description
Solid Phase Synthesis With the Solid Phase Consisting of Two Different Sa~bstrates The present invention relates to the synthesis of substances on a solid phase, said solid phase consisting of two different substrates, a reaction system suitable for this purpose and a process comprising a solid phase synthesis and an analytical process by which both the chemical structure and the biological characteristics of the synthesised substance are analysed.
The synthesis of molecules on a solid phase is especially advantageous when a number of consecutive revac;tion steps is required to build up the molecule, because the solid phase permits easy separation of the respective intermediates obtained from the reactants and automation of the synthesis. In particular, the solid phase synthesis is the standard synthesis for producing polypeptides and polynucleotides. In order to provide the largest possible surface at a given reaction volume, porous solid phases are generally used.
In the last few years, the significance of the solid phase synthesis has increased considerably, especially due its application in forming chemical libraries.
For example, such chemical libraries have been produced by the so-called "split-mix" process wherein micro-spheres, so-called beads, are split over several reaction vessels, reunited after the first step of the synthesis, for example attachment of the first substituent, and split once again for the second variable substituent. Repeating this :process provides a technically simple way to generate thousands or millions of different molecules, only one specific molecule species being lc7cated on each bead. In order to form a sufficient quantity of a substance and for easy handling of the solid phase, porous beads are generally used as the support material. For biological screening of the synthesised substance theM so-called FACS (fluorescence assisted cell sorting) process is generally used directly on the bead, but this process works only for certain receptor-ligand pairs. Alternatively, the substance may be removed from the bead by dissolution and subjected to an assay in solute form; this results in several substances being mixed which is a disadvantage (Felder, Chimia 48, pp. 531 - 541, 1994).
In particular, multi-parallel syntheses wherein a specific species is synthesised per reaction vessel or solid reaction surface, respectively, have been proposed as an alternative to the "split-mix" process. Even though the number of species which may be synthesised is smaller vis-a-vis the "split-mix" technique, this process has the advantage that the identity of the synthesised product may be verified at all times due to its fixed position and that a larger quantity may be synthesised. As a rule, tl-~e synthesis proceeds on a support having the largest possible surface, for example porous resin substrates. For biological assays, the product of the synthesis must therefore be removed from the support by dissolution once again so that the disadvantages associated with screening liquid samples, for exarr~ple artefacts caused by intermolecular interactions, remain. If the synthesis is carried out on a non-porous planar support instead, the amount synthesised is t:oo small for many chemical analyses, especially in case of miniaturised surf°aces.
Against this background, the objective of the invention was to provide a simple solid phase synthesis permitting both the synthesis of a quantity of the sub-stance sufficient for chemical identification of the substance obtained and greater sensitivity of detection of the biological activity of the substance obtained vis-a-vis the prior art.
According to the invention, this object is achieved by the solid phase synthesis of substances in a reaction vessel containing a liquid phase, characterised in that, for the synthesis, the solid phase comprises a substrate reversibly intro-duced into the liquid phase and all or part of the wall of the reaction vessel.
Preferably the part of the: vessel wall used as the solid phase for synthesis according to the invention :is the base of the reaction vessel. This base is preferably not porous and, especially preferably, not porous and planar.
The substrate reversibly introduced into the liquid phase is preferably one or more beads, e.g. beads made of a resin, for example a chemically modified polystyrene basic resin. ~:'hese beads permit easy handling of the substrate after completion of the synthesis on the one hand and have a larger surface on the other so that the amount ~f the synthesised substance becomes very large at a given volume. Alternatively, the substrate reversibly introduced into the liquid phase may also be the tip of a needle, for example a plastic needle, as used already in mufti-parallel ,,yntheses (Geysen et al., Proc. Natl. Acad. Sci.
U.S.A. 81, 3998, 1984).
The synthesis of molecules on a solid phase is especially advantageous when a number of consecutive revac;tion steps is required to build up the molecule, because the solid phase permits easy separation of the respective intermediates obtained from the reactants and automation of the synthesis. In particular, the solid phase synthesis is the standard synthesis for producing polypeptides and polynucleotides. In order to provide the largest possible surface at a given reaction volume, porous solid phases are generally used.
In the last few years, the significance of the solid phase synthesis has increased considerably, especially due its application in forming chemical libraries.
For example, such chemical libraries have been produced by the so-called "split-mix" process wherein micro-spheres, so-called beads, are split over several reaction vessels, reunited after the first step of the synthesis, for example attachment of the first substituent, and split once again for the second variable substituent. Repeating this :process provides a technically simple way to generate thousands or millions of different molecules, only one specific molecule species being lc7cated on each bead. In order to form a sufficient quantity of a substance and for easy handling of the solid phase, porous beads are generally used as the support material. For biological screening of the synthesised substance theM so-called FACS (fluorescence assisted cell sorting) process is generally used directly on the bead, but this process works only for certain receptor-ligand pairs. Alternatively, the substance may be removed from the bead by dissolution and subjected to an assay in solute form; this results in several substances being mixed which is a disadvantage (Felder, Chimia 48, pp. 531 - 541, 1994).
In particular, multi-parallel syntheses wherein a specific species is synthesised per reaction vessel or solid reaction surface, respectively, have been proposed as an alternative to the "split-mix" process. Even though the number of species which may be synthesised is smaller vis-a-vis the "split-mix" technique, this process has the advantage that the identity of the synthesised product may be verified at all times due to its fixed position and that a larger quantity may be synthesised. As a rule, tl-~e synthesis proceeds on a support having the largest possible surface, for example porous resin substrates. For biological assays, the product of the synthesis must therefore be removed from the support by dissolution once again so that the disadvantages associated with screening liquid samples, for exarr~ple artefacts caused by intermolecular interactions, remain. If the synthesis is carried out on a non-porous planar support instead, the amount synthesised is t:oo small for many chemical analyses, especially in case of miniaturised surf°aces.
Against this background, the objective of the invention was to provide a simple solid phase synthesis permitting both the synthesis of a quantity of the sub-stance sufficient for chemical identification of the substance obtained and greater sensitivity of detection of the biological activity of the substance obtained vis-a-vis the prior art.
According to the invention, this object is achieved by the solid phase synthesis of substances in a reaction vessel containing a liquid phase, characterised in that, for the synthesis, the solid phase comprises a substrate reversibly intro-duced into the liquid phase and all or part of the wall of the reaction vessel.
Preferably the part of the: vessel wall used as the solid phase for synthesis according to the invention :is the base of the reaction vessel. This base is preferably not porous and, especially preferably, not porous and planar.
The substrate reversibly introduced into the liquid phase is preferably one or more beads, e.g. beads made of a resin, for example a chemically modified polystyrene basic resin. ~:'hese beads permit easy handling of the substrate after completion of the synthesis on the one hand and have a larger surface on the other so that the amount ~f the synthesised substance becomes very large at a given volume. Alternatively, the substrate reversibly introduced into the liquid phase may also be the tip of a needle, for example a plastic needle, as used already in mufti-parallel ,,yntheses (Geysen et al., Proc. Natl. Acad. Sci.
U.S.A. 81, 3998, 1984).
It is an essential feature of the invention that, in each step of the synthesis, both the substrate reversibly introduced into the liquid phase and the part of the ves-sel wall of the reaction vessel acting as the solid phase are present in such a chemically modified state that both components of the solid phase may immo-bilise the same molecules .and preferably have an almost identical reactivity.
A
large number of resins far the solid phase synthesis is available commercially (e.g. from Calbiochem-f~Iovabiochem GmbH, Schwalbach), which may be used according to the invention as substrates that rnay be reversibly introduced into the liquid phase. Examples include the Merrifield resin (chloromethyl-derived polystyrene resin), hydroxy-functionalised resins, amino-function-alised resins, trityl resins, arylsilyloxy resins, carboxy-functionalised resins, aldehyde-functionalised resins, thiol-functionalised resins and carbonate resins to which different molecules having different reactive groups may be attached directly or via a spacer in a~ manner known to the person skilled in the art depending on the reactive group. If a certain resin has been selected as substrate capable of reversible introduction for the synthesis according to the invention, care must be taken to ensure that all or part of the vessel wall of the reaction vessel acting as the solid phase bears functional groups permitting immobilisation of the same molecules as on the substrate capable of reversible introduction. Preferably, the reversibly introduced substrate and the part of the vessel wall of the reaction vessel acting as the solid phase have the same functional groups. Suitable methods for functionalising the solid phase are well known to the person skilled in the art. For example, surfaces may generally be functionalised by silanisation or the Langmuir-Blodgett technique. If the part of the vessel wall of the reaction vessel acting as the solid phase is a glass base, for example, and if an amino-functionalised resin has been selected as the substrate capable of reversible introduction, the amino function on the glass surface may be generated by derivatisation, for example with aminopropyl trimethoxysil:rne. If, on the other hand, the base of the reaction vessel is an acrylic glass base, for example, and a carboxy-functionalised resin has been selected as the substrate capable of reversible introduction, the carboxy function on the acrylic glass surface may be applied by a Langmuir-Blodgett film as disclosed in DE C 4332003, for example.
When adding the synthesis component for the solid phase synthesis to a solid phase prepared in such a rn,anner care should be taken to ensure that the amount of this synthesis ~;omponent is added in excess to the functional groups bonded to the solid phase. (liven sufficient reaction time, this ensures that both components of the solid phase react fully even in case of differences of reac-tivity between the functional groups on the substrate reversibly introduced into the liquid phase and on thc; vessel wall of the reaction vessel. This makes sure that the same final molecule is synthesised on the vessel wall of the reaction vessel and in the reversibly introduced substrate. Also, care must be taken to ensure sufficient rinsing between the individual steps of the synthesis. For this purpose, the upper opening of tile reaction vessel is preferably provided with a screen which ensures that the reversibly introduced substrate remains in the reaction vessel during the rinsing step. If the sidewall of the reaction vessel is used as the solid phase, the; reaction solutions may also be siphoned off through a porous base o:F the reaction vessel.
As opposed to the mufti-parallel synthesis known from the prior art, the inven-tion ensures that the substance is not only synthesised on a porous support firmly anchored to the reaction vessel, but also on the vessel wall of the reaction vessel and a substrate reversibly introduced into the liquid phase which may easily be removed from the vessel.
The special advantages of the synthesis method according to the invention re-side in the analysis of both the physico-chemical and the biological character-istics of the synthesised substance which is greatly simplified in comparison with the prior art.
Without any previous reaction and separation steps, a large number of biolo-gical assays may be carried out directly with the synthesis product prepared according to the invention and present on the solid phase. The substrate reversibly introduced into the liquid phase is transferred from the reaction vessel to another container when the synthesis is complete. The synthesis products remaining in the reaction vessel, for example at the bottom of the reaction vessel, may be then be directly subjected to a biological assay. The selection of the biological assay is not limited and includes all assays suitable for analysing biological or biochemical characteristics, for example. In par-ticular, these include chemiluminescence methods, surface plasmon resonance methods, fluorescence methods and surface techniques utilising acoustic chan-ges (acoustic sensors). The advantages of the invention are particularly evident in biological assays which ihave the purpose of determining the interactions of the synthesised substance having receptors directly in a cell membrane or the ligand-receptor interactions which generate a signal too weak to be measured in conventional assays.
Interactions of the synthesised substance with receptors directly in a cell mem-brane may be analysed with cell assays comprising a microscopic observation of the cells through thin transparent substrates. In that case the base of the reaction vessel is selectcvd in such a manner that it is suitable for the cell assay in question. Preferably, the base of the reaction vessel is a glass substrate such as a quartz glass base in case of a fluorescence assay with, for example, calcium imaging. In addition to the facilitated process through the direct synthesis on the bottom of the reaction vessel, the cell assay carried out in accordance with the synthesis of the invention also has improved sensitivity, because all the molecules are present on the surface in a uniform orientation or the orientation may be controlled by the synthesis, respectively.
In addition, the synthesis of the invention is particularly advantageous in cases where an analysis of ligand interactions is required, because conventional assays would not be sufficiently sensitive or too unspecific for such an analy-sis. Owing to the drastic reduction of the detection volume in case of detection of a substance present as a thin surface film, assays have become possible which are more sensitive by several orders of magnitude than pertinent volume methods and which even permit detection of single molecules. Such an assay is described in the German patent application 198 22 542.4 and, in addition to determining absolute cor~cc,ntrations, pernlits determination of affinity constants and binding kinetics which often are a gauge for the specificity of the interaction. Preferably, the base of the reaction vessel for the synthesis accord-ing to the invention is a duartz glass base in that case, too.
Especially when the biological assay shows an interesting result, the chemical identity of the tested compound may be determined in a second step by analys-ing the solid phase removed from the reaction vessel earlier. Classical chemi-cal techniques of analysis such as GC/MS, TOF/MS, MALDI and NMR are considered for this purpose, but micro-sequencing may also be carried out depending of the type of the synthesised substance.
In a preferred embodiment of the invention, the reaction vessel is a well of a microtiter plate, for example a 96, 384 or 1536 microtiter plate. Such a micro-titer plate permits conducting up to 1536 different chemical syntheses in paral-lel in each well by adding different reactants. In addition, microstructurised systems such as systems on the basis of wafer technology are reaction vessels suitable for use according to the invention.
In addition, the invention provides a reaction system suitable for the solid phase synthesis according t:o the invention. The reaction system according to the invention comprises a rnicrotiter plate filled with a liquid phase, character-ised in that each of the wells of the microtiter plate additionally comprise a substrate reversibly introduced into the liquid phase, all or part of each of the substrate and the vessel wall of the pertinent well bearing functional groups for immobilising the same molecules.
Example 400 mg of polystyrene beads (diameter 200 Vim) bearing 200 ~mol of amine groups are dissolved in 5 rnl of dimethyl formamide (DMF) and placed into a well of a microtiter plate the base of which consists of a glass plate having a thickness of 170 ~m and provided with a thin layer of ethoxy aminosilane by known methods [e.g. S. Sec:ger et al. in: Synthetic Microstructures in Biologi-cal Research, Ed.: J. Schnur, M. Peckerar, Plenum Publishing Corporation, p.
53 - 66 (19!2)). Then 1 mrnol of Fmoc amino acid, 1 mmol of dicylohexyl carbodiimide (DCC), 1 mrnol ofN-hydroxybenzotriazole (HOBt) and 1 mmol of dimethyl aminopyridine (DMAP) are added and incubated at room temper-ature over night.
After rinsin;~ with DMF, the Fmoc protective group is cleaved by 30 minutes of incubation with 20 % piperidine in DMF at room temperature and a new reaction cycle for preparing a solid phase-coupled dipeptide begins. The cycle is repeated six times so that: a hexapeptide results. The side chain protective groups are finally removed with 25 % trifluoroacetic acid in dichloromethane (30 min., room temperature) and rinsed.
Depending on the sequence of the amino acids used, corresponding hexapep-tides are generated in the individual wells of the plate. The beads are now removed and may be subjected to chemical analysis methods. The hexapep-tides bound on the glass base of the microtiter plate may easily be investigated for protein bondings by adding fluorescence-labelled target protein molecules and scanning the base with a fluorescence scanner.
A
large number of resins far the solid phase synthesis is available commercially (e.g. from Calbiochem-f~Iovabiochem GmbH, Schwalbach), which may be used according to the invention as substrates that rnay be reversibly introduced into the liquid phase. Examples include the Merrifield resin (chloromethyl-derived polystyrene resin), hydroxy-functionalised resins, amino-function-alised resins, trityl resins, arylsilyloxy resins, carboxy-functionalised resins, aldehyde-functionalised resins, thiol-functionalised resins and carbonate resins to which different molecules having different reactive groups may be attached directly or via a spacer in a~ manner known to the person skilled in the art depending on the reactive group. If a certain resin has been selected as substrate capable of reversible introduction for the synthesis according to the invention, care must be taken to ensure that all or part of the vessel wall of the reaction vessel acting as the solid phase bears functional groups permitting immobilisation of the same molecules as on the substrate capable of reversible introduction. Preferably, the reversibly introduced substrate and the part of the vessel wall of the reaction vessel acting as the solid phase have the same functional groups. Suitable methods for functionalising the solid phase are well known to the person skilled in the art. For example, surfaces may generally be functionalised by silanisation or the Langmuir-Blodgett technique. If the part of the vessel wall of the reaction vessel acting as the solid phase is a glass base, for example, and if an amino-functionalised resin has been selected as the substrate capable of reversible introduction, the amino function on the glass surface may be generated by derivatisation, for example with aminopropyl trimethoxysil:rne. If, on the other hand, the base of the reaction vessel is an acrylic glass base, for example, and a carboxy-functionalised resin has been selected as the substrate capable of reversible introduction, the carboxy function on the acrylic glass surface may be applied by a Langmuir-Blodgett film as disclosed in DE C 4332003, for example.
When adding the synthesis component for the solid phase synthesis to a solid phase prepared in such a rn,anner care should be taken to ensure that the amount of this synthesis ~;omponent is added in excess to the functional groups bonded to the solid phase. (liven sufficient reaction time, this ensures that both components of the solid phase react fully even in case of differences of reac-tivity between the functional groups on the substrate reversibly introduced into the liquid phase and on thc; vessel wall of the reaction vessel. This makes sure that the same final molecule is synthesised on the vessel wall of the reaction vessel and in the reversibly introduced substrate. Also, care must be taken to ensure sufficient rinsing between the individual steps of the synthesis. For this purpose, the upper opening of tile reaction vessel is preferably provided with a screen which ensures that the reversibly introduced substrate remains in the reaction vessel during the rinsing step. If the sidewall of the reaction vessel is used as the solid phase, the; reaction solutions may also be siphoned off through a porous base o:F the reaction vessel.
As opposed to the mufti-parallel synthesis known from the prior art, the inven-tion ensures that the substance is not only synthesised on a porous support firmly anchored to the reaction vessel, but also on the vessel wall of the reaction vessel and a substrate reversibly introduced into the liquid phase which may easily be removed from the vessel.
The special advantages of the synthesis method according to the invention re-side in the analysis of both the physico-chemical and the biological character-istics of the synthesised substance which is greatly simplified in comparison with the prior art.
Without any previous reaction and separation steps, a large number of biolo-gical assays may be carried out directly with the synthesis product prepared according to the invention and present on the solid phase. The substrate reversibly introduced into the liquid phase is transferred from the reaction vessel to another container when the synthesis is complete. The synthesis products remaining in the reaction vessel, for example at the bottom of the reaction vessel, may be then be directly subjected to a biological assay. The selection of the biological assay is not limited and includes all assays suitable for analysing biological or biochemical characteristics, for example. In par-ticular, these include chemiluminescence methods, surface plasmon resonance methods, fluorescence methods and surface techniques utilising acoustic chan-ges (acoustic sensors). The advantages of the invention are particularly evident in biological assays which ihave the purpose of determining the interactions of the synthesised substance having receptors directly in a cell membrane or the ligand-receptor interactions which generate a signal too weak to be measured in conventional assays.
Interactions of the synthesised substance with receptors directly in a cell mem-brane may be analysed with cell assays comprising a microscopic observation of the cells through thin transparent substrates. In that case the base of the reaction vessel is selectcvd in such a manner that it is suitable for the cell assay in question. Preferably, the base of the reaction vessel is a glass substrate such as a quartz glass base in case of a fluorescence assay with, for example, calcium imaging. In addition to the facilitated process through the direct synthesis on the bottom of the reaction vessel, the cell assay carried out in accordance with the synthesis of the invention also has improved sensitivity, because all the molecules are present on the surface in a uniform orientation or the orientation may be controlled by the synthesis, respectively.
In addition, the synthesis of the invention is particularly advantageous in cases where an analysis of ligand interactions is required, because conventional assays would not be sufficiently sensitive or too unspecific for such an analy-sis. Owing to the drastic reduction of the detection volume in case of detection of a substance present as a thin surface film, assays have become possible which are more sensitive by several orders of magnitude than pertinent volume methods and which even permit detection of single molecules. Such an assay is described in the German patent application 198 22 542.4 and, in addition to determining absolute cor~cc,ntrations, pernlits determination of affinity constants and binding kinetics which often are a gauge for the specificity of the interaction. Preferably, the base of the reaction vessel for the synthesis accord-ing to the invention is a duartz glass base in that case, too.
Especially when the biological assay shows an interesting result, the chemical identity of the tested compound may be determined in a second step by analys-ing the solid phase removed from the reaction vessel earlier. Classical chemi-cal techniques of analysis such as GC/MS, TOF/MS, MALDI and NMR are considered for this purpose, but micro-sequencing may also be carried out depending of the type of the synthesised substance.
In a preferred embodiment of the invention, the reaction vessel is a well of a microtiter plate, for example a 96, 384 or 1536 microtiter plate. Such a micro-titer plate permits conducting up to 1536 different chemical syntheses in paral-lel in each well by adding different reactants. In addition, microstructurised systems such as systems on the basis of wafer technology are reaction vessels suitable for use according to the invention.
In addition, the invention provides a reaction system suitable for the solid phase synthesis according t:o the invention. The reaction system according to the invention comprises a rnicrotiter plate filled with a liquid phase, character-ised in that each of the wells of the microtiter plate additionally comprise a substrate reversibly introduced into the liquid phase, all or part of each of the substrate and the vessel wall of the pertinent well bearing functional groups for immobilising the same molecules.
Example 400 mg of polystyrene beads (diameter 200 Vim) bearing 200 ~mol of amine groups are dissolved in 5 rnl of dimethyl formamide (DMF) and placed into a well of a microtiter plate the base of which consists of a glass plate having a thickness of 170 ~m and provided with a thin layer of ethoxy aminosilane by known methods [e.g. S. Sec:ger et al. in: Synthetic Microstructures in Biologi-cal Research, Ed.: J. Schnur, M. Peckerar, Plenum Publishing Corporation, p.
53 - 66 (19!2)). Then 1 mrnol of Fmoc amino acid, 1 mmol of dicylohexyl carbodiimide (DCC), 1 mrnol ofN-hydroxybenzotriazole (HOBt) and 1 mmol of dimethyl aminopyridine (DMAP) are added and incubated at room temper-ature over night.
After rinsin;~ with DMF, the Fmoc protective group is cleaved by 30 minutes of incubation with 20 % piperidine in DMF at room temperature and a new reaction cycle for preparing a solid phase-coupled dipeptide begins. The cycle is repeated six times so that: a hexapeptide results. The side chain protective groups are finally removed with 25 % trifluoroacetic acid in dichloromethane (30 min., room temperature) and rinsed.
Depending on the sequence of the amino acids used, corresponding hexapep-tides are generated in the individual wells of the plate. The beads are now removed and may be subjected to chemical analysis methods. The hexapep-tides bound on the glass base of the microtiter plate may easily be investigated for protein bondings by adding fluorescence-labelled target protein molecules and scanning the base with a fluorescence scanner.
Claims (9)
1. A solid phase synthesis of substances in a reaction vessel containing a liquid phase, characterised in that, for the synthesis, the solid phase comprises a substrate reversibly introduced into the liquid phase and all or part of the vessel wall of the reaction vessel.
2. A solid phase synthesis according to claim 1, characterised in that the reaction vessel comprises a non-porous base as part of the wall of the vessel which serves as the solid phase for the synthesis.
3. A solid phase synthesis according to any of the claims 1 and 2, characterised in that all or part of the vessel wall of the reaction vessel and the reversibly introduced substrate bear the same functional groups.
4. A solid phase synthesis according to any of the claims 2 and 3, characterised in that the base is a quartz glass base.
5. A solid phase synthesis according to any of the previous claims, characterised in that. the substrate reversibly introduced into the liquid phase is one or more beads.
6. A solid phase synthesis according to any of the claims 1 to 4, characterised in that the substrate reversibly introduced into the liquid phase is a needle-head.
7. A solid phase synthesis according to any of the previous claims, characterised in that the reaction vessel is a well in a microtiter plate.
8. A process comprising (a) a solid phase synthesis according to any of the previous claims and (b) an analytical process, comprising (b1) the analysis of the chemical structure of the synthesised substance by analysing the substance synthesised on the substrate reversibly introduced into the liquid phase, and (b2) the analysis of the biological characteristics of the synthesised substance by analysing the substance synthesised on the vessel wall of the reaction vessel.
9. A reaction system comprising a microtiter plate filled with a liquid phase, characterised in that the wells of the microtiter plate additionally comprise a substrate reversibly introduced into the liquid phase, all or part of each of the substrate and the vessel wall of the well in question bearing functional groups for immobilising the same respective molecules.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19844988.7 | 1998-09-30 | ||
DE19844988A DE19844988A1 (en) | 1998-09-30 | 1998-09-30 | Parallel solid phase synthesis |
PCT/EP1999/007203 WO2000018792A1 (en) | 1998-09-30 | 1999-09-29 | Solid phase synthesis, whereby the solid phase consists of two different substrates |
Publications (1)
Publication Number | Publication Date |
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CA2347923A1 true CA2347923A1 (en) | 2000-04-06 |
Family
ID=7882889
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002347923A Abandoned CA2347923A1 (en) | 1998-09-30 | 1999-09-29 | Solid phase synthesis with the solid phase consisting of two different substrates |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP1117678B1 (en) |
JP (1) | JP2002525377A (en) |
CN (1) | CN1324366A (en) |
AT (1) | ATE264866T1 (en) |
AU (1) | AU6197699A (en) |
CA (1) | CA2347923A1 (en) |
DE (2) | DE19844988A1 (en) |
WO (1) | WO2000018792A1 (en) |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE8816749U1 (en) * | 1988-08-23 | 1990-05-10 | Boehringer Ingelheim Kg, 6507 Ingelheim, De | |
DE3935572A1 (en) * | 1989-10-25 | 1991-05-02 | Biotechnolog Forschung Gmbh | METHOD FOR PEPTID SYNTHESIS AND SUPPORT FOR THIS |
EP0586600B1 (en) * | 1991-05-24 | 1996-07-10 | The President And Fellows Of Harvard College | Parallel sequential reactor |
JPH06220084A (en) * | 1993-01-23 | 1994-08-09 | Shimadzu Corp | Peptide synthesizer |
US5763263A (en) * | 1995-11-27 | 1998-06-09 | Dehlinger; Peter J. | Method and apparatus for producing position addressable combinatorial libraries |
WO1998041534A2 (en) * | 1997-03-19 | 1998-09-24 | Biosepra Inc. | Multifunctional ceramic particles as supports for solid phase and combinatorial synthesis |
DE19723469A1 (en) * | 1997-06-04 | 1998-12-10 | Dirk Dr Vetter | Assay reactor e.g. for synthesis of peptide(s), oligo-nucleotide(s) |
-
1998
- 1998-09-30 DE DE19844988A patent/DE19844988A1/en not_active Withdrawn
-
1999
- 1999-09-29 JP JP2000572250A patent/JP2002525377A/en not_active Withdrawn
- 1999-09-29 CA CA002347923A patent/CA2347923A1/en not_active Abandoned
- 1999-09-29 DE DE59909256T patent/DE59909256D1/en not_active Expired - Fee Related
- 1999-09-29 AU AU61976/99A patent/AU6197699A/en not_active Abandoned
- 1999-09-29 AT AT99948888T patent/ATE264866T1/en not_active IP Right Cessation
- 1999-09-29 EP EP99948888A patent/EP1117678B1/en not_active Expired - Lifetime
- 1999-09-29 WO PCT/EP1999/007203 patent/WO2000018792A1/en active IP Right Grant
- 1999-09-29 CN CN99812577A patent/CN1324366A/en active Pending
Also Published As
Publication number | Publication date |
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WO2000018792A1 (en) | 2000-04-06 |
JP2002525377A (en) | 2002-08-13 |
EP1117678A1 (en) | 2001-07-25 |
ATE264866T1 (en) | 2004-05-15 |
CN1324366A (en) | 2001-11-28 |
DE59909256D1 (en) | 2004-05-27 |
EP1117678B1 (en) | 2004-04-21 |
DE19844988A1 (en) | 2000-04-13 |
AU6197699A (en) | 2000-04-17 |
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