DE10118698A1 - Immobilization method and arrangement of connections produced therewith on a planar surface - Google Patents

Abstract

The invention relates to a method for immobilizing compounds on a surface, comprising the steps DOLLAR A - providing a compound to be immobilized as the first reaction partner and a surface as the second reaction partner, the first reaction partner being a first reactive group and the second reaction partner being a first reactive group comprises, and DOLLAR A - reacting the two reactants DOLLAR A whereby it is provided that a heterocyclic ring system is formed when the two reactants are reacted.

Description

The present invention relates to a method for immobilizing compounds in special of biomolecules, thereby producing planar surfaces and arrangements and use of the planar surface and the arrangement in a method for loading tuning the substrate specificity of an enzymatic activity.

With the increasing availability of sequence information from the different ge nomprojjekte won the arrangement of nucleic acid fragments with high density on egg nem carrier material, so-called chips or biochips, of great importance, their full However, potential only with the availability of newer synthesis techniques and miniaturization could be exhausted and led to a variety of applications. Next Nucleic acids became natural products or libraries thereof, but also arrangements of Oligopeptides and proteins are applied to such chips. As carrier materials for this Arrangements were made from cellulose (D.R. Englebretsen, D.R.K. Harding; 1994, High yield, directed immobilization of a peptide-ligand onto a beaded cellulose support, Pept. Res., 7,  322-326, R. Frank, 1992, Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support, Tetrahedron, 48, 9217-9232; A. Kramer and J. Schneider-Mergener, Methods in Molecular Biology, vol 87: Combinatorial Peptide Library Protocols, pp. 25-39, edited by: S. Cabilly; Humana Press Inc., Totowa, NJ), glass (S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas; 1991, Light-directed, spati ally addressable parallel chemical synthesis, Science, 251, 767-773 J. Robles, M. Beltran, V. Marchan, Y. Perez, I. Travesset, E. Pedroso, A. Grandas; 1999, Towards nucleopeptides con taining any trifunctional amino acid, Tetrahedron, 55, 13251-13264), nitrocellulose (S.J. Hawthorne, M. Pagano, P. Harriott, D. W. Halton, B. Walker; 1998, The synthesis and utiliza tion of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors, anal. Biochem., 261, 131-138), PTFE membranes, gold (B.T. Houseman, M. Meksich; 1998, Effi client solid-phase synthesis of peptide-substituted alkanethiols for the preparation of substrates that support the adhesion of cells, J. Org. Chem., 63, 7552-7555), titanium oxide (SJ. Xiao, M. Textor, ND. Spencer, M. Wieland, B. Keller, H. Sigrist; 1997, Immobilization of the cell adhesive peptide ARG-GLY-ASP-CYS (RGDC) on titanium surfaces by covalent chemical attachment, J. Materials Science-Materials in Medicine, 8, 867-872), silicon oxide and modes used polypropylene surfaces.

With the increasing importance of proteomics and their biotechnological application focus on peptides and proteins. Generally they are proteins and mostly their enzymatic activities, which are common to almost all biochemical reactions half and outside the cell. The use of arrangements from Nuklein acids with which either the messenger RNA (mRNA), which is currently active in the cell ve genes were generated, or DNA copies of this mRNA are detected Although of great importance, the information available with it is from a number of Reasons not sufficient to make the processes both intracellular and extracellular Understand processes and of them in various biotechnological applications to make use of. One reason is that the amount of mRNA in a cell often not correlated with the corresponding amount of protein produced in the cell. except Once made, proteins can be modified by slight chemical modifications in the Cell (post-translational modifications) significantly in their enzymatic activity (and to be influenced in their biological function). So there is a need a parallel analysis of the enzymatic activity of as many proteins as possible, in particular  Enzymes. Such an approach allows u. a. the very quick determination of the Substrate specificity of a defined enzyme, which in turn is an important requirement for the design of knowledge-based inhibitors, or the selective testing of pharmaceuticals ka or drug candidates, especially in the context of predicting side effects gene.

Basically, immobilization has a number of advantages. This includes under among other things the increased stability of the immobilized compounds. For example If nucleic acids or proteins are immobilized, one regularly observes that their Half-life significantly increased when exposed to different media, also with those media which have a nuclease or protease activity. One reason for the be observed increased half-life may certainly be due to the reduced accessibility of the nuc linseed acids or protein for the corresponding degradative enzyme activities.

Another advantage of immobilization is the reusability of the immobilized Links. For example, it is possible to immobilize compounds Support materials that are used in the context of chromatographic processes after passage of a medium that has to be cleaned up or under the influence of immobilized It is necessary to modify the connections and, if necessary, to reuse them the. Such processes are, for example, affinity chromatography or biocon version and biotransformation.

Another application of immobilized compounds or arrangements from Nuk Linseed acids or proteins is the application area of the so-called bio mentioned at the beginning crisps. In these there is a well-defined correlation between the type of immobilized Molecule and its positioning or arrangement on a surface required.

There are basically two methods, biomolecules, such as oligoeptides or nuc linseed acids, with a surface (planar support or resin beads) or a biopoly mer (protein surface and thus bioconjugate formation). On the one hand, before manufactured biomolecules (and purified) biomolecules for the immobilization reaction, the ge directional or non-directional, can be used and on the other hand, these biomoles coolers can be synthesized step by step directly on an appropriate surface. In the state In the art, for example, peptides on cellulose (R. Frank, 1992, Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane  support, Tetrahedron, 48, 9217-9232; A. Kramer and J. Schneider-Mergener, Methods in Mo lecular Biology, vol 87: Combinatorial Peptide Library Protocols, pp. 25-39, edited by: S. Ca billy; Humana Press Inc., Totowa, NJ; Töpert, F., Oires, C., Landgraf, C., Oschkinat, H. and Schneider-Mergener, J., 2001, Synthesis of an array comprising 837 variants of the hYAP WW protein domain) or on polypropylene (WO 92/04366) or on glass (S. P. A. Fodor, J.L. Read, M.C. Pirrung, L. Stryer, A.T. Lu, D. Solas; 1991, Light-directed, spatially addressable parallel chemical synthesis, Science, 251, 767-773, J.P. Pellois, W. Wang, X.L. Gao; 2000 Peptide synthesis based on t-Boc chemistry and solution photogenerated acids, J Comb. Chem., 2, 355-360) or on chitin (W. Neugebauer, R.E. Williams, J.R. Barbier, R. Brze zinski, G. Willick; 1996, Peptide synthesis on chitin, Int. J. Pept. Prot. Res., 47, 269-275) or to Separose (R. Gast, J. Glokler, M. Hoxter, M. Kiess, R. Frank, W. Tegge; 1999, Method for determining protein kinase substrate specificities by the phosphorylation of pep tide libraries on beads, phosphate-specific staining, automated sorting, and sequencing, anal. Biochem., 276, 227-241).

Nucleic acids could also be synthesized by direct synthesis on suitable surfaces, such as glass (SPA Fodor, JL Read, MC Pirrung, L. Stryer, AT Lu, D. Solas; 1991, Light directed, spatially addressable parallel chemical synthesis, Science, 251 , 767-773, J. Robles, M. Beltran, V. Marchan, Y. Perez, I. Travesset, E. Pedroso, A. Grandas; 1999, Towards nu cleopeptides containing any trifunctional amino acid, Tetrahedron, 55, 13251- 13264), can be directly synthesized. It is understandable for the person skilled in the art that, as a result of the not always complete yields of the individual synthesis steps or coupling steps, certain heterogeneities can arise in the different amino acid sequences in the sense described above. In particular in syntheses that require many reaction steps, as is the case with the synthesis of amino acid sequences (one coupling reaction and one deprotection per amino acid unit and generally one reaction at the end of the synthesis for the simultaneous cleavage of all protective groups of the side chain functions) a problem. For example, in the synthesis of an amino acid sequence consisting of 20 amino acid units or 40 amino acid units and an assumed average yield of 95% for the necessary 41 or 81 reaction steps, the expected theoretical yield is only 0.95 ⁴¹ = 0.122 (12.2 %) or 0.95 ⁸¹ = 0.0157 (1.57%). Even with an assumed average yield of 99%, there are only 66.2% and 44.3% for the examples listed above.

The application of immobilization technology, for example in the field of biochips and especially the use of biochips to determine the substrate specificity of En zymes such as kinases, phosphatases, acetyltransferases, glycosyltransferases, Farnesyltransferases, sulfatases, sulfotransferases or proteases based on defined arrangement of peptides differing in their amino acid sequence, who the large number due to these limitations in addition to the desired amino acid sequence number of other amino acid sequences, which are characterized by the absence of one or more distinguish amino acid building blocks. Exactly this, the expert as a failure or Ab breakage known by-products, may be the result of the Inku with a modification of the amino acid sequences arranged on the surface, strongly distort enzymatic activity or make interpretation of the results more difficult. In order to avoid these disadvantages, the immobilization of cleaned, Biomolecules may be helpful. Biomolecules such as, for example, have been used in the prior art Peptides or nucleic acids, on different surfaces such as glass (S. P. A. Fodor, J.L. Read, M.C. Pirrung, L. Stryer, A.T. Lu, D. Solas; 1991, Light-directed, spatially addressable parallel chemical synthesis, Science, 251, 767-773, J.P. Pellois, W. Wang, X.L. Gao; 2000 Peptide synthesis based on t-Boc chemistry and solution photogenerated acids, J. Comb. Chem., 2, 355-360, J. Robles, M. Beltran, V. Marchan, Y. Perez, I. Travesset, E. Pedroso, A. grandas; 1999, Towards nucleopeptides containing any trifunctional amino acid, tetrahe dron, 55, 13251-13264), cellulose (D.R. Englebretsen, D.R.K. Harding; 1994, High yield, directed immobilization of a peptide-ligand onto a beaded cellulose support, Pept. Res., 7, 322-326, R. Frank, 1992, Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support, Tetrahedron, 48, 9217-9232; A. Kramer and J. Schneider-Mergener, Methods in Molecular Biology, vol 87: Combinatorial Peptide Library Protocols, pp. 25-39, edited by: S. Cabilly; Humana Press Inc., Totowa, NJ), Nitrozel lulose (S. J. Hawthorne, M. Pagano, P. Harriott, D. W. Halton, B. Walker; 1998, The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibi tors, anal. Biochem., 261, 131-138), titanium oxide (SJ. Xiao, M. Textor, ND. Spencer, M. Wie land, B. Keller, H. Sigrist; 1997, Immobilization of the cell adhesive peptide ARG-GLY- ASP-CYS (RGDC) on titanium surfaces by covalent chemical attachment, J. Materials Sci ence-Materials in Medicine, 8, 867-872) or Gold (B.T. Houseman, M. Meksich; 1998, Efficient  solid-phase synthesis of peptide-substituted alkanethiols for the preparation of substrates that support the adhesion of cells, J. Org. Chem., 63, 7552-7555).

A further distinction of immobilization can be made based on the orientation or binding the individual immobilized compound relative to the surface become. A distinction can be made here between specific and non-specific immobilization become. In the case of non-specific immobilization, the is carried out in an immobilization event Contact between the compound to be immobilized and the surface on or on the the connection is immobilized in different ways. In other words, it rea are either different reactive contained in the compound to be immobilized Groups with the surface or different ones react on the surface reactive groups with a reactive group of the compound to be immobilized (cross-linking). As a result, a population of immo is formed bilized compounds that are heterogeneous in terms of their binding to the surface. This different type of immobilization can result in an often undesirable lead different behavior of the immobilized connection. For example, at the Immobilization of a substrate for a kinase on or on a surface using of the amino groups contained within this substrate several (depending on the number of the amino groups present in the compound to be immobilized) Best reaction and thus the final orientation of the connection on the surface hen. Are involved in the subsequent incubation of this immobilized compound (Kinase substrate) with a biological flux containing at least one kinase activity liquid one or more of these amino groups for the effective formation of an Em zym / substrate / complex required, such non-specific immobilization can lead to this ren, that only a small population of the immobilized substrates in the right kind and Are anchored in such a way that the measurement signal is below the detection limit. So that is a specific or directed immobilization is of great advantage. Here takes place in an immobilization event, the contact between the compound to be immobilized and the surface on or on which the connection is immobilized, in each case in the same Chen way and all connections are in a defined and predictable Ori entation bound on or to the surface.  

This type of immobilization is usually a covalent immobilization, with a chemoselective reaction between the compound to be immobilized and the surface (the carrier material). For this purpose, a number of reactions can be used which are known to those skilled in the art as such (GA Lemieux and CR Bertozzi, 1998, chemoselective ligation reactions with proteins, oligosac charides and cells, TIBTECH, 16, 506-513) and in Fig . 1 are shown. The chemoselective reaction can basically be caused by two mechanisms. In the case of a mechanism, the compound to be immobilized has a reactive group which reacts specifically with the surface. This can be accomplished in that the said reactive group is present only once on the compound to be immobilized. In the second mechanism, the reactive group may be present several times, but the groups differ in their reactivity, so that only the presence of certain reaction conditions such as pH or the like leads to a reaction with the surface.

The use of immobilized compounds such as amino acid sequences in the form of Biochips require that both the connections and the link between the surface and the immobilized compound the respective test or investigation conditions. There is also an increasing need in technology after quantifying the interaction between the immobilized compound and an agent contained in a sample by means of the immobilized compound examined and quantified if necessary. This in turn presupposes that the actual loading density of the surface with the immobilized compound is known or can be determined non-destructively.

The present invention was therefore based on the object of a method for immobilization To provide connections that meet the needs described above will be right.

According to the invention the object is achieved by a method for immobilizing compounds on a surface comprising the steps

• - Providing a compound to be immobilized as the first reaction part ner and a surface as a second reactant, the first reactant first reactive group and the second reaction partner comprises a first reactive group, and
• - Implementation of the two reaction partners

characterized in that when the two reactants are reacted a hetero cyclic ring system is formed.

In one embodiment it is provided that the heterocyclic ring system between the first and the second reaction partner is formed.

In a further embodiment it is provided that the heterocyclic ring system consists of Share the first reactive group of the first reactant and the first reactive Group of the second reactant is formed.

Basically, in the method according to the invention there is the possibility that the hete rocyclic ring system without loss of atoms (addition) or with the escape of at least at least one molecule (condensation and / or substitution) arises.

In one embodiment it is provided that the heterocyclic ring system from 3-12 A toms, where 1-7 atoms are either nitrogen, sulfur or oxygen. Further it is preferred that the heterocyclic ring system consisting of 3 to 12 atoms consists of 1 to 7 heteroatoms, where the heteroatoms are selected from the group, the stick includes substance, sulfur and oxygen.

In a still further embodiment it is provided that the heterocyclic ring system is selected from the group consisting of thiazoles, aminothiazoles, imidazoles, benzimidazoles and indo le, tetrahydroisoquinoline, tetrahydro-β-carboline, dihydroquinazoline, oxidation products from ortho-amino-benzoic acid derivatives and pyrazoles.  

In one embodiment it is provided that the heterocyclic ring system comprises one of the following structural elements 1, 2, 3, 4 or 5, preferably 2, 3, 4 or 5:

wherein the substituents are selected independently of one another and wherein
X is O, S or NH, preferably NH;
A is a ring system of 4 to 8 atoms, preferably aryl;
YS or O, preferably O;
R ¹ -R ³ alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles or biomolecule
R ⁴ -R ^{9 is} alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, biomolecules or H, D, T.

Unless otherwise stated, the following terms are intended to mean this:
Alkyl: branched and unbranched C _1-20 alkyl, C _3-20 cycloalkyl,
preferably: branched and unbranched C _1-12 alkyl, C _3-12 cycloalkyl,
and more preferably: branched and unbranched C _1-6 alkyl, C _3-6 cycloalkyl;
Alkenyl: branched and unbranched C _2-20 alkenyl, branched and unbranched C _1-20 alkyl-OC _2-20 alkenyl, C _1-20 (-O / SC _2-20 ) _2-20 alkenyl, aryl-C _2-20 alkenyl, branched and unbranched heterocyclyl-C _2-20 alkenyl, C _3-20 cycloalkenyl,
preferably: branched and unbranched C _2-12 alkenyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkenyl, and
more preferably: branched and unbranched C _2-6 alkenyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _2-8 alkenyl;
Alkynyl: branched and unbranched C _2-20 alkynyl, branched and unbranched C _1-20 (-O / SC _2-20 ) _2-20 alkynyl,
preferably: branched and unbranched C _2-12 alkynyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkynyl, and
more preferably: branched and unbranched C _2-6 alkynyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _2-8 alkynyl;
Cycloalkyl: bridged and non-bridged C _3-40 cycloalkyl,
preferably: bridged and unbridged C _3-26 cycloalkyl, and more preferably: bridged and unbridged C _3-15 cycloalkyl;
Aryl: substituted and unsubstituted mono- or multi-linked phenyl-, pentalen-, azulene-, anthracenyl-, indacene-, acenaphthylene, fluorene, phenalen, phenanthrene,
Preferred: substituted and unsubstituted mono- or multi-linked phenyl, penalen, azulene, anthracenyl, indene, indacene, acenaphtylene, fluorene systems;
preferably: substituted and unsubstituted mono- or multi-linked phenyl, pentalen, anthracenyl systems and their partially hydrogenated derivatives;
Heteroatoms: N, S, O, Se, P, B,
preferably: N, S, O, Se, and
more preferably: N, S, O;
Heterocycles: unsaturated and saturated 3-15-membered mono-, bi- and tricyclic rings with 1-7 heteroatoms,
preferably: 3-10-membered mono-, bi- and tricyclic rings with 1-5 heteroatoms, and
more preferably: 5-, 6- and 10-membered mono-, bi- and tricyclic rings with 1-3 heteroatoms;
Biomolecule: nucleic acids, genes, chromosomal nucleic acids, cDNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides, protein domains, proteins, sugars, poly sugars, lipids, polylipids, natural products or combinations of two or more of these compounds.
In addition to the above-mentioned groups or molecules, ie alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, biomolecules or natural substances may have substituents, more particularly 0 to 30, preferably 0 to 10 and preferably 0 to 5 of the following Substituent, which can occur either individually or in any combination: fluorine, chlorine, bromine, iodine, hydroxyl, amide, ester, acid, amine, acetal, ketal, thiol, ether, phosphate, sulfate, sulfoxide, peroxide, sulfonic acid, thioether , Nitrile, ureas and carbamate.
Preferred substituents are: fluorine, chlorine, bromine, hydroxyl, amide, ester, acid, A min. Ether, phosphate, sulfate, sulfoxide, thioether, nitrile, urea, carbamate.
Particularly preferred substituents are: chlorine, hydroxyl, amide, ester, acid, ether, nitrile.

In one embodiment it is provided that the reactive group is selected from the Group containing thioamines, thiocarbonyls, thioureas, hydroxylamino, alpha- Isothiocyanatoketones, anthranilic acid amides, anilines, aldehydes, ketones, 1,2-diketones, ami ne, aminooxy derivatives, arylhydrazines, arylamines, heteroarylamines, ortho-amino Includes benzoic acid, hydrazines, 2-cyano aldehydes, imines and acid halides.

In one embodiment it is provided that the first reactive group is to be immobilized is a reactive group which is selected from the group consisting of thioamide, Aniline, thiourea, thiocarbonyl, anthranilic acid, anthranilic acid amide and hydroxylamino contains.

In a further embodiment it is provided that the first reactive group of the upper is a reactive group which is selected from the group consisting of carbonyl, keto and Includes aldehyde.  

In a still further embodiment it is provided that the conversion is a chemoselect reactive reaction.

Finally, it is provided in one embodiment that the immobilization is a ge is directed immobilization.

In one embodiment it is provided that the heterocyclic ring formed is in distinguishes its absorption / emission behavior from the educts.

In a further embodiment it is provided that the connection to be immobilized is selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides, proteins indomains, proteins, sugar, poly sugar, lipids, polylipids, natural products and low molecular weight lare connections.

In a preferred embodiment it is provided that the surface is a planar surface area is.

In one embodiment it is provided that the planar surface is a Chip acts.

In a further preferred embodiment it is provided that the surface is a material al comprises, which is selected from the group consisting of silicates, ceramics, glass, metals, organi cal carrier materials, cellulose, nitrocellulose, PTFE membranes, polypropylene, gold, Includes titanium dioxide and silicon oxide.

In a still further preferred embodiment it is provided that the planar upper surface is a non-porous surface.

In a preferred embodiment it is provided that the implementation of the immobilized sizing connection with the surface under microwave radiation.  

In a further aspect of the invention, the object is achieved by a method for Production of an arrangement comprising at least two different connections at least two different locations on a planar surface, the first connection extension at a first point on the planar surface and the second connection at a second position of the planar surface according to one of the methods according to the invention immo is accounted for.

In a still further aspect, the task is solved by a planar surface adjustable according to one of the methods according to the invention.

Finally, the task is covered in a further aspect by a planar surface send a compound covalently immobilized on it, whereby between the immobilized Connection and the surface a heterocyclic ring system is arranged, the first is formed by the immobilization process.

In one embodiment of the planar surface it is provided that the heterocyclic Ring system consists of 3-12 atoms, where 1-7 atoms are either nitrogen or sulfur Are oxygen.

In a further embodiment it is provided that the heterocyclic ring systems are selected from the group consisting of thiazoles, aminothiazoles, imidazoles, benzimidazoles, Indoles, tetrahydroisoquinolines, tetrahydro-β-carbolines, dihydroquinazoline, oxidation pro products of ortho-amino-benzoic acid derivatives and pyrazoles include.

In a still further embodiment, the heterocyclic ring system comprises one of the following structural elements 1, 2, 3, 4 or 5:

wherein the substituents are selected independently of one another and wherein
X is O, S or NH, preferably NH;
A is a fused ring system of 4 to 8 atoms, preferably aryl;
YS or O, preferably O;
R ^1- R ³ alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles or biomolecule;
R ⁴ -R ^{9 is} alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, biomolecules or H, D, T.

In a preferred embodiment it is provided that the heterocyclic ring system comprises the structural element 2 or 3. In a very particularly preferred embodiment it is envisaged that the heterocyclic ring system comprises structural element 4 or 5.

In a further aspect of the invention, the object is achieved by an invented Process according to the invention for producing an arrangement that can be produced.

In a still further aspect of the invention, the object is achieved by an arrangement which comprises a planar surface according to the invention. In a preferred embodiment form is the arrangement for a by the inventive method Production of an arrangement producible arrangement.

In a further aspect, the object is achieved by using an arrangement according to the invention or a planar surface according to the invention in a method for determining the substrate specificity of an enzymatic activity, comprising the following steps:

• - Provide a set of amino acid sequences on the planar surface surface of a carrier material, the amino acid sequences being immobilized in a directed manner,
• - Contacting and / or incubating an enzymatic activity with the the set of amino acid sequences, and
• - Detection of a reaction between one of the immobilized amino acids sequences and the enzymatic activity,

in which

• - The planar surface is a planar surface according to the invention or a planar surface Surface of an arrangement according to the invention is, and
• - When implementing the enzymatic activity with the set of amino acids The molecular weight of at least one of the amino acid sequences is changed.

In one embodiment it is provided that the change in molecular weight is caused by Formation or cleavage of a covalent bond on one of the amino acid sequences follows, preferably at that amino acid sequence that with the enzymatic activi activity reacts. It is particularly preferred if the detection of the reaction on or under Use of the amino acid sequence immobilized on the surface of the carrier material he follows.

In one embodiment it is provided that the reaction is detected by detection the change in molecular weight takes place.

In a further embodiment it is provided, characterized in that the after by means of a detection method which is selected from the group, the car radio includes graphics, plasmon resonance spectroscopy and fluorescence spectroscopy.  

In a still further embodiment it is provided that the enzymatic activity is selected from the group consisting of kinases, sulfotransferases, glycosyltransferases, Ace tyltransferases, farnesyltransferases, palmityltransferases, phosphatases, sulfatases, esters ases, lipases, acrylases and proteases.

Unless otherwise stated, natural substances should in particular in particular a compound that can be isolated from animal or plant organisms, or that fully synthetic analogs with a molecular weight below 1000 kDa understood become.

The present invention is based on the surprising finding that by means of The inventive method an immobilization of compounds, especially of Amino acid sequences can take place and the immobilized amino acid sequences as a result of the connection to be immobilized and the parent surface heterocyclic ring system is particularly stable in terms of resolution solution of the immobilization (acid and base stability of the immobilization). Continue to have the inventors surprisingly found that using the Immo the immobilization efficiency, which is based on knowledge of the concentration the initial concentrations used from the loading or immobilization density can be calculated, controlled. The immobilization density in turn is ahead setting for the implementation of quantitative studies, as described at the beginning.

Without wishing to be bound in the following, the determination of the immobilization seems density is essentially based on the fact that between the immobilization the compound to be immobilized and the surface to which the compound is immobilized is to be formed, forms a heterocyclic ring system. As a result of the absorption properties The newly formed heterocyclic ring system can be directly linked to the one site amount of the compound immobilized on the surface can be inferred. It is in the frame the knowledge of the expert in the field that the reactive group of the immobilisie renden compound and the reactive group of the surface, and optionally another group to be added by a third reactant or a second reactive Group of the compound to be immobilized or a second reactive group of the surface  can originate (where all of the above reactive groups or parts thereof) leads to the formation of the heterocyclic ring system) thus derivatized is that the heterocyclic ring system that forms as a result of the immobilization Chromophore contains or represents that allows specific detection. The proof typically takes place by means of UV-Vis, fluorescence or infrared spectroscopy

In a preferred embodiment of the immobilization method according to the invention is a directional immobilization. When directed immobilization should ensure that under the respective reaction conditions essentially only a special link between the connection to be immobilized, which is is preferably an amino acid sequence, and the surface is produced. In the case of immobilization of amino acid sequences, the choice of reactive depends Group on the side of the amino acid sequences essentially from the single sequence.

It is within the scope of the present invention that the immobilization of several different amino acid sequences is provided with a terminal structure which is uniform to all amino acid sequences and that this terminal structure is made available for the specific reaction with the surface, in particular an activated surface, and during immobilization is used. The amino or carboxyl groups contained in the amino acid sequence are typically not impaired in the chemoselective reactions. Examples of suitable reactions are the formation of amide bonds from thioesters and 1,2-aminothiols ( FIG. 1E), the formation of thioamide bonds from dithioesters and 1,2-aminothiols, the formation of oximes from amino-oxy compounds and aldehydes ( FIG 1A, R ⁴ = H), the formation of oximes from amino-oxy compounds and ketones (Fig. 1A, R ⁴ is not H), the formation of hydrazones from hydrazines and aldehydes (Fig. 1C, R ⁴ = H.) , the formation of hydrazone from hydrazides and ketones, ( Fig. 1C, R ⁴ not H), the formation of thiosemicarbazones from thiosemicarbazides and aldehydes ( Fig. 1B, R ⁴ = H), the formation of thiosemicarbazones from thiosemicarbazides and ketones ( Fig . 1B, R ⁴ is not H), the formation of thioesters of thiocarboxylates and alpha-Halocarbonylen (Fig. 1D), and succinimides and maleimides from mercaptans (Fig. 1F).

In the context of the present invention, the following heterocyclic ring systems and the reactive groups forming them are particularly preferred. The reactive groups are first and second reactive groups of the two reactants. In principle, it is possible that each of the first or second reactive groups described herein is that of the compound to be immobilized or that of the surface. The selection of which reactive group is present on the compound to be immobilized and which is present on the surface depends on the type of compound to be immobilized or the surface available for immobilization. The decisive factor in the selection of the corresponding groups is the ability to form a heterocyclic ring system. The following heterocyclic ring systems are particularly preferred:
NEN imidazoles from 1,2-diketones and amines, benzimidazoles from phenylenediamine derivatives and aldehydes, indoles from aryl hydrazines and aldehydes, tetrahydroisoquinolines from Arylami and aldehydes, tetrahydro-β-carboline and related Tetrahydroimidazopyridine of heteroaryl amines and aldehydes, Dihydroquinazoline as well as their oxidation products of ortho-amino -Benzoic acid derivatives and aldehydes, pyrazoles from hydrazines and 2-cyan aldeydes, cycloaddition of two olefins, cycloaddition of olefins and imines, cycloaddition of imines and acid halides.

In a preferred embodiment of the immobilization method according to the invention It is provided that in the connection to be immobilized or conjugated contain an aniline function which is arranged so that it has a heterocycle with a corresponding reaction partner (surface or other biomolecule) can form.

In a further embodiment it is provided that in or to be immobilized conjugating compound contains a thioamide function which is arranged so that a heterocycle with a corresponding reactant (surface or other Biomolecule) can form.

In a further embodiment it is provided that in or to be immobilized conjugating compound contains a thiourea function which is arranged so that they have a heterocycle with a corresponding reactant (surface or at whose biomolecule) can form.  

Directed immobilization is intended to supplement what has already been said was carried out for this purpose, in particular understood that in the case of immobilization an amino acid sequence each amino acid sequence via a defined reactive group or Accumulation of reactive groups is bound to the surface. As a result of this bin The specificity of the application ensures that the individual A amino acid sequences are in an energetically preferred state, so that the such immobilized amino acid sequences in largely similar secondary and There are tertiary structures.

It is within the scope of the present invention that the amino acid sequences in situ on the Surface of the arrangement are synthesized, here all possible shapes are conceivable are, d. H. sequential addition of the individual amino acids that make up the amino acid sequence acids, as well as the use of block synthesis techniques involving groupings of amino acids are joined together and then the individual blocks sequentially are lined up and the blocks or rows of them are then immobilized are or are added to already immobilized amino acid sequences. Decide is in any case the formation of a heterocyclic ring system as a result of Immobilization.

The fact that various aspects of the immobilization process disclosed herein were explained based on the immobilization of amino acid sequences, the Of content, since the guidelines and procedures outlined here make sense also according to all other compounds to be immobilized or immobilized ten or find application.

The immobilization method according to the invention can also be used as a conjugation method is drawn or executed. Conjugation should be understood to mean in particular who the directional linkage of different biomolecules, these molecules sufficiently differentiate in size so that one of these biomolecules as the surface can be viewed and the other biomolecule (s) on the surface of the first represents the biomolecule (s) to be immobilized. Such conjuga tion methods can be used for the regioselective labeling of biomolecules with mostly small ones Compounds that are suitable as probes in subsequent experiments serve. Examples  for such probes are fluorescent labels, electron spin resonance Labels, radioactive labels and biotinylations.

Furthermore, it is within the scope of the present invention that the reactive group of the upper surface of the carrier material on a compound that is itself immobilized is arranged or contained. This compound is also referred to as primary immobilized herein Connection called. This primarily immobilized compound is preferably flat if to form a heterocyclic ring system on the surface of a carrier terials bound.

The invention is explained below with reference to examples and figures, which make up further features, embodiments and advantages result. It shows

FIG. 1 is an overview of different chemoselective reactions;

Figure 2 shows an overview of various Immobilisierungsreak invention that lead to the formation of a thiazole ring.

Fig. 3 carry an illustration of various combinations of thioamides as ers ter reactive group of the compound to be immobilized and the first reactive group of the surface, the rule for the formation of heterocyclic ring systems;

FIG. 4 shows the synthesis and immobilisation of an ortho-amino-benzoyl-peptide functionalized aldehyde on a surface to form a substituted 2,3,4a, 8a-tetrahydro-1H-quinazolin-4-one;

Fig. 5 immobilizing a biomolecule on a surface ne by ei has been modified also to form a heterocyclic ring system duri Fende primary immobilization;

Fig. 6 is the result of the incubation of a modified glass surface with egg ner kinase; and

Fig. 7 shows the result of incubation of a modified glass surface with egg ner kinase.

The individual figures are explained in more detail below.

Fig. 1 shows an overview of various chemoselective reactions according to the prior art: A) aldehydes (R ⁴ = H) or ketones (R ⁴ not H) and amino-oxy compounds react to oximes, B) aldehydes (R ⁴ = H) or ketones (R ⁴ not H) and thiosemicarbazides react to thiosemicarbazones, C) aldehydes (R ⁴ = H) or ketones (R ⁴ not H) and hydrazides react to hydrazone D) thiocarboxylates and halocarbonyls react to thioesters, E) thioesters and β-aminothiols react to β-mercaptoamides, F) mercaptans and maleimides react to succinimides.

Fig. 2 shows an overview of various inventive immobilization reactions, which lead to the formation of a thiazole ring. In Fig. 2A, is brominated on the solid phase to an alpha bromo ketone (IV) is assumed to be alpha-ketone (IX). An either protected or unprotected peptide or biomolecule, which carries either a C-terminal or N-terminal or a thio amide function (Va) at a defined position within the biomolecule, immobilized to form a thiazole (VI). In Fig. 2B the Immobilization is a thiourea derivative (Vb) either a protected or unsaturated present peptide or biomolecule with a protecting immobilized on the solid phase present alpha-bromo ketone (IV) to form a aminothiazole (VIII), respectively. Finally, starting from an alpha-bromoketone (IV), FIG. 2C describes the immobilization of an amino-oxy-peptide or biomolecule. This immobilization represents an example of chemoselective immobilization, in which a chemoselective reaction is only made possible by applying specific reaction conditions, here pH 5. The al pha bromoketone is converted to an al pha isothiocyanato ketone (X) as in the first alternative described in Example 9 and this is combined with the amino oxypeptide or biomolecule (XI) to form a hydroxylaminothiazole (XII) with the intermediate formation of a thiourea. implemented. The radicals R ⁴ and R ⁵ represent alkyl, alkenyl, alkynyl, cycloalkyl or aryl radicals or heterocycles or surfaces or H, D or T, where alkyl is branched and unbranched C _1-20 -Alkyl, C _3-20 -cycloalkyl, preferably for branched and unbranched C _1-12 -alkyl, C _3-12 -cycloalkyl and particularly preferably for branched and unbranched C _1-6 -alkyl, C _3-6 -cycloalkyl- Leftovers. Alkenyl stands for branched and unbranched C _2-20 alkenyl, branched and unbranched C _1-20 alkyl-OC _2-20 alkenyl, C _1-20 (-O / S- C _2-20 ) _2-20 alkenyl , Aryl-C _2-20 alkenyl, branched and unbranched heterocyclyl-C _2-20 alkenyl, C _3-20 cycloalkenyl, preferably for branched and unbranched C _2-12 alkenyl, branched and unbranched C _1-12 ( -O / SC _2-12 ) _2-12 alkenyl, particularly preferred for branched and unbranched C _2-6 alkenyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _2-8 alkenyl radicals; Alkynyl represents branched and unbranched C _2-20 alkynyl, branched and unbranched C _1-20 (-O / SC _2-20 ) _2-20 alkynyl, preferably branched and unbranched C _2-12 alkynyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkynyl, particularly preferred for branched and unbranched C _2-6 alkynyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _{2- 8} alkynyl radicals; Cycloalkyl stands for bridged and non-bridged C _3-40 cycloalkyl, preferably for bridged and non-bridged C _3-26 cycloalkyl, particularly preferably for bridged and non-bridged C _3-15 cycloalkyl radicals, aryl for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, azulenyl, anthracenyl, in dacenyl, acenaphtyl, fluorenyl, phenalenyl, phenanthrenyl, preferably for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl -, Azulenyl, anthracenyl, indenyl, indacenyl, acenaphthyl, fluorenyl, particularly preferably for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, anthracenyl residues, and their partially hydrogenated derivatives. Heterocycles can be unsaturated and saturated 3-15-membered mono-bi and tricyclic rings with 1-7 heteroatoms, preferably 3-10-membered mono-bi and tricyclic rings with 1-5 heteroatoms and particularly preferably: 5, 6 and 10- link mono-bi and tricyclic rings with 1-3 heteroatoms.

In addition, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, Biomolecule or natural product 0 to 30 (preferably 0 to 10, particularly preferably 0 to 5) the following substituents occur individually or in combination with one another; Fluorine, chlorine, Bromine, iodine, hydroxyl, amide, ester, acid, amine, acetal, ketal, thiol, ether, phosphate, sulfate, Sulfoxide, peroxide, sulfonic acid, thioether, nitrile, urea, carbamate, the following be Preferred are: fluorine, chlorine, bromine, hydroxyl, amide, ester, acid, amine, ether, phosphate, sul fat, sulfoxide, thioether, nitrile, urea, carbamate, and particularly preferably: chlorine, hy droxyl, amide, ester, acid, ether, nitrile.

Fig. 3 shows a representation of various combinations of thioamides as the first reactive group in the compound to be immobilized and various first reactive groups of the surface, which lead to the immobilization of biomolecules with the formation of heterocyclic ring systems. For example, a biomolecule, in particular a peptide, is used as the compound to be immobilized. Referring to FIG. 3A it comes using a thioamide function of the peptide (XVII) with a 1,2-aminothiol function of the surface (X = S, XVIIIa) to form a thiazoline (XIXa) or together with a 1, 2-amino alcohol function of the surface (X = O, XVIIIb) to form an oxazoline (XIXb) or together with a 1,2-diamino function of the surface (X = NH, XVIIIc) to form an imidazoline (XIXc). In Fig. 3B it is evident that the reaction using a thioamide function of the peptide (XX) together with an ortho-phenylenediamine function (XXI) on the surface leads to the formation of a benzimidazole (XXII). In FIG. 3C is clear that using a thioamide function of the peptide (XXIII) together with an alpha-halo-keto function (XXIV, Y = Cl, Br, J, preferably Br, Cl; more preferably Br) on the surface of biomolecules can be immobilized to form thiazoles (XXV). Finally, Fig. 3D shows that using a thioamide function of the peptide (XXVI) together with a hydrazide function (XXVII) can form oxadiazoles on the surface. The radicals R ⁴ -R ⁹ represent alkyl, alkenyl, alkynyl, cycloalkyl or aryl radicals, or heterocycles or surfaces or H, D or T, where alkyl is branched and unbranched C _{1- 20} alkyl, C _3-20 cycloalkyl, preferably branched and straight branch te C _1-12 alkyl, C _3-12 cycloalkyl, and particularly preferably represents branched and unbranched C _{1- 6} alkyl, C _3-6 cycloalkyl -Remains stands. Alkenyl stands for branched and unbranched C _2-20 alkenyl, branched and unbranched C _1-20 alkyl-OC _2-20 alkenyl, C _1-20 (-O / SC _2-20 ) _{2- 20} alkenyl, aryl- C _2-20 alkenyl, branched and unbranched heterocyclyl-C _2-20 alkenyl, C _3-20 cycloalkenyl, preferably for branched and unbranched C _2-12 alkenyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkenyl, particularly preferred for branched and unbranched C _2-6 alkenyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _2-8 alkenyl radicals; Alkynyl stands for branched and unbranched C _2-20 alkynyl, branched and unbranched C _1-20 (-O / SC _{2- 20} ) _2-20 alkynyl, preferably for branched and unbranched C _2-12 alkynyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkynyl, particularly preferred for branched and unbranched C _2-6 alkynyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _2-8 alkynyl radicals; Cycloalkyl is bridged and non-bridged C _3-40 cycloalkyl, preferably bridged and bridged C _3-26 cycloalkyl, more preferably bridged and non-bridged C _{3- 15} cycloalkyl radicals, aryl represents substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, azulenyl, anthracenyl, indacenyl, acenaphthyl, fluorenyl, phenalenyl, phenanthrenyl, preferably for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, azulenyl, anthracene Indenyl, indacenyl, acenaphtyl, fluorenyl, particularly preferably for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, anthracenyl residues, and their partially hydrogenated derivatives. Heterocycles can be unsaturated and saturated 3-15-membered mono-bi and tricyclic rings with 1-7 heteroatoms, preferably 3-10-membered mono-bi and tricyclic rings with 1-5 heteroatoms and particularly preferably: 5, 6 and 10 -linked mono-bi and tricyclic rings with 1-3 heteroatoms.

In addition, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, Biomolecule or natural product 0 to 30 (preferably 0 to 10, particularly preferably 0 to 5) the following substituents occur individually or in combination with one another; Fluorine, chlorine, Bromine, iodine, hydroxyl, amide, ester, acid, amine, acetal, ketal, thiol, ether, phosphate, sulfate, Sulfoxide, peroxide, sulfonic acid, thioether, nitrile, urea, carbamate, the following be Preferred are: fluorine, chlorine, bromine, hydroxyl, amide, ester, acid, amine, ether, phosphate, sul fat, sulfoxide, thioether, nitrile, urea, carbamate, and particularly preferably: chlorine, hy droxyl, amide, ester, acid, ether, nitrile.

Fig. 4 shows the immobilization of a biomolecule to form a substituted 2,3,4a, 8a-tetrahydro-1H-quinazolin-4-one (XIII). Starting from an amino-modified solid phase, a building block is added to form a covalent bond. After the step-by-step synthesis of the biomolecule on the solid phase, this is by adding isatoic acid (XIV) to form an ortho-amino-benzoyl biomolecule (XV), which is subsequently separated from the solid phase by the action of acid as an ortho-amino-benzoyl biomolecule. Amide (XVI) is split off. The ortho-amino-benzoyl biomolecule amide (XVI) obtained is brought into contact with a glass support whose surface has an aldehyde function, and a substituted 2,3,4a, 8-tetrahydro-1H- quinazolin-4-ones (XIII).

Fig. 5 shows a specific embodiment of the immobilization of a biomolecule in which the surface is modified by a duri fenden also to form a heterocyclic ring system reaction. Starting from a surface modified with a thioamide (Z ¹ = CR ⁴ R ⁵ , XXIXa) or a thiourea (Z ¹ = NR ⁴ , XXIXb), treatment with 1,4-dibromo-2,3-diketobutane (XXX ) immobilized bromoacetylated thiazoles (Z ¹ = CR ⁴ R ⁵ , XXXIa) or bromoacetylated aminothiazoles (Z ¹ = NR ⁴ , XXXIb). These functions can be used for an immobilization reaction of a biomolecule provided with a thioamide function (Z ² = CR ⁴ R ⁵ , Va) or thiourea function (Z ² = NR ⁴ , Vb). Depending on the combination of the derivatized biomolecule and the surface modified in a primary immobilization reaction, XXXIIa (Z ¹ = NR ⁴ , Z ² = NR ⁴ ), XXXIIb (Z ¹ = CR ⁴ R ⁵ , Z ² = NR ⁴ ), XXXIIc (Z ¹ = CR ⁴ R ⁵ , Z ² = CR ⁴ R ⁵ ) or XXXIId (Z ¹ = NR ⁴ , Z ¹ = CR ⁴ R ⁵ ). The radicals R ⁴ and R ⁵ represent alkyl, alkenyl, alkynyl, cycloalkyl or aryl radicals, or heterocycles or surfaces or H, D or T, where alkyl is for branched and unbranched C _{1 -20} alkyl, C _3-20 cycloalkyl, preferably for branched and unbranched C _1-12 alkyl, C _3-12 cycloalkyl and particularly preferably for branched and unbranched C _1-6 alkyl, C _3-6 cycloalkyl -Remains stands. Alkenyl stands for branched and unbranched C _2-20 alkenyl, branched and unbranched C _1-20 alkyl-OC _2-20 alkenyl, C _1-20 (- O / SC _2-20 ) _2-20 alkenyl, aryl -C _2-20 alkenyl, branched and unbranched heterocyclyl-C _2-20 alkenyl, C _3-20 cycloalkenyl, preferably for branched and unbranched C _2-12 alkenyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkenyl, particularly preferred for branched and unbranched C _2-6 alkenyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _2-8 alkenyl radicals; Alkynyl stands for branched and unbranched C _2-20 alkynyl, branched and unbranched C _1-20 (-O / SC _2-20 ) _2-20 alkynyl, preferably for branched and unbranched C _2-12 alkynyl, branched and unbranched C _1-12 (-O / SC _2-12 ) _2-12 alkynyl, particularly preferred for branched and unbranched C _2-6 alkynyl, branched and unbranched C _1-6 (-O / SC _2-8 ) _{2- 8} alkynyl radicals; Cycloalkyl stands for bridged and unbridged C _3-40 cycloalkyl, preferably for bridged and unbridged C _3-26 cycloalkyl, particularly preferably for bridged and unbridged C _3-15 cycloalkyl radicals, aryl for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, azulenyl, anthracenyl, in dacenyl, acenaphtyl, fluorenyl, phenalenyl, phenanthrenyl, preferably for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl -, Azulenyl, anthracenyl, indenyl, indacenyl, acenaphthyl, fluorenyl, particularly preferably for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, anthracenyl residues, and their partially hydrogenated derivatives. Heterocycles can be unsaturated and saturated 3-15-membered mono-bi and tricyclic rings with 1-7 heteroatoms, preferably 3-10-membered mono-bi and tricyclic rings with 1-5 heteroatoms and particularly preferably: 5, 6 and 10- membered mono-, bi- and tricyclic rings with 1-3 heteroatoms.

In addition, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, Biomolecule or natural product 0 to 30 (preferably 0 to 10, particularly preferably 0 to 5) the following substituents occur individually or in combination with one another; Fluorine, chlorine, Bromine, iodine, hydroxyl, amide, ester, acid, amine, acetal, ketal, thiol, ether, phosphate, sulfate, Sulfoxide, peroxide, sulfonic acid, thioether, nitrile, urea, carbamate, the following be Preferred are: fluorine, chlorine, bromine, hydroxyl, amide, ester, acid, amine, ether, phosphate, sul fat, sulfoxide, thioether, nitrile, urea, carbamate, and particularly preferably: chlorine, hy droxyl, amide, ester, acid, ether, nitrile.

Fig. 6 A glass surface modified with Leu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide (cf. Example 5 and Fig. 5, XXXIIc) (amino acid sequence was dissolved in 200 mM sodium phosphate buffer pH 5.5 and was at RT 1 nL each of this solution in an arrangement of 70 rows and 168 columns (a total of 11,760 spots) was applied to the bromketo-functionalized glass surfaces ( FIG. 5, XXXIa) by means of a nano plotter from Ge sim. The spot-to-spot The glass surfaces treated in this way were then treated with a microwave for 2 min and then incubated for 3 hours at RT (cf. Example 12)), which was initially preincubated with 10 ml of 100 μM ATP solution in 50 mM sodium phosphate buffer pH 7.5 for 10 minutes , The modified glass surface was then covered with a second glass surface and, in the resulting space, protein kinase A (10 U / mL) together with ATP / γ ³² P-ATP mixture (100 µM / mL; 100 µCi / mL) using capillary force brought in. After incubation at 25 ° C. for 30 minutes, the phosphorylation of the corresponding peptides was detected using a phosphorimage from FUJIFILM (cf. Example 13). It becomes clear that on the one hand the linkage of the immobilized kinase substrate is tolerated by protein kinase A and on the other hand that the modification of the glass surfaces proceeds smoothly and without major fluctuations in the immobilization density. Furthermore, it is clear that the resolution of the phosphor oil used here is sufficient to analyze more than 11,000 measuring points per biochip.

Fig. 7 A to the peptide thioxoCit-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH Example 11 and ₂ (Fig. 2B, Vb, R ⁴ = H) modified glass surface (see FIG.. 2B , VIII, R ⁴ = H) was first pre-incubated with 10 ml of 100 µM ATP solution in 50 mM sodium phosphate buffer pH 7.5 for 10 minutes. The modified glass surface was then covered with a second glass surface and, in the resulting space, protein kinase A (10 U / mL) together with an ATP / γ ³² P-ATP mixture (100 µM / mL; 100 µCi / mL) Capillary force introduced. After incubation at 25 ° C. for 30 minutes, the phosphorylation of the corresponding peptides was detected using a phosphorimage from FUJIFILM (cf. Example 14). It becomes clear that on the one hand the linkage of the immobilized kinase substrate is tolerated by protein kinase A and on the other hand that the modification of the glass surfaces proceeds smoothly and without major fluctuations in the immobilization density. Wei terhin is clear that the resolution of the phosphor oil used here is sufficient to analyze more than 950 measuring points per biochip.

The following examples relate to the functionalization of glass, the surface of which is required as a surface for immobilization (Examples 1 to 9), the immobilization of various peptides provided with a reactive group on a surface (Examples 10 to 12) and the analysis of kinase mediated peptide modification using the peptides immobilized according to the invention. The abbreviations listed below were used:
Ala = L-alanine
Arg = L-arginine
ATP = adenosine 5'-triphosphate
Ala = alanine, 3-aminopropionic acid
Boc = tertiary butoxycarbonyl
cDNA = complementary DNA
Cit = L-citrulline
DCM = dichloromethane
DIC = N, N'-diisopropylcarbodiimide
DMF = N, N'-dimethylformamide
DNA = deoxyribonucleic acid
DTT = 1,4-dithio-DL-threitol
EGTA = ethylene glycol bis (2-aminoethyl) -N, N, N ', N'-tetraacetic acid
Fmoc = 9-fluorenylmethoxycarbonyl
Gly = glycine
HBTU = O- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate
HPLC = high-performance liquid chromatography
L = liter
Leu = L-leucine
M = molar
MBHA = methylbenzhydrylamine
MBP = myelin basic protein
MeOH = methanol
mL = milliliters
mM = millimolar
mRNA = messenger RNA
Pbf = 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
PNA = peptide nucleic acid
PTFE = polytetrafluoroethylene
RNA = ribonucleic acid
RP = reversed phase
RT = room temperature
SDS = sodium lauryl sulfate
Ser = L-serine
tBu = tertiary butyl
TFA = trifluoroacetic acid
THF = tetrahydrofuran
Tris = 2-amino-2-hydroxymethyl-1,3-propanediol
Tween20 = polyoxyethylene sorbitant monolaurate (trademark from Atlas Chemie)

The following reagents and solvents were used:
Bromine, tert-butyl methyl ether, 1,3-diisopropylcarbodiimide, N, N-diisopropylethylamine, vinegar, glycerol, urea, 40% hydroxylamine solution, piperidine, triethylamine, dichloromethane, diethyl ether, N, N-dimethylformamide, ethanol , Methanol, and tetrahydrofuran are from Merck Eurolab (Darmstadt, Germany). Oxalyl chloride, sodium thiocyanate, trifluoroacetic acid, dimethyl sulfoxide, thioacetamide, Lawesson's reagent, formic acid, and thiourea were purchased from Fluka (Deisenhofen, Germany). Adenosine 5'-triphosphate, 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, sodium chloride, magnesium chloride, 1,4-dithio-DL-threitol, sodium lauryl sulfate, polyoxyethylene sorbitan monolaurate, and ethylene glycol bis - (2-aminoethyl) -N, N, N ', N'-tetraacetic acid are from Sigma (Taufkirchen, Germany). The Rink-Amid-MBHA resin, (benzotriazol-1-yl) - N, N, N ', N'-tetramethyluronium hexafluorophosphate, as well as all Fmoc-amino acid pentafluorophenyl esters were obtained from Novablochem (Bad Soden, Germany). Cellulose membranes "Whatman 50" (Whatman Maidstone, UK) were used for the SPOT synthesis.

Chromatography and physical data

RP-18 HPLC-MS analyzes were performed by chromatography using a Hew lett Packard Series 1100 system (degasser G1322A, quaternary pump G1311A, automatic sampler G1313A, thermostatted column compartment G 1316A, variable UV detector G1314A) and coupled ESI- MS (Finnigan LCQ ion trap mass spectrometer) performed. The separation was carried out on RP-18 column material (Vydac 218 TP5215, 2.1 × 150 mm, 5 µm, C18, 300 A with guard column) at 30 ° C and a flow of 0.3 mL / min using a linear gradient for all chromatograms ( 5-95% B within 25 min, with A: 0.05% TFA in water and B: 0.05% TFA in CH ₃ CN). The UV detection took place at = 220 nm.

Preparative HPLC was carried out using a system from Merck / Hitachi (quaternary pump L-6250, variable UV detector L- / 400, interface D-7000, software: HPLC system manager D-7000 for NT 4.0) using a column from Merck Eurolab (LiChrospher 100, RP18, 10 × 250 mm) with a solvent flow of 6.0 mL / min. The solvent system used was composed of components A (H ₂ O / 0.1 vol% TFA) and B (CH ₃ CN / 0.1 vol% TFA).

Devices for the production of soluble peptides

The peptides used for immobilization were used as C-terminal peptide amides of a parallel synthesis machine "Syro" (MultiSynTech, Witten, Germany) after the Standard Fmoc protocol synthesized on Rink amide MBHA resin. All peptides obtained after cleavage from the resin and cleavage of all protective groups, by means of HPLC-MS analyzed and showed the desired molecular ion signals. After subsequent HPLC Purification, the peptides were lyophilized and stored at -20 ° C.

The peptides used for the immobilization (13-mer peptides of the proteins MBP, casein, Histone H1) were automatically generated using the device using the standard SPOT synthesis method Autospot AMS 222 (Abimed, Langenfeld, Germany) using the control software Autospot XL Ver. 2.02 carried out. The washing steps took place in stainless steel dishes (Merck Eurolab), which were moved on a rocking table.

example 1 Aldehyde functionalization of aminopropylsilylated glass surfaces

4-carboxybenzaldehyde (Fluka, 21873) was dissolved 0.3 M in DMF. The resulting Wed was activated by the addition of half an equivalent of DIC for 15 min at RT. Aminopropylsilylated glass surfaces cleaned with compressed air (2.5 × 7.5 cm; Sigma, Silane- Prep ™, S4651) were covered with the activated carboxybenzaldehyde solution thus obtained layers and incubated for three hours at RT. Then the glass treated in this way surfaces washed five times with 30 mL DMF each for 3 minutes at RT. After three The glass surfaces were washed three minutes each time with 30 mL DCM at RT dried and stored at 4 ° C until further use.

Example 2 Ketone functionalization of aminopropylsilylated glass surfaces

Levulinic acid (Fluka, 61380) was dissolved 0.3 M in DMF. The resulting mixture was activated by the addition of half an equivalent of DIC for 15 min at RT. With compressed air cleaned aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651) were covered with the levulinic anhydride solution thus obtained and three  Incubated for hours at RT. The glass surfaces treated in this way were then washed five times washed with 30 mL DMF each for 3 minutes at RT. After washing three times, three each Minutes with 30 mL DCM each at RT, the glass surfaces were dried and dried until stored for further use at 4 ° C.

Example 3 Thioamide functionalization of aminopropylsilylated glass surfaces ( Fig. 5, XXIXa)

Succinic acid monothioamide was dissolved in DM 0.2 M. The resulting mixture was activated by the addition of half an equivalent of DIC for 15 min at RT. Aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651) cleaned with compressed air were covered with the modified succinic anhydride solution thus obtained and incubated for three hours at RT. The glass surfaces treated in this way ( FIG. 5, XXIXa) were then washed five times with 30 mL DMF each for 3 minutes at RT. After washing three times for three minutes each with 30 mL DCM at RT, the glass surfaces were dried and stored at 4 ° C. until further use.

Example 4 Bromoketone functionalization of aminopropylsilylated glass surfaces

1,4-dibromo-2,3-diketobutane (Aldrich, D3,916-9) ( Fig. 5. XXX) was 0.2 M dissolved in DMF 0.1% triethylamine. Aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651) cleaned with compressed air were overlaid with solution and incubated at RT for seven hours. The glass surfaces treated in this way were then washed five times with 30 mL DMF each for 3 minutes at RT. After washing three times for three minutes each with 30 mL DCM at RT, the glass surfaces were dried and stored at 4 ° C until further use.

Example 5 Bromoketone functionalization of thioamide-modified glass surfaces (see FIG. 5)

The structures shown in this example (XXXIa) enable a simple and with gu surface modification)  

Aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651, see Example 3) reacted with succinic acid monothioamide were treated with a 0.1 M 1,4-dibromo-2,3-diketobutane solution ( FIG. 5, XXX) overlaid in ethanol and incubated for three hours at RT. The glass surfaces treated in this way ( FIG. 5, XXXIa) were then washed five times with 30 ml of ethanol each for 3 minutes at RT. After washing three times for three minutes each with 30 mL DCM at RT, the glass surfaces were dried and stored at 4 ° C. until further use.

Example 6 Bromoketone functionalization of phenylthiourea-modified glass surfaces (see FIG. 5)

The structures shown in this example (XXXIb) allow a simple and with gu surface modification)

Aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651) reacted with 4-carboxyphenylthiourea ( FIG. 5, XXIXb) were treated with a 0.1 M 1,4-dibromo-2,3-diketobutane solution ( FIG . 5, XXX) overlaid in ethanol and incubated for three hours at RT. The glass surfaces treated in this way ( FIG. 5, XXXIb) were then washed five times with 30 ml of ethanol each for 3 minutes at RT. After three washes for three minutes each with 30 mL DCM at RT, the glass surfaces were dried and stored at 4 ° C until further use.

Example 7 Bromopyruvic acid functionalization of aminopropylsilylated glass surfaces

This surface modification shows that it is possible to use even very small structures Conversion of an amino-functionalized into a bromoketone-functionalized glass surface used. Bromopyruvic acid is the smallest possible compound that both the carboxyl function necessary for the amide bond formation and that for the subsequent immobilization of the biomolecule necessary alpha-bromo-keto function contains.  

Sodium pyruvate was converted into the corresponding acid chloride with oxalyl chloride. With Compressed air cleaned aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane- Prep ™, S4651) were covered with the solution thus obtained and 5 hours at RT incubated. The glass surfaces treated in this way were then five times with 30 mL DMF washed at RT for 3 minutes each. After three washes, three minutes each The glass surfaces were dried with 30 mL methanol and DCM at RT each. The thus obtained pyruvic acid-modified glass surfaces were replaced by one hour treatment with a solution of 0.1 mL bromine in 10 mL glacial acetic acid Bromopyruvic acid-modified glass surfaces transferred. After three times The were washed three minutes each with 30 mL methanol and DCM at RT Glass surfaces dried and stored at 4 ° C until further use.

Example 8 Bromacetophenone functionalization of aminopropylsilylated glass surfaces (see FIGS . 2A, IX)

4-Acetylbenzoic acid (Fluka, 00932) was dissolved 0.3 M in DMF. The resulting mixture was activated by the addition of half an equivalent of DIC for 15 min at RT. Aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651) cleaned with compressed air were layered with the 4-acetylbenzoic anhydride solution thus obtained and incubated for 3 hours at RT. The glass surfaces treated in this way ( FIG. 2, IX) were then washed five times with 30 mL DMF each for 3 minutes at RT. After washing three times each for three minutes each with 30 mL methanol and DCM at RT, the glass surfaces were dried. The glass surfaces modified in this way were transferred to the broma cetophenone-modified glass surfaces ( FIG. 2, IV) by treatment with a solution of 0.1 ml of bromine in 10 ml of glacial acetic acid for one hour. After washing three times each for three minutes each with 30 mL methanol and DCM at RT, the glass surfaces were dried and stored at 4 ° C. until further use.

Example 9 Thiocyanato-acetophenone functionalization of aminopropylsilylated glass surfaces (see Fig. 2C)

Bromacetophenone-modified ( Fig. 2, IV, see Example 8), aminopropylsilylated glass surfaces (2.5 × 7.5 cm; Sigma, Silane-Prep ™, S4651) were overlaid with a 0.1 M solution of sodium thiocyanate in ethanol and five hours incubated at 50 ° C. The glass surfaces treated in this way ( FIG. 2, X) were then washed five times with 30 ml of ethanol in each case for 3 minutes at RT. After washing three times for three minutes each with 30 mL DCM at RT, the glass surfaces were dried and stored at 4 ° C. until further use.

Example 10 Immobilization of anthraniloyl peptides on aldehyde-functionalized glass surfaces (see FIG. 4)

• a) The peptides used for immobilization (13 mer peptides each, which cover the entire Primary structure of the proteins MBP, casein and histone H1 represent) were according to Stan dard-SPOT synthesis methods (R. Frank, Tetrahedron, 48, 1992, pp. 9217-9232; A. Kramer and J. Schneider-Mergener, Methods in Molecular Biology, vol 87: Combinatorial Peptide Libra ry Protocols, pp. 25-39, edited by: S. Cabilly; Humana Press Inc., Totowa, NJ) on cellulose as C-terminal peptide amides synthesized. Appropriately protected Fmoc Amino acid pentafluorophenyl ester dissolved in DMF and spotted 1 µL each. The dome lation reaction was carried out twice at RT for 25 min each. The spin-off of the Fmoc Protection group was carried out using 20% piperidine in DMF for 20 min at RT. To the last Fmoc cleavage was carried out by the N-termini of the cellulose-bound peptides a five hour incubation with a saturated solution of isatoic acid in DMF at 50 ° C in the corresponding ortho-aminobenzoylated derivatives are transferred. The secession of the perma no protective groups (tBu for serine, threonine, tyrosine, glutamic acid and aspartic acid; Boc for lysine; Trityl for asparagine, glutamine, cysteine, histidine and Pbf for arginine) was carried out by treatment for two hours with 97% trifluoroacetic acid at RT. Then were the cellulose-bound peptides washed with DCM, MeOH and diethyl ether and in Va vacuum dried. The peptides were cleaved from the cellulose using of ammonia gas 24 hours at RT. The spots with the physically adsorbed peptides were punched out and transferred to 96-well microtiter plates. After removing the peptides with The samples were filtered in 200 µL 20% methanol under ultrasound conditions, in 384well microtiter plates transferred, lyophilized and at -20 ° C until further use stored.  
• b) The ortho-aminobenzoyl peptides present in microtiter plates were dissolved in 200 mM sodium phosphate buffer pH 6.0 (final concentration of the peptides 0.5 mM), which contained 15% by volume DMSO. Subsequently, 0.01 .mu.L of this solution was applied at RT to the aldehyde-modified glass surfaces (cf. Example 1 and FIG. 4) using a Gesim nano plotter and incubated at RT for four hours. After washing three times at RT with 100 mL distilled water each, the modified glass surfaces ( FIG. 4, XIII) were incubated with 30 mL of a 40% aqueous solution of hydroxylamine for 30 minutes at RT to deactivate the remaining aldehyde functions. The glass surfaces were then washed five times for 3 minutes each with 50 mL water at RT and then twice each for 3 minutes each with 50 mL methanol at RT. The glass surfaces treated in this way were dried and stored at 4 ° C. until further use.

Example 11 Immobilization of peptides containing thioxocitrulline on bromacetophenone-functionalized glass surfaces (cf. FIG. 2B)

• a) The peptide used for immobilization thioxoCit-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ ( Fig. 2B, Vb, R ⁴ = H) was fixed to the solid according to standard methods of Fmoc-based chemistry Phase synthesized as a C-terminal peptide amide. Appropriately protected Fmoc amino acids were activated with one equivalent of HBTU and three equivalents of diisopropylamine in DMF and coupled to Rink-Amind-MBHA resin in DMF. The Fmoc protective group was cleaved using 20% piperidine in DMF for 30 min at RT. The permanent protective groups (tBu for serine and Pbf for arginine) were split off and the polymer was simultaneously detached by treatment with 97% trifluoroacetic acid at RT for two hours. The resulting mixture was filtered and the filtrate was precipitated by adding tert-butyl methyl ether. The precipitate was separated off and purified by means of HPLC on RP18 material with acetonitrile / water mixtures (0.1% trifluoroacetic acid). The fractions containing the desired product were lyophilized and stored at -20 ° C. until further use.
• b) The peptide thioxoCit-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ ( Fig. 2B, Vb, R ⁴ = H) was dissolved in 200 mM sodium phosphate buffer pH 5.5 (final concentration 1 mM) containing 30 vol% glycerol. Then, at RT, 1 nL of this solution was applied in an arrangement of 70 rows and 168 columns (11760 spots in total) to the bromacetophenone-functionalized glass surfaces (FIGS . 2B, IV, see Example 8) using a Gesim nano plotter. The spot-to-spot distance was 0.3 mm. The glass surfaces treated in this way ( FIG. 2B, VIII, R ⁴ = H) were then incubated for 3 hours at RT. After washing three times at RT with 100 mL of distilled water each, the modified glass surfaces were incubated with 30 mL of a 3% aqueous solution of thioacetamide for 30 minutes at RT to deactivate the remaining bromoacetophenone functions. The glass surfaces were then washed five times for 3 minutes each with 50 mL water at RT and then twice each for 3 minutes each with 50 mL methanol at RT. The glass surfaces treated in this way were dried and stored at 4 ° C. until further use.

Example 12 Immobilization of thioamide-containing peptides on bromketone-functionalized glass surfaces (see FIG. 5)

• a) The peptide used for immobilization Leu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide (Kemptide-thioamide) ( Fig. 5, Va) was according to standard methods of Fmoc-based chemistry on the solid phase as a C-terminal Peptide thioxoamide synthesized. Fmoc-Gly-OH bound to Rink-Amid-MBHA resin was refluxed with Lawesson's reagent in THF for 3 hours. The resin was then washed with THF and DCM, shaken for 1 hour with DMF and then washed with DMF, DCM and methanol. After removing the Fmoc protective group with 50% morpholine in DMF (40 min), the correspondingly protected Fmoc amino acids were coupled with one equivalent of HBTU and three equivalents of diisopropylamine in DMF. The Fmoc protective group was cleaved using 20% piperidine in DMF for 30 min at RT. The permanent protective groups (tBu for serine and Pbf for arginine) were split off and the polymer was simultaneously detached by treatment with 97% trifluoroacetic acid at RT for two hours. The resulting mixture was filtered and the filtrate was precipitated by adding tert-butyl methyl ether. The precipitate was separated off and purified by means of HPLC on RP18 material with acetonitrile / water mixtures (0.1% trifluoroacetic acid). The fractions containing the desired product were lyophilized and stored at -20 ° C. until further use.
• b) The peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide ( Fig. 5, Va) was dissolved in 200 mM sodium phosphate buffer pH 5.5 (final concentration 1 mM) containing 50 vol% glycerol. Then, at RT, 1 nL of this solution was applied in an arrangement of 70 rows and 168 columns (11760 spots in total) to the bromketone-functionalized glass surfaces (cf. Example 5 and Fig. 5, XXXIa) by means of a Gesim nano plotter. The spot-to-spot distance was 0.3 mm. The glass surfaces treated in this way were then treated with a microwave for 2 min and then incubated at RT for 3 hours. After washing three times at RT with 100 mL of distilled water each, the modified glass surfaces ( Fig. 5, XXXIIc) were incubated with 30 mL of a 3% aqueous solution of thioacetamide for 30 minutes at RT to deactivate the remaining alpha-bromoketone functions. The glass surfaces were then washed five times for 3 minutes each with 50 mL water at RT and then twice each for 3 minutes each with 50 mL methanol at RT. The glass surfaces treated in this way were dried and stored at 4 ° C. until further use.

Example 13 Analysis of kinase-mediated peptide modifications on modified glass surfaces ( Fig. 5, XXXII)

A glass surface modified with the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide (see Example 12 and Fig. 5, XXXIIc) was washed with 10 ml of 100 µM ATP in kinase buffer (50 mM Tris-HCl, 150 mM NaCl, 30 mM MgCl ₂ , 4 mM DTT, 2 mM EGTA, pH 7.5) incubated at RT for 10 minutes. The glass surface was dried and then a second, unmodified glass surface of the same dimensions was placed on the peptide-modified glass surface. Then 50 µL of a mixture of protein kinase A (Sigma, P26452, 10 U / mL), 100 µM / mL ATP and 100 µCi / mL γ- ³² P-ATP (Amersham, 9.25 mBq / 250 µCi / 25 µL, Activity <5000 Ci / mmol) in kinase buffer (50 mM Tris-HCl, 150 mM NaCl, 30 mM MgCl ₂ , 4 mM DTT, 2 mM EGTA, pH 7.5) by capillary forces in the gap caused by the on the mo second glass surface is formed, abandoned. Then was incubated at RT in an almost water-saturated atmosphere for 30 min. In order to reduce the background caused by non-specific binding of ATP or kinase molecules to the glass surfaces, the modified glass surface was washed as follows:

• 3 times 3 minutes at RT with wash buffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)
• - 2 times 3 minutes at RT with 1 M NaCl solution
• - 2 times 3 minutes at RT with distilled water
• - 2 times 3 minutes at RT with 80% formic acid in ethanol
• - 2 times 3 minutes at RT with distilled water
• - 2 times 5 minutes at 50 ° C with a solution containing 6 M urea, 2 M thiourea and 1% SDS contained
• - 3 times 3 minutes at RT with distilled water
• - 3 times 3 minutes at RT with methanol

After the glass surface had dried, the amount of radioactive phosphate incorporated in the glass surface-bound peptides was determined by means of a PhosphorImager system (FLA-3000, FUJIFILM) (cf. FIG. 6).

Example 14 Analysis of kinase-mediated peptide modifications on modified glass surfaces ( FIG. 2B, VIII, R ⁴ = H)

A glass surface modified with the peptide thioxoCit-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ ( FIG. 2B, Vb, R ⁴ = H) (cf. Example 11 and FIG. 2B, VIII, R ⁴ = H) was incubated with 10 ml of 10 μM ATP in kinase buffer (50 mM Tris-HCl, 150 mM NaCl, 30 mM MgCl ₂ , 4 mM DTT, 2 mM EGTA, pH 7.5) at RT for 10 minutes. The glass surface was dried and then a second, non-modified glass surface of the same dimensions was placed on the peptide-modified glass surface. Then 50 µL of a mixture of protein kinase A (Sigma, P26452, 10 U / mL), 100 µM / mL ATP and 100 µCi / mL γ- ³² P-ATP (Amersham, 9.25 mBq / 250 µCi / 25 µL, Activity <5000 Ci / mmol) in kinase buffer (50mM Tris-HCl, 150mM NaCl, 30mM MgCl ₂ , 4mM DTT, 2mM EGTA, pH 7.5) by capillary forces in the gap caused by those modified on the Glass surface lying on second glass surface is formed, abandoned. The mixture was then incubated at RT in an almost water-saturated atmosphere for 30 min. In order to reduce the background caused by non-specific binding of ATP or kinase molecules to the glass surfaces, the modified glass surface was washed as follows:

• 3 times 3 minutes at RT with wash buffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)
• - 2 times 3 minutes at RT with 1 M NaCl solution
• - 2 times 3 minutes at RT with distilled water
• - 2 times 3 minutes at RT with 80% formic acid in ethanol
• - 2 times 3 minutes at RT with distilled water
• - 2 times 5 minutes at 50 ° C with a solution containing 6 M urea, 2 M thiourea and 1% SDS contained
• - 3 times 3 minutes at RT with distilled water
• - 3 times 3 minutes at RT with methanol

After the glass surface had dried, the amount of radioactive phosphate incorporated in the glass surface-bound peptides was determined using a phosphorus-lean system (FLA-3000, FUJIFILM) (cf. FIG. 7).

The drawings disclosed in the foregoing description, claims, and 00340 00070 552 001000280000000200012000285910022900040 0002010118698 00004 00221 Features of the invention can be used both individually and in any combination Realization of the invention in its various embodiments may be essential.

Claims (29)

1. A method of immobilizing compounds on a surface comprising the steps

• - Providing a compound to be immobilized as a first reaction partner and a surface as a second reaction partner, the first reaction partner comprising a first reactive group and the second reaction partner comprising a first reactive group, and
• - Implementation of the two reaction partners

characterized in that a heterocyclic ring system is formed when the two reactants are reacted. 2. The method according to claim 1, characterized in that the heterocyclic ring system is formed between the first and second reactants. 3. The method according to claim 1 or 2, characterized in that the heterocyclic Ring system from parts of the first reactive group of the first reaction partner and the first th reactive group of the second reactant is formed. 4. The method according to any one of claims 1 or 3, characterized in that the hete rocyclic ring system from the group consisting of thiazoles, aminothiazoles, imidazoles, benzimida zole, indoles, tetrahydroisoquinolines, tetrahydro-β-carbolines, dihydroquinazoline, oxidati on products from ortho-amino-benzoic acid derivatives and pyrazoles. 5. The method according to any one of claims 1 to 4, characterized in that the heterocyclic ring system comprises the following structural elements:
wherein the substituents are selected independently of one another and wherein
X is O, S or NH, preferably NH;
A is a ring system of 4 to 8 atoms, preferably aryl;
YS or O, preferably O;
R ¹ -R ³ alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles or biomolecule
R ⁴ -R ^{9 is} alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, biomolecules or H, D, T. 6. The method according to any one of claims 1 to 5, characterized in that the reacti ve group is selected from the group consisting of thioamines, thiocarbonyls, thioureas, Hydroxylamino, alpha-isothiocyanatoketones, anthranilic acid amides, anilines, aldehydes, keto ne, 1,2-diketones, amines, aminooxy derivatives, arylhydrazines, arylamines, heteroarylamines, ortho-amino-benzoic acid, hydrazines, 2-cyano-aldehydes, imines and acid halides summarizes. 7. The method according to any one of claims 1 to 6, characterized in that the first reactive group of the compound to be immobilized is a reactive group consisting of is selected from the group consisting of thioamide, aniline, thiourea, thiocarbonyl, anthranilic acid, Contains anthranilic acid amide and hydroxylamino. 8. The method according to any one of claims 1 to 7, characterized in that the first surface reactive group is a reactive group selected from the group which includes carbonyl, keto and aldehyde. 9. The method according to any one of claims 1 to 8, characterized in that the order is a chemoselective reaction. 10. The method according to any one of claims 1 to 9, characterized in that the im mobilization is directed immobilization. 11. The method according to any one of claims 1 to 10, characterized in that the ent standing heterocyclic ring differs from the E in its absorption / emission behavior products differs. 12. The method according to any one of claims 1 to 11, characterized in that the to immobilizing compound is selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides, protein domains, proteins, sugar, poly sugar, lipids, polylipi de, natural products and low molecular weight compounds.   13. The method according to any one of claims 1 to 12, characterized in that the O surface is a planar surface. 14. The method according to claim 13, characterized in that it is the planar Surface is a chip. 15. The method according to any one of claims 1 to 14, characterized in that the O surface comprises a material selected from the group consisting of silicates, ceramics, Glass, metals, organic carrier materials, cellulose, nitrocellulose, PTFE membranes, Includes polypropylene, gold, titanium dioxide and silicon oxide. 16. The method according to any one of claims 13 to 15, characterized in that the planar Surface is a non-porous surface. 17. The method according to any one of claims 1 to 16, characterized in that the order placement of the connection to be immobilized with the surface under microwave radiation development takes place. 18. A method for producing an arrangement comprising at least two different ones Connections at at least two different locations on a planar surface, thereby characterized in that the first connection at a first point on the planar surface and the second connection at a second location on the planar surface after one of the ver drive is immobilized according to one of the preceding claims. 19. Planar surface can be produced by a method according to one of the preceding the claims. 20. Planar surface comprising a compound covalently immobilized thereon a heterocyclic ring system between the immobilized compound and the surface is arranged, which is only formed by the immobilization process. 21. Planar surface according to claim 19 or 20, characterized in that  the heterocyclic ring system is selected from the group consisting of thiazoles, aminothiazoles, Imidazoles, benzimidazoles, indoles, tetrahydroisoquinolines, tetrahydro-β-carbolines, dihydro quinazoline, oxidation products from ortho-amino-benzoic acid derivatives and pyrazoles. 22. Planar surface according to one of claims 19 to 21, characterized in that the heterocyclic ring system comprises the following structural elements:
wherein the substituents are selected independently of one another and wherein
X is O, S or NH, preferably NH;
A is a fused ring system of 4 to 8 atoms, preferably aryl;
YS or O, preferably O;
R ¹ -R ³ alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles or biomolecule
R ⁴ -R ^{9 is} alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, biomolecules or H, D, T. 23. Arrangement producible according to claim 18. 24. The arrangement, in particular according to claim 23, comprising a planar surface any of the preceding claims.   25. Use of an arrangement according to one of the preceding claims or a planar surface according to one of the preceding claims in a method for determining the substrate specificity of an enzymatic activity comprising the following steps:

• Provision of a set of amino acid sequences on the planar surface of a carrier material, the amino acid sequences being immobilized in a directed manner,
• Contacting and / or incubating an enzymatic activity with that with the set of amino acid sequences, and
• Detection of a reaction between one of the immobilized amino acid sequences and the enzymatic activity,

characterized in that

• - The planar surface is a planar surface according to one of claims 1 to 17 or a planar surface of an arrangement according to one of claims 23 to 24, and
• - When the enzymatic activity is implemented with the set of amino acids, there is a change in the molecular weight of at least one of the amino acid sequences.

26. The method according to claim 25, characterized in that the change in the mole molecular weight by forming or breaking a covalent bond to one of the ami no acid sequences takes place, preferably on that amino acid sequence that with the enzymatic activity reacts. 27. The method according to claim 25 or 26, characterized in that the detection of Reaction is carried out by detecting the change in molecular weight.   28. The method according to any one of claims 25 to 27, characterized in that the Detection is carried out by a detection method that is selected from the group, the Auto includes radiography, plasmon resonance spectroscopy and fluorescence spectroscopy. 29. The method according to any one of claims 25 to 28, characterized in that the en zymatic activity is selected from the group consisting of kinases, sulfotransferases, glyco syltransferases, acetyltransferases, farnesyltransferases, palmityltransferases, phosphatases, Includes sulfatases, esterases, lipases, acrylases and proteases.