WO2003014138A2

WO2003014138A2 - Method for immobilizing compounds by means of nitron formation and arrangement for immobilizing compounds

Info

Publication number: WO2003014138A2
Application number: PCT/EP2002/008671
Authority: WO
Inventors: Frank Osterkamp; Mike Schutkowski
Original assignee: Jerini Ag
Priority date: 2001-08-03
Filing date: 2002-08-01
Publication date: 2003-02-20
Also published as: WO2003014138A3; WO2003014138A8; AU2002336944A1; DE10138092A1

Abstract

The invention relates to a method for immobilizing compounds on a surface, comprising the following steps: provision of a compound which is to be immobilized as a first reaction partner and provision of a surface as a second reaction partner, the first reaction partner comprising a first reactive group and the second reactive partner comprising a first reactive group; and reacting both reaction partners, whereby a nitron group is formed during the reaction of both reaction partners and the surface is a planar surface.

Description

Process for immobilizing Nerbindungen and arrangement of immobilized Nerbindungen

The present invention relates to processes for immobilizing compounds on a surface, in particular polymer compounds, a structure comprising a planar surface with a compound immobilized thereon, processes for producing an arrangement comprising at least two different compounds, preferably polymeric compounds, at at least two different locations planar surface, a planar surface that can be produced with it, a method for determining the substrate specificity of an enzymatic activity and a method for cleaning a compound, in particular a polymeric compound.

With the ever-increasing availability of sequence information from the various genome projects, the arrangement of nucleic acid fragments with high density on a carrier material, so-called chips or biochips, became very important, but their full potential could only be exploited with the availability of newer synthesis techniques and miniaturization a variety of applications. In addition to nucleic acids, natural products or libraries thereof, but also arrangements of oligopeptides and proteins were applied to such chips. Cellulose (DR Englebretsen, DRK 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. Krarner 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), Glas (SPA Fodor, JLRead, MCPirrung, L.Stryer, ATLu, D.Solas; 1991, Light-directed, spatially addressable parallel chemical synthesis, Science, 251, 767-773 J. Robles, M. contribution, V. Marchan, Y. Perez, I. Travesset, E. Pedroso, A. Grandas; 1999, Towards nucleopeptides containing any trifunctional amino acid, tetrahedron , 55, 13251-13264), nitrocellulose (SJ Hawthorne, M. Pagano, P. Harriott, DW Haiton, B. Walker; 1998, The synthesis a nd utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors, anal. Biochem., 261, 131-138), PTFE membranes, gold (BT 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), 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 modified polypropylene surfaces.

With the increasing importance of proteomics and their biotechnological application, peptides and proteins are becoming the focus of interest. In general, it is proteins and mostly their enzymatic activities that enable almost all biochemical reactions inside and outside the cell. The use of arrays of nucleic acids, with which either the messenger RNA (mRNA) generated by genes that are currently active in the cell or DNA copies of this mRNA are detected, is of great importance, but is the one obtainable with it Information is insufficient for a number of reasons to understand the processes of both intracellular and extracellular processes and to use them in various biotechnological applications. One reason is that the amount of mRNA in a cell often does not correlate with the corresponding amount of protein produced in the cell. In addition, proteins that have been produced can be significantly influenced in their enzymatic activity (and thus in their biological function) by slight chemical modifications in the cell (post-translational modifications). It is therefore necessary to carry out a parallel analysis of the enzymatic activity of as many proteins as possible, in particular enzymes. Such an approach allows u. a. the very fast determination of the substrate specificity of a defined enzyme, which in turn is an important prerequisite for the design of knowledge-based inhibitors, or the selective testing of drugs or drug candidates, especially in the context of predicting side effects.

Basically, immobilization has a number of advantages. This includes the increased stability of the immobilized compounds. If, for example, nucleic acids or proteins are immobilized, it is regularly observed that their half-life increases significantly when exposed to various media, including media that have a nuclease or protease activity. One reason for the observed increased half-life may certainly be the reduced accessibility of the nucleic acids or protein for the corresponding degradative enzyme activities. Another advantage of immobilization is the reusability of the immobilized compounds. For example, by immobilizing compounds on support materials that are used in the context of chromatographic processes, after passing through a medium that is to be purified or modified under the influence of the immobilized compounds, and optionally regenerating the support material, this support material can be added again use. Examples of such processes are affinity chromatography or bioconversion and biotransformation.

Another application of immobilized compounds or arrangements of e.g. B. Nucleic acids or proteins are the biochips mentioned at the beginning. These require a precisely defined correlation between the type of immobilized molecule and its positioning or arrangement on a surface.

There are basically two methods of connecting biomolecules, such as oligopeptides or nucleic acids, to a surface (planar support or resin beads) or a biopolymer (protein surface and thus bioconjugate formation). On the one hand, prefabricated (and purified) biomolecules can be used for the immobilization reaction, which can be directional or non-directional, and on the other hand these biomolecules can be synthesized step by step directly on a corresponding surface. In the prior 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 Molecular Biology, vol 87: Combinatorial Peptide Library Protocols, pp. 25-39, edited by: S. Cabilly; 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 (SPA Fodor, JLRead, MCPirrung, L.Stryer, ATLu, D.Solas; 1991, Light-directed, spatially addressable parallel chemical synthesis, Science, 251, 767-773, JP Pellois, W. Wang, XL Gao; 2000, Peptide synthesis based on t-Boc chemistry and solution photo-generated acids, J. Comb. Chem., 2, 355-360) or on chitin (W. Neugebauer, RE Williams, JR Barbier, R. Brzezinski, G. Willick; 1996, Peptide synthesis on chitin, Int. J. Pept. Prot. Res., 47, 269-275) or an 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 peptide 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, JLRead, MCPirrung, L.Stryer, ATLu, D.Solas; 1991, Light-directed, spatially addressable parallel chemical synthesis, Science, 251, 767 -773, J. Robles, M. contribution, V. Marchan, Y. Perez, I. Travesset, E. Pedroso, A. Grandas; 1999, Towards nucleopeptides containing any trifunctional amino acid, Tetrahedron, 55, 13251-13264 ), can be synthesized directly. 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. This can be the case 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 removal of all protective groups of the side chain functions) Pose 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 required 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%, only 66.2% and 44.3% result for the examples listed above.

When using immobilization technology, for example in the field of biochips and in particular when using biochips to determine the substrate specificity of enzymes such as kinases, phosphatases, acetyltransferases, glycosyltransferases, farnesyltransferases, sulfatases, sulfotransferases or proteases, which depend on defined arrangements Peptides differing in their amino acid sequence, due to these limitations, in addition to the desired amino acid sequence, a large number of further amino acid sequences will be present, which are characterized by the lack of one or more amino acid building blocks. Exactly these by-products, known to those skilled in the art as faulty or terminating sequences, can, under certain circumstances, strongly falsify the result of the incubation with an enzymatic activity which modifies the amino acid sequences arranged on the surface, or make it difficult to interpret the results. In order to avoid these disadvantages, the immobilization of biomolecules that have been cleaned as far as possible can be helpful. In the prior art, biomolecules, such as peptides or nucleic acids, on various surfaces such as glass (SPA Fodor, JLRead, MCPirrung, L.Stryer, ATLu, D.Solas; 1991, Light-directed, spatially addressable parallel chemical synthesis, Science , 251, 767-773, JP Pellois, W. Wang, XL Gao; 2000, Peptide synthesis based on t-Boc chemistry and solution photogenerated acids, J. Comb. Chem., 2, 355-360, J. Robles, M Contribution, V. Marchan, Y. Perez, I. Travesset, E. Pedroso, A. Grandas; 1999, Towards nucleopeptides containing any trifunctional amino acid, Tetrahedron, 55, 13251-13264), cellulose (DR Englebretsen, DRK 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), nitrocellulose (SJ. Hawthorne, M. Pagano, P. Harriott, DW Haiton, B. Walker; 1998, The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone in - hibitors, Anal. Biochem., 261, 131-138), 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) or Gold (BT 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 differentiation of the immobilization can be made based on the orientation or binding of the individual immobilized compound relative to the surface. A distinction can be made here between specific and non-specific immobilization. In the case of non-specific immobilization, the contact between the compound to be immobilized and the surface on or on which the compound is immobilized takes place in different ways in an immobilization event. In other words, either different reactive groups contained in the compound to be immobilized react with the surface or different reactive groups present on the surface react with a reactive group of the compound to be immobilized (crosslinking). As a result, a population of immobilized compounds is formed which are heterogeneous in terms of their surface binding. This different type of immobilization can lead to an often undesirable different behavior of the immobilized Connection. For example, when immobilizing a substrate for a kinase on or on a surface using the amino groups contained within this substrate, several (depending on the number of amino groups present in the compound to be immobilized) options for the reaction and thus the final orientation of the There is a connection on the surface. If, during the subsequent incubation of this immobilized compound (kinase substrate) with a biological fluid containing at least one kinase activity, one or more of these amino groups are required for the effective formation of an enzyme substrate / complex, such non-specific immobilization can lead to only one small population of the immobilized substrates is anchored in the correct manner and thus the measurement signal is below the detection limit. Therefore, a specific or directed immobilization is of great advantage. In an immobilization event, the contact between the compound to be immobilized and the surface on or on which the connection is immobilized takes place in the same way, and all connections are bound to or on the surface in a defined and predictable orientation.

This type of immobilization is usually a covalent immobilization, with a chemoselective reaction between the compound to be immobilized and the surface (of the carrier material). A number of reactions can be used for this purpose, which are known to those skilled in the art as such (GA Lemieux and CR Bertozzi, 1998, chemoselective ligation reactions with proteins, oligosaccharides and cells, TIBTECH, 16, 506-513) and in FIG. 1 are shown. The chemoselective reaction can basically be caused by two mechanisms. In one mechanism, the compound to be immobilized has a reactive group that 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 selective reaction with the surface.

The use of immobilized compounds such as amino acid sequences in the form of biochips makes it necessary for both the compounds and the link between the surface and the immobilized compound to pass the respective test or investigation. conditions. Furthermore, there is an increasing need in the art for a quantification of the interaction between the immobilized compound and an agent which is contained in a sample which is to be examined and, if appropriate, quantified by means of the immobilized compound. 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 providing a method for immobilizing compounds which meets the needs described above. The invention was also based on the object of providing a method which allows simple immobilization of compounds with simultaneous possible modification of the linker used for the immobilization, in particular after it has been installed between the surface and the compound to be immobilized.

Another object of the invention was to provide a process for the purification of compounds, a reliable separation of by-products formed during the synthesis thereof, in particular termination sequences, being ensured.

According to the invention, the object is achieved in a first aspect by a method for immobilizing compounds on a surface, which comprises 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

React the two reactants

comprises, a nitron group being formed when the two reactants react and the surface being a planar surface. In a second aspect, the object is achieved by a method for immobilizing polymeric compounds on a surface, the individual polymeric compounds being composed of a plurality of subunits and the method comprising the following steps:

a) providing a polymeric compound to be immobilized in solution as the first reaction partner and a surface as the second reaction partner, the first reaction partner comprising a first reactive group and the second reaction partner comprising a first reactive group; and

b) reacting the two reactants to form a nitron group, in particular between the polymeric compound and the surface.

In one embodiment of the method according to the invention it is provided that the nitron group is formed between the first and the second reaction partner.

In a further embodiment of the method according to the invention it is provided that the nitron group is formed from parts of the first reactive group of the first reaction partner and the first reactive group of the second reaction partner.

In a still further embodiment of the method according to the invention it is provided that the first reactive group of the compound to be immobilized is a reactive group which is selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals.

In one embodiment of the method according to the invention it is provided that the first reactive group of the surface is a reactive group which is selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines, aldehydes, ketones, acetals, ketals, thiocarbonyl compounds, thioketals and thioacetals includes.

In a preferred embodiment of the method according to the invention it is provided that the reaction is a chemoselective reaction. In a particularly preferred embodiment of the method according to the invention it is provided that the immobilization is a directed immobilization.

In a very particularly preferred embodiment of the method according to the invention it is provided that the compound to be immobilized is selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides, protein domains , Proteins, sugars, poly sugars, lipids, polylipids, coenzymes, natural products and low molecular compounds as well as derivatives and analogs of the above-mentioned groups.

In one embodiment of the method according to the invention it is provided that at least one of the subunits is selected from the group consisting of amino acids, nucleotides, carbohydrates, amino alcohols, polyalcohols, diols, terpenes, lipids, olefins, epoxies, haloalkanes, alcohols, polyamines, diamines, amines , Aminothiols, mercapto alcohols, polythiols, dithiols and thiols.

In a further embodiment of the method according to the invention it is provided that the surface is a planar surface.

In yet another embodiment of the method according to the invention it is provided that the planar surface is a chip, preferably the surface of a chip.

In a preferred embodiment of the method according to the invention, it is provided that the surface comprises a material that is selected from the group consisting of silicates, ceramics, glass, metals, organic carrier materials, cellulose, nitrocellulose, chitin, PTFE, polypropylene, polystyrene, copolymers, Gold, platinum, silver, copper, silicon, titanium dioxide, magnetic materials, magnetizable materials and silicon oxide.

In a particularly preferred embodiment of the method according to the invention, it is provided that the planar surface is a non-porous surface. In a very particularly preferred embodiment of the method according to the invention it is provided that the reaction of the compound to be immobilized with the surface takes place under microwave radiation.

In a more preferred embodiment of the method according to the invention it is provided that the nitron structure has a structure according to formula (I)

in which

the radical X is the surface, in particular a surface of a solid phase support, and at least one of the radicals Y or Z is an immobilized compound; or

at least one of the radicals Y or Z is a surface, in particular a surface of a solid phase support, and the radical X is an immobilized compound.

In an even more preferred embodiment of the method according to the invention it is provided that at least Z or Y or X is a surface and the second reaction partner comprises the structure FR ¹ -, wherein

R ^{1 is} selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals and

F is a compound functionalized with R ¹ or a material functionalized with R ¹ .

In an even more preferred embodiment of the method according to the invention it is provided that Z or Y or X is an immobilized compound and the first reaction partner comprises the structure GR ² -, wherein R ^{2 is} selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals and

G is a compound functionalized with R ² .

In one embodiment of the method according to the invention it is provided that the immobilized compound has a structure G -G -, wherein

G ^{1 is} selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, PNA, peptides, of which polypeptides, protein domains, proteins, sugars, poly sugars, lipids, polylipids, coenzymes, Includes natural products, and low molecular weight compounds and derivatives and analogues thereof, and

G ^{2 is} a connecting element which connects G ¹ and the nitron group directly and the connecting element can be split selectively at the point of connection to G ¹ or selectively between G ¹ and the nitron group. '

In a further embodiment of the method according to the invention it is provided that the connecting element G ^{2 is} selected from the group which additionally comprises functionalized protective groups, the protective groups preferably being those for amines, alcohols, diols, carboxylic acids, carbonyl groups and thiols.

In a third aspect, the object is achieved by a structure which comprises a planar surface, preferably a surface of a solid phase support, with a compound immobilized thereon, and the structure comprising a nitron group.

In one embodiment of the structure according to the invention it is provided that the immobilized compound is a polymeric compound. In a preferred embodiment of the structure according to the invention it is provided that the nitron group is arranged between the surface and the immobilized compound.

In a more preferred embodiment of the structure according to the invention it is provided that a connecting element is arranged between the nitron group and the immobilized connection.

In a fourth aspect, the object is achieved by a method for producing an arrangement which comprises at least two different compounds, in particular polymeric compounds, at at least two different locations on a planar surface, the first connection at a first location on the planar surface and the second Connection is immobilized at a second location on the planar surface by one of the methods according to the invention.

In a further aspect, the object is achieved by a planar surface which can be produced by one of the methods according to the invention.

In another aspect, the object is achieved according to the invention by a planar surface which comprises a compound covalently immobilized thereon, in particular a polymeric compound, a nitron group being arranged between the immobilized compound and the surface.

In a seventh aspect, the object is achieved according to the invention by an arrangement comprising at least two different compounds, preferably polymeric compounds, at at least two different locations on a planar surface which can be produced by the method according to the invention.

In one embodiment, the arrangement comprises the planar surface according to the invention.

In an eighth aspect, the object is achieved according to the invention by an arrangement comprising at least two different compounds, preferably polymeric compounds, at at least two different locations on a planar surface which comprises the planar surface according to the invention. In a ninth aspect, the object is achieved according to the invention by a method for determining the substrate specificity of an enzymatic activity, which comprises the following steps:

Providing a set of amino acid sequences on the planar surface of a carrier material, the amino acid sequences being directionally immobilized,

Contacting and / or incubating an enzymatic activity with the set of amino acid sequences, and

Detection of a reaction between one of the immobilized amino acid sequences and the enzymatic activity,

in which

the planar surface is a planar surface according to the invention or a planar surface of an arrangement according to the invention, and

when the enzymatic activity is implemented with the set of amino acids, the molecular weight of at least one of the amino acid sequences is changed.

In one embodiment of the method according to the invention for determining the substrate specificity of an enzymatic activity, it is provided that the reaction is detected on or using the amino acid sequence immobilized on the surface of the carrier material.

In a preferred embodiment of the method according to the invention for determining the substrate specificity of an enzymatic activity, it is provided that the change in molecular weight takes place by the formation or cleavage of a covalent bond to one of the amino acid sequences, in particular to the amino acid sequence which reacts with the enzymatic activity. In a particularly preferred embodiment of the method according to the invention for determining the substrate specificity of an enzymatic activity, it is provided that the reaction is detected by detecting the change in the molecular weight.

In a very particularly preferred embodiment of the method according to the invention for determining the substrate specificity of an enzymatic activity, it is provided that the detection is carried out by a detection method which is selected from the group consisting of autoradiography, plasmon resonance spectroscopy, fluorescence spectroscopy, UV spectroscopy, infrared spectroscopy, Includes electron microscopy, atomic force microscopy and scanning tunneling microscopy.

In a further embodiment it is provided that the units to be detected are introduced in a subsequent reaction via specifically binding or specifically reacting, preferably labeled reagents, which are selected from the group consisting of antibodies, enzymes, proteins, chelators, synthetic receptors, amines and Thiols includes.

In yet another embodiment of the method according to the invention for determining the substrate specificity of an enzymatic activity, it is provided that the enzymatic activity is selected from the group consisting of kinases, sulfotransferases, glycosyltransferases, acetyltransferases, farnesyltransferases, palmityltransferases, phosphatases, sulfatases, esterases, lipases , Acetylases and proteases.

In a tenth aspect, the object is achieved by a method for purifying a compound, in particular a polymeric compound, which comprises the following steps:

Providing a mixture of compounds, the mixture being composed such that the mixture contains at least one chemical compound to be immobilized which is provided with one or more reactive groups, in particular a polymeric compound, together with one or more reactive groups. n) includes non-containing by-products,

Immobilizing the chemical compound containing the reactive group (s) on a surface, in particular a solid phase support, by reaction of the reactive ve (n) group-containing chemical compound with the surface to form one or more covalent bond (s) between the compound and the surface, wherein between the surface and the immobilized compound at least one nitron grouping is formed by the reaction or at least present is and

Remove the non-immobilized chemical compound (s) and the non-immobilized by-products.

In one embodiment it is provided that the method according to the invention for cleaning a compound comprises a further step:

Cleaving off the immobilized compound from the surface, in particular by cleaving a covalent bond other than the one formed in the second step.

In a further embodiment it is provided that the surface of the method according to the invention for cleaning a compound comprises a reactive group.

In yet another embodiment it is provided that the one or more covalent bonds are formed by reaction of the reactive group (s) on the compound with one or more of the reactive group (s) on the surface.

In one embodiment it is provided that the reactive group is selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals.

In a preferred embodiment it is provided that the reactive group is contained in the compound several times, preferably 2 times to 50 times, preferably 2 to 10 times.

In an even more preferred embodiment of the method according to the invention for cleaning a connection, it is provided that the structure according to the invention is formed as a result of the one or more covalent bond (s) between the connection and the surface. In one embodiment of the method according to the invention for cleaning a connection, it is provided that the surface is planar.

In a further embodiment of the method according to the invention for cleaning a compound, it is provided that the surface consists of a material selected from the group consisting of solid phase supports for synthesis, activated carbon, inorganic particles, magnetic particles and magnetizable particles, materials suitable for chromatography or solid phase extraction , Silicon dioxide, glass, cellulose, chitin, cotton, polyolefins, titanium oxide, gold, platinum, silver, copper, halogenated polyolefins such as PVDF or PVC, and polytetrafluoroethylene and ceramic.

In yet another embodiment of the method according to the invention for purifying a compound, it is provided that the compound is selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides, Protein domains, proteins, sugars, poly sugars, lipids, polylipids, coenzymes, natural products, and low-molecular compounds and derivatives and analogues thereof.

In a particularly preferred embodiment of the method according to the invention for purifying a compound, it is provided that the compound is a polypeptide which comprises 2 to 1000 amino acids, preferably 3 to 200 amino acids, preferably 4 to 100 amino acids and even more preferably 5 to 20 amino acids.

In one embodiment of the method according to the invention for cleaning a compound, it is provided that one or more of the reactive groups is / are connected directly or indirectly to the terminal subunit of the compound to be immobilized, in particular the polymeric compound to be immobilized.

In a further embodiment of the method according to the invention for purifying a compound, it is provided that one or more of the reactive groups is / are connected directly or indirectly to the side chain of the N-terminal amino acid of the polypeptide. In a still further embodiment of the method according to the invention for purifying a compound, it is provided that the compound, in particular the peptide or polypeptide, is a substrate for an enzymatic activity which is selected from the group consisting of kinases, sulphotransferases, glycosyl transferases, acetyl transferases, Farnesyltransferases, palmityltransferases, phosphatases, sulfatases, esterases, lipases, acetylases, proteases.

In one embodiment of the method according to the invention for purifying a compound, it is provided that the reaction is a chemoselective reaction.

In a preferred embodiment of the process according to the invention for purifying a compound, the reaction is carried out within a period of 0.1 second to 2 hours, preferably within a period of 1 second to 1 hour and preferably within less than 10 minutes.

The great breadth of application of the present invention is based on the surprising finding that conditions have been found for the immobilization reactions which make it possible to immobilize in relation to a large number of other functional groups which may be present in the substrate, in particular those which can typically occur in peptides or proteins to lead chemoselectively. The prior art describes processes which carry out the reaction in anhydrous solvents. The reaction is carried out with the addition of dehydrating auxiliaries and or using at least one reactant in high concentration (> 10 mM) or in large excess. In contrast, the immobilization reaction according to the invention in its various embodiments and uses is preferably used in aqueous media at an acidic pH in the range between 1 and 6.5, preferably between 2 and 5.5 and particularly preferably between 3 and 4.5, if appropriate using an organic co-solvent (such as MeOH, DMSO or glycerin). Under these conditions e.g. no amino functions possibly present in the compound to be immobilized, but only N-hydroxy-substituted amines which are incorporated into the compound to be immobilized or are present in the compound for the purpose of immobilization.

The above reaction conditions apply to any immobilization reaction disclosed herein, including such immobilization reactions as described in the context of the process according to the invention for the purification of a compound, in particular a polymeric compound.

The invention is further based on the surprising finding that the use or formation of the nitron group, preferably used as a linker or element of a linker structure, ensures or possible immobilization that is particularly stable in storage, in particular with regard to the influence of oxygen and Humidity. Overall, such an immobilization is also characterized in that it is stable, in particular, under conditions such as are typically used when carrying out biological tests (English assays) and at the same time can be produced from comparatively readily available starting components.

Another surprising advantage of using a nitron group or nitron linker in the sense described above is that the course of the immobilization reaction can be followed in a very simple manner. This can be done by spectroscopic methods, in particular FT-LR and UV / NIS spectroscopy, whereby in the ER spectroscopy Νitrone show a very characteristic strong absorption in the range between 1200 and 1300 cm ^"1. These spectroscopic methods are also for spatially resolved quantification of the Immobilization efficiency suitable.

The use of a Νitron group for immobilization also has surprising advantages or applications in that, owing to the inherent lability of the Νitron group towards dipolarophiles in the sense of 1,3-dipolar cycloadditions, i.e. after immobilization with the formation of the Νitron group, a large number of modified, in particular heterocyclic linkers can be produced. By specifically changing the linker structure used, the signal / noise ratio can often be optimized in tests of in-immobilized compounds compared to an enzymatic activity.

In particular in the process according to the invention for the purification of compounds, the immobilization by formation of a Gruppeitron group allows an easy separation of by-products which have arisen especially in the synthesis of the polymeric compounds to be immobilized, in particular the removal of the termination sequences obtained in the oligomer synthesis. The subsequent release of the compound from the solid phase using of volatile or at least easily removable reagents is ensured by the installation of a selectively cleavable connecting element.

The present invention relates generally to compounds which contain the group having the general structural formula (I) and their use as a linker, in particular as a linker for the immobilization of compounds on solid phase supports.

The use of structure (I), referred to herein as the nitrone group, as a linker in the sense mentioned above brings decisive advantages, in particular in the production of molecular arrays on solid, preferably planar, supports and in solid-phase screening thereof, but also in preparative purification of complex synthesis mixtures or of complex compositions, for example biological samples. These linkers can also be used to produce materials for affinity chromatography. The nature of the connection to be immobilized can be freely selected within wide limits.

With reference to structure (I), the following combinations between the position of the (solid phase) surface and the immobilized compound are possible:

X is either a surface, in particular a surface of a solid phase support, and one of the radicals Y or Z independently of one another is an immobilized compound, or at least one of the radicals Y or Z is a surface, in particular a surface of a solid phase support, and the rest X is an immobilized Connection.

The surface (denoted as X or Y or Z in (I)) consists of a preferably solid support F which is functionalized with R ¹ . This means that on the surface of F there is at least one set, ie a type, of reactive groups R ¹ , which are preferably covalently linked to F. R ¹ preferably comprises the functional groups of Hydroxylamines, preferably N-substituted hydroxylamines, aldehydes, ketones, acetals, ketals, thiocarbonyl compounds, thioketals and thioacetals.

The compound to be immobilized, as contained in X or Y or Z in the linker structure according to the invention, which is shown in (I), can be composed of a group rank G which is functionalized with a reactive group R, where R the compound classes of the hydroxylamines, preferably N-substituted hydroxylamines, aldehydes, ketones, acetals, ketals, thiocarbonyl compounds, thioketals and thioacetals.

During the immobilization, the reactive groups of the surface (R ¹ ) react in a preferably chemoselective reaction with the corresponding reactive group of the compound (R ² ) to be immobilized. For example, corresponding hydroxylamine-functionalized compounds (GR) to be immobilized can react with the carbonyl- or thiocarbonyl-functionalized surfaces (FR ¹ ) with the formation of nitrone. Likewise, corresponding hydroxylamine derivatives, which are presented on a surface, can react as a reactive group with the carbonyl / thiocarbonyl functionalities of a compound (R ² ) to be immobilized with the formation of nitron.

A large number of functionalized or functionalizable materials can be used as materials for the carrier material F or for the surface in the context of the present invention. These materials are, for example, compounds such as silicates, ceramics, glass, metals, organic carrier materials, cellulose, nitrocellulose, PTFE, polypropylene, polystyrene, copolymers, gold, platinum, silver, copper, silicon, titanium dioxide, silicon oxide, magnetizable and magnetic materials. These and other materials can be used either alone or in combination with each other or with other materials.

The immobilized compound in general and specifically the immobilized compound G can be selected from a large number of compounds and classes of compounds. So G can be: nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, PNA, peptides, polypeptides, protein domains, proteins, sugars, poly sugars, lipids, polylipids, coenzymes, natural products, and low molecular weight compounds. Furthermore, G can be selected from the group of compounds which can be obtained or derived from the above-mentioned compounds and classes of compounds by derivatization. Furthermore, the immobilized compound or G can be selected from the NEN. Furthermore, the immobilized compound or G can be selected from the group of compounds, combinations of one or more of the compounds or classes of compounds mentioned in the above groups. Such compounds are also referred to herein as hybrid compounds. Examples include glycopeptides or glycolipids, PNAs and the like.

Another embodiment of the present invention provides that the immobilized compound, which is also referred to herein as G, has a structure G ^! -G ² , where G ^{2 is} a

1 9

Is connecting element that connects G and R and is selectively cleavable after immobilization, preferably at the point of connection to G ¹ . After immobilization for the purpose of preparative cleaning (or for analytical purposes), such connecting elements allow the preferably residue-free removal of the immobilized compound from the solid phase support. Preferred connecting elements are modified standard protective groups which are modified by R ² at a suitable position, in particular protective groups for amines, alcohols, diols, carboxylic acids, carbonyl groups and thiols (TW Greene, PG M Wuts: Protective Groups in Organic Synthesis, 2nd edition , John Wileys & Sons New York, 1991). Depending on the type of modified protective group that is selected, the lability of the bond to specific reaction conditions can be adjusted. 2 shows, by way of example, corresponding modified protective groups which are unstable towards acids or light or bases. The examples shown are only intended to clarify the principle, but in no way represent a limitation of the protection and disclosure content.

With regard to the configuration of G ¹ , what has been said here regarding the configuration of the immobilized connection in general and specifically the immobilized connection G applies.

The term planar surface denotes surfaces that are essentially two-dimensionally aligned. In particular, it is not provided according to the present invention that the surface is a spherical surface or an essential part of such. In the configuration of the planar surface, it is preferred that the distinct locations at which a connection is immobilized are not, or at least not significantly, separated from another distinct location on the surface by a three-dimensional structure. The term “non-porous surface” is intended to refer to those surfaces in which capillary forces do not or essentially do not occur on the surface.

“Directed immobilization” is to be understood here in particular to mean that each immobilized or immobilized compound is or is bound to the surface via a defined reactive group or accumulation of reactive groups. It is, for example, the reactive group important for the immobilization Selectively attached to the N-terminus of a peptide, directed immobilization using this N-terminal reactive group ensures that all side chain functionalities are available for subsequent, e.g. biological tests on the immobilized compound and have not been modified by the immobilization ,

The term “arrangement” is understood here in particular to mean that each specific immobilized compound is immobilized at a specific location on a surface. Preferably, each of these locations can be identified or addressed on the surface. The locations are thus about distinct locations where essentially each species of the immobilized compound is immobilized. In other words, there is a map from which the location of each of the immobilized compounds on the surface can be deduced. As a rule, each is immobilized Connection is immobilized at a certain location not just one molecule, but a large number of identical molecules (depending on the density of functionalization on the surface, this is typically between 1 frnol and 1 μmol / cm ² ).

As a further measure in the configuration of the arrangements according to the invention, it can be provided that those places or areas of the surface that are not provided with an immobilized connection are blocked. The blocking ensures that during or after the chemoselective reaction of the compound to be immobilized with the possibly functionalized surfaces, unreacted but still reactive groups or groups on the surface are inactivated.

This blocking reaction is particularly necessary for downstream biological tests, since otherwise an added enzymatic activity or other components of a biological sample used, for example, are unspecific with these, not yet blocked, reactive groups can react on the surface and possibly provide a large background signal. Such non-specific reactions with surfaces are a common cause of unfavorable signal-to-noise ratios in biochemical analyzes. Compounds which are not sterically demanding, react very well with the groups to be blocked and generate surface properties which are as favorable as possible are suitable for this blocking. The selection of these compounds will depend on the type of sample or interaction partner that interacts with one of the amino acid sequences. The compound is made hydrophilic if it is known that the enzymatic activity preferably binds non-specifically to hydrophobic surfaces and hydrophobic if it is known that the enzymatic activity preferably binds non-specifically to hydrophilic surfaces. It is known to the person skilled in the art that a biomolecule, such as a protein, requires a three-dimensional, precisely defined structure for the correct biological function. This tertiary structure is clearly dependent on the environment. A protein in water, as a hydrophilic solvent, has a tendency to hide all, or rather, as many, hydrophobic groups as possible inside. If such a protein gets into a more hydrophobic environment (hydrophobic surface), the protein can therefore be refolded or unfolded and thus inactivated. On the other hand, proteins are known that are naturally present within (hydrophobic) biomembranes. Such proteins would fold over when they came into contact with a hydrophilic surface and thereby denatured or inactivated. In such a case, a hydrophobic surface is desirable.

In a preferred embodiment of the arrangement according to the invention and its various uses and applications, it is provided that the various immobilized compounds are amino acid sequences, ie peptides, oligopeptides, polypeptides or proteins, in particular are substrates or possible substrates of enzymatic activities which are contained in samples as interaction partners , to which the arrangement according to the invention is exposed. Here, enzymatic activities are generally understood to mean those enzymatic activities which are distinguished by the fact that they transfer an atomic group, a molecule or a group of molecules to a molecule. Here, enzymatic activities are to be understood in particular as kinases, sulfotransferases, glycosyltransferases, acetyltransferases, farnesyltransferases, palmityltransferases, phosphatases, sulfatases, esterases, lipases, acetylases and proteases. The enzymatic activity is accordingly, if appropriate, one or more of the amino acid sequences of the arrangement, that is to say one or more of the Amino acid sequences on the chip, change in terms of molecular weight. Such a change in molecular weight may be a decrease or increase in it, and may result in further changes in the physicochemical properties of the amino acid sequences or the distinct locations where a species of amino acid sequence is located.

Evidence that one or more of the different amino acid sequence species has a binding event and, if the interaction partner of the amino acid sequence is or carries an enzymatic activity, an enzymatic reaction to the respective amino acid sequence can be made using various techniques take place that are known to the expert. So it is possible to carry out a cleavage reaction, e.g. B. mediated by a protease by changing the fluorescence of a suitable substrate molecule bound to the surface. In principle, all reactions in which the molecule bound to the surface is changed in the molecular weight by transferring further molecules (co-substrates) can be followed by introducing a radioactive label into the co-substrate. For this, after the reaction has taken place, the radioactivity built into the modified molecule bound to the surface must be quantified. With the aid of such radioactive labels, all transferases, such as, for example, kinases, acetyltransferases, farnesyltransferases and glycosyltransferases, can be characterized with regard to the enzymatic activity. Alternatively, previously non-existent reactive groups resulting from the respective enzymatic conversion on the respective amino acid sequence can be detected by means of subsequent specific reactions. For example, a mercapto function obtained after enzymatic conversion can be detected by means of a subsequent reaction with Ellman's reagent. The special groups introduced by the enzymatic activity, which are not otherwise present in the amino acid sequences, can also be produced by specifically binding, e.g. B. fluorescent-labeled reagents can be detected. Phosphate monoesters resulting from a kinase reaction can e.g. B. selectively made visible by a fluorescence-labeled, synthetic chelator (R. Gast, J. Glökler, M. Höxter, M. Kieß, R. Frank, W. Tegge; 1999, Methods for Determining Protein Kinase Substrates by the Phosporylation of Peptide Libraries on Beads, Phosphate-Specific Staining, Automated Sorting, and Sequencing, Analytical Biochemistry, 276, 227-241) It is within the scope of the present invention that the arrangement comprises a certain number of different amino acid species. It can be provided that the same amino acid species is present at several distinct locations on the surface or the carrier material. On the one hand, this enables an internal standard to be implemented, and on the other hand, possible marginal effects can also be displayed and recorded.

Another aspect of the present invention is concerned with a method for purifying a compound, in particular a polymeric compound.

Beginning with the development of automated peptide synthesizers, there have been great advances in the production of peptides and peptide derivatives. It is foreseeable that the known methods of peptide synthesis would benefit enormously from an improvement in the quality of the raw product. It is understandable for the person skilled in the art that, due to the not always complete yields of the individual synthesis steps, certain heterogeneities can arise in the different amino acid sequences in the sense described above. Especially in syntheses that require many reaction steps, such as in poly / oligomer synthesis, e.g. when peptides (amino acid sequences) are synthesized (one coupling reaction and one deprotection per amino acid unit and generally one reaction at the end of the synthesis for the simultaneous deprotection of all protective groups of the side chain functions), this can be a problem.

This means that one or more purification steps are necessary for all applications in which peptides are required in a defined purity, which are usually very time-consuming and labor-intensive compared to the actual automated synthesis. Such methods are gel filtration and HPLC and / or combinations thereof (RB Merrifield, J. Am. Chem. Soc, 85, 2149 (1963)). Affinity chromatography can be a very effective method for some special peptides or polypeptides. However, in the case of affinity purifications, it should be noted that peptides which lack only one or a few amino acids usually also react with these materials used in affinity chromatography and therefore lead to impurities in the resulting mixture. To solve this problem, it has been proposed to introduce a blocking reaction (such as acetylation with acetic anhydride) after each condensation (coupling) step, so as to prevent the further extension of incomplete peptide sequences and thus the formation of incorrect sequences. In the course of a In such a synthesis, the desired target peptide sequence and a large number of so-called termination sequences are formed after the coupling of the last amino acid, which lack at least one amino acid and which are modified by the corresponding blocking reactions at the N-terminus. Since only the desired target peptide sequence contains a free N-terminus at the end, this can be used for a peptide remediation. Some methods for peptide purification based on this principle are state of the art. (For example: R. Camble, R. Garner and GT Young, Nature, 217, 247 (1968); K. Suzuki, Y. Sasaki and N. Endo, Chem. Pharm. Bull., 24, 1 (1976); DS Kemp and DG Roberts, Tetrahedron Lett., 4269 (1975); T. Weiland, C. Birr and H. Wissenbach, Angew. Chem., Int. Ed., Engl, 8, 764 (1969), H. Wissman and R. Geiger, Angew. Chem., Int. Ed., Engl, 9, 908 (1970); RB Merrifield and AE Bach, J Org. Chem. 43, 4808 (1975); TJ Lobl, RM Deibel and AW Yen, Anal Biochem., 170, 502 (1988); H. Ball, C. Grecian, SBH Kent and P. Mascagni, in "Peptides", JE Rivier and GR Marshall, Eds., ESCOM, Leiden 1990 pp 435; Funakoshi, S ., et al., "Chemoselective One-Step Purification Method for Peptides Synthesized by the Solid-Phase Technique," Proc. Nat. Acad. Sci. USA, 88, 6981 (1991). However, none of these methods is simple and effective This is a purification procedure and mostly complicated separation processes are used, and there is also a state-of-the-art method in which only the desired one is included at the N-terminus a cysteinyl-methionine dipeptide modified target peptide sequence is bound to a special mercury column via the free cysteine-SH function. After separation of the termination sequences by simple washing, the methionine-target peptide sequence binding is cleaved by bromocyan treatment (DE Krieger, BW Erikson and RB Merrifield, Proc. Nat. Acad. Sci. USA, 73, 3160 (1976)). However, this method is not suitable for target peptide sequences that contain cysteines or methionine.

The methods according to the invention allow the immobilization and simultaneous or subsequent purification of biomolecules, such as peptides, and thus the production of peptide arrays. This process can be easily automated by using simple pipetting robots. In particular, the problems described above of the rapid separation of termination sequences from the desired target peptide sequence are solved by the following steps or measures:

The provision of a mixture of compounds in which the compound to be purified (target compound Z) is equipped with a reactive group which enables it to form nitrone. higt, and the by-products to be separated do not contain them. Such mixtures are easily accessible in solid phase polymer synthesis by the blocking method (see FIG. 3). This process was explained above using the example of peptide synthesis and analogously also applies to other polymer syntheses (such as DNA or RNA synthesis). A building block is either attached directly to the unblocked free terminus of the polymer; which carries the reactive group important for the immobilization, or a building block which enables the attachment of the reactive groups in a preferably one-step reaction.

In example 6, the second variant for the introduction of the N-substituted hydroxylamine was realized: After the bromoacetamide modification of the free N-terminus of the peptide, a nucleophilic substitution was carried out with the formation of the N-hydroxyglycine derivative. An equivalent alternative would be the use of a building block carrying the reactive group, e.g. B. a Boc-protected N-hydroxyglycine to modify the free N-terminus of the target peptide. The typically subsequent reactions such as the elimination of any protective groups which may be present and the elimination of the mixture from the solid phase used for the synthesis are dealt with in the same way by the target compound and by-product.

The target compound which is selectively marked or addressable by the reactive group is then selectively immobilized on the surface from the reaction mixture to form nitrone. The by-products that are not covalently bound to the surface can be removed by washing away. The target compound Z is thus present on the surface in a cleaned or purified form. This process is shown schematically in FIG. 4. Are on several points of a planar surface such. B. immobilized various peptides, this method can be used not only to carry out the array production, but also to perform a high-throughput cleaning of the array.

So far, the potential of so-called spot synthesis of peptides (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) are not fully exploited for array production because they are cleaned large number of different peptides on such a small scale appeared disproportionately expensive. Since the blocking reaction in spot synthesis raller way can be carried out efficiently, as well as. B. cleavage of the Fmoc group, the method according to the invention offers a way to combine spot synthesis and peptide array production with simultaneous purification of the peptides.

For a process for the preparative purification of polymeric compounds, spherical surfaces which are highly loaded (> 1 mmol / g) and are typically used as scavenger resins are preferably used for immobilization with the formation of nitrone. In this embodiment of the method, preferably only one species of a polymeric target compound is immobilized per surface, but this in a correspondingly larger preparative amount (1 nmol to 0.1 mol, preferably 100 nmol to 10 mmol, particularly preferably 1 μmol to 1 mmol) , The necessary cleavage of the purified polymeric compound after immobilization is prepared by installing one of the connecting elements G described above (see also FIG. 2), which cleaves the immobilized compound between the nitron group and G ¹ , preferably at the point of attachment G ¹ , enables. The entire process is shown schematically in FIG. 5, the removal of the reagents used for the cleavage is carried out, if necessary, according to standard methods known to the person skilled in the art, such as in the case of volatile reagents by concentration or freeze-drying, or in other cases by solid phase extraction or precipitation of the target product. In

9 9

6 is illustrated using the example of a base-labile group G (FIG. 6A) and an acid-labile group G (FIG. 6B), which structure the cleaned immobilized polymers in the region of the nitron group and the group G ² can have and under what conditions a release of the immobilized connections is possible.

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

1 shows an overview of different chemoselective reactions;

2 shows an overview for various configurations of the structure G -

G ² -R ² , in the case of (A) for an acid-labile linkage, in the case of (B) for a photolabile linkage and in the case of (C) for a base-labile linkage; 3 shows a schematic representation of the possible by-products in the step-by-step synthesis of polymeric compounds on a solid phase using blocking reactions;

4 shows a schematic representation of a method for directional immobilization or purification of target compounds, starting from a mixture of compounds resulting analogously to FIG. 3;

5 shows a schematic illustration of the cleaning method according to the invention;

6 shows two examples of immobilized, polymeric compounds with different structures of the cleavable connecting element and their selective cleavage;

7 shows some variants of the modification of cellulose surfaces;

8 shows the synthesis of an amino acid sequence suitable for immobilization and its immobilization with formation of a nitrone;

9 shows the immobilization of an amino acid sequence to form a nitrone and the subsequent release for analytical purposes of a modified amino acid sequence to obtain the nitrone;

10 shows an HPLC chromatogram and three mass spectra of a synthesis

Crude product of an amino acid sequence suitable for immobilization;

Fig. 11 is an HPLC chromatogram and two mass spectra one after

Immobilization of the modified amino acid sequence released again; and 12 shows the result of the incubation of a modified glass surface with proteininase A.

Fig. 1. An overview of various chemoselective reactions according to the prior art is shown: A) aldehydes (R ⁴ = H) or ketones (R ⁴ not H) and amino-oxy compounds react to form 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 hydrazones, D) aldehydes (R ⁴ = H) or Ketones (R ⁴ not H) and 1,2-aminothiols react to thiazolines (X = S) and 1,2-amino alcohols to oxazolines (X = O) and 1,2-diamines react to imidazolines (X = NH) , E) thiocarboxylates and α-halocarbonyls react to thioesters, F) thioesters and ß-aminothiols react to ß-mercaptoamides, F) mercaptans and maleinimides react to succinimides. The radical R ¹ represents alkyl, alkenyl, alkynyl, cycloalkyl or aryl radicals, or heterocycles or surfaces, and the radicals R ⁴ -R ⁶ 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 _1-0 -alkyl, C _3-2 o-cycloalkyl, preferably for branched and unbranched C _1-12 - Alkyl, C _3-12 cycloalkyl and particularly preferably branched and unbranched C _1-6 alkyl, C _3-6 cycloalkyl radicals. Alkenyl represents branched and unbranched C ₂ - ₂ 0- alkenyl, branched and unbranched C ₁ .20-alkyl-O-C2.20- alkenyl, C _1-20 (-O / SC _2-20) _2-2 oalkenyl, aryl-C _2-20 alkenyl, branched and unbranched cyclyl- hetero C _2-20 alkenyl, C _3-2 o-cycloalkenyl, preferably represents branched and unbranched C ₂ -12- alkenyl, branched and unbranched C _{_1-12} (-O / S-C2-i ₂₎ ₂ -i ₂ alkenyl, particularly preferably branched and unbranched C _2-6 alkenyl, branched and unbranched C _1-6 (-O / SC _2-8) ₂ - ₈ alkenyl - leftovers; Alkynyl represents branched and unbranched C ₂ -20- alkynyl, branched and unbranched C. ₁ ₂₀ (-O / SC ₂ - _2θ ) ₂ - ₂ oalkynyl, preferably for branched and unbranched C _2-2 alkynyl, branched and unbranched C ₁ - ₁₂ (-O / S ^ -u ^ -alkynyl, particularly preferably for branched and unbranched C ₂ - ₆ - alkynyl branched and unbranched Cι _-6 (-O / S-C2 _-8) ₂ - ₈ alkynyl radicals; cycloalkyl is bridged and non-bridged C _3-40 cycloalkyl, preferably bridged and non-bridged C _3-26 cycloalkyl, particularly preferably bridged and non-bridged C _3-15 cycloalkyl radicals, aryl stands for substituted and unsubstituted mono- or multi-linked phenyl, pentalenyl, azulenyl, Anthracenyl, indacenyl, acenaphtyl, fluorenyl, phenalenyl, phenanthrenyl, preferably for substituted and unsubstituted mono- or multi- linked phenyl, pentalenyl, azulenyl, anthracenyl, indenyl, indacenyl, acenaphtyl, Fluorenyl, particularly preferred 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, the following substituents can occur on the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroatoms, heterocycles, biomolecule or natural product 0 to 30 (preferably 0 to 10, particularly preferably 0 to 5) 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 being preferred : Fluorine, chlorine, bromine, hydroxyl, amide, ester, acid, amine, ether, phosphate, sulfate, sulfoxide, thioether, nitrile, urea, carbamate, and particularly preferably: chlorine, hydroxyl, amide, ester, acid, ether , Nitrile.

Fig. 2 Shown are some examples of possible structural formulas of the selectively cleavable connecting element G ² , on the one hand with a reactive group capable of nitrone formation in bold (generally R, which in the examples is representative of other groups disclosed herein as hydroxylamino or aldehyde group are formed) and, on the other hand, are linked via a selectively cleavable bond to the only schematically shown compound to be immobilized, referred to herein as G ¹ . The selectively cleavable bond is interrupted by a serpentine line.

Part (A) of FIG. 2 shows acid-labile configurations of G ² . For linking to hydroxy functionalities on the compound to be immobilized, e.g. B. a hydroxylamine-modified tetrahydropyranyl protecting group (THP) as shown in (a) is suitable (R ² = HONH-). The aldehyde-modified (R ² = H- (C = O) -) tert-butyloxycarbonyl protective group (BOC) shown in (b) is similarly acid labile, and is suitable for linking to an amino functionality in the compound to be immobilized. The nitro-substituted benzylamine shown in part (B) of FIG. 2, which is additionally hydroxylamine-substituted, would be photolabile. Suitable base-labile connecting elements are the hydroxylamine (a) or aldehyde-substituted (b) 9-fluorenyl-methoxycarbonyl protective groups (Fmoc) shown in part (C). 3 shows a schematic representation of the possible by-products in the step-by-step synthesis of polymeric compounds using blocking reactions. Solid phase compounds are generally synthesized in several steps. When displaying biopolymers, such as peptides, DNA, RNA or PNA, several chemically very similar reactions have to be carried out, which do not always take place completely, ie in addition to the desired target compound Z ¹ , a large number of by-products are formed, which are caused by chain termination (termination sequences) or characterized by the lack of one or more building blocks within the chain (faulty sequences). In order on the one hand to prevent the occurrence of incorrect sequences and on the other hand to make the termination sequences chemically or physically distinguishable from the desired target compound Z ¹ , the synthesis can be carried out (a) in such a way that an additional reaction occurs after each reaction step (condensation or addition step) takes place, which chemically modifies the unreacted starting material, but not the intermediate product leading to the desired target compound Z ¹ . These modifications or blockages are represented by black dots at the terminus of the termination sequences A ¹ to A ⁴ . After repeating this procedure, a mixture consisting of the desired target compound Z ^λ and a large number of modified termination sequences A ^] -A ^{4 is obtained} . Since mixtures prepared by this process only contain a reactive group in the desired target compound Z ¹ , this can be selectively modified with suitable reagents which allow the attachment of a reactive group R ² (step b). This is usually followed by further steps (c), which are dealt with both by the target compound and by the termination sequences and relate to the isolation of the reaction mixture, such as the elimination of any protective groups still present and the elimination of the compounds from the solid phase support used for the synthesis ,

FIG. 4 shows a schematic representation of a method for the directed immobilization or purification of target compounds, starting from a mixture of compounds resulting from FIG. 3, consisting of the desired target compound Z ² , which has been selectively modified with suitable reagents, and a large number of termination sequences A'-A who do not wear this modification. This mixture is reacted with a surface which bears a reactive grouping which in turn allows a chemoselective reaction with the modified target compound Z ² to form a nitrone (a). The unmodified termination sequences A -A ⁴ can be separated from the immobilized target compound Z ³ (b), for example by simple processes such as washing, wiping, centrifuging, etc. The target compound Z ³ immobilized on the surface can be used for other purposes such as e.g. B. downstream biological tests can be used immobilized. This is particularly advantageous, for example, if the surface is a planar surface, in which several different amino acid sequences may be attached to different distinct locations on the planar surface.

FIG. 5 shows a schematic representation of a method for the directed immobilization and preparative purification of target compounds, starting from a mixture of compounds consisting of the desired target compound Z ² , which results analogously to FIG. 3, and which is selective with suitable reagents with the introduction of a selectively cleavable linker element

9 1

G and a reactive group was modified, and a large number of termination sequences A - A ² , which do not carry this modification. This mixture is reacted with a surface which bears a reactive grouping which allows a chemoselective reaction with the modified target compound Z ² to form a nitrone (a). The unmodified termination sequences A -A ² can be separated from the immobilized target compound Z ³ (b) by simple processes such as washing, wiping, centrifuging, etc. by selective splitting of the connecting element G (c), preferably at the one Binding indicated by the arrow, and optional further isolation steps, such as concentration, freeze-drying or solid phase extraction, the target compound Z is obtained in its unmodified form. Possible configurations of G ² are described in FIG. 2. The splitting step c) is described in more detail in FIG. 6 using two examples.

6 shows, by way of example, two structures of polymers immobilized on a surface via nitrone groups, which allow the release of the desired target compound. 6A shows a structure which enables the desired target compound to be released as an amine by treatment with bases, in particular amines, preferably secondary amines or mixtures which contain appropriate bases (a). In contrast, FIG. 6B shows a structure which allows the desired target compound to be released as alcohol by treatment with acids, in particular acetic acid, sulfonic acid derivatives or a large number of Lewis acids or mixtures which contain the corresponding acids (b). 7 shows some variants of the modification of cellulose surfaces. The hydroxyl groups of cellulose can in a multi-stage reaction (T. Ast, N. Heine, L. Germeroth, J. Schneider-Mergener, H. Wenschuh; efficient assembly of peptomers on continous surfaces, Tetrahedron Lett., 1999, 40, 4317-4318 ) are modified in such a way that amino groups that can be used for further reactions are present (a), a photolabile element (PL) being introduced between these amino groups and the surface (I), which can be obtained by irradiation with UV light from a meandering line broken bond can be split. By reacting surface I with either 4-carboxybenzaldehyde (b, see example 2b) or levulinic acid (c, see example 4), surfaces II and III are obtained, for example.

FIG. 8 shows the synthesis and immobilization of an amino acid sequence (IN) on a surface which has aldehyde functions (II), with the formation of a substituted nitrone. Starting from an amino-modified solid phase, the first building block is added to form a covalent bond. After the step-by-step synthesis of the amino acid sequence on the solid phase using the blocking method by means of acetylation after each coupling step, this amino acid sequence is replaced by a) bromoacetylation, b) subsequent nucleophilic substitution by means of hydroxylamine and c) cleavage from the solid phase and removal of all side chain protective groups by means of TFA into those for immobilization appropriate amino acid sequence IN transferred. The Ν-hydroxy-glycine-labeled amino acid sequence thus obtained is brought into contact with a suitable aldehyde-functionalized carrier (II) (d) and the amino acid sequence is immobilized on the surface of this carrier with the formation of a nitrone.

9 shows the immobilization of an amino acid sequence (V) on a surface which has aldehyde functions (II), with the formation of a substituted nitrone, and the subsequent release of a modified amino acid sequence to obtain the nitrone. The carrier II is brought into contact with compound V and the amino acid sequence is immobilized on the surface of the carrier II with the formation of a nitrone. Due to the photolabile structural element contained in II (cf. FIGS. 7, 1), the immobilized amino acid sequence can be released again by means of light while maintaining the nitron structure as a modified amino acid sequence (VI) (cf. Example 6b). The modified amino acid quenz can be analyzed using various methods such as HPLC, CE, NMR, ER, UN-Nis spectroscopy and mass spectrometry.

Fig. 10 shows the HPLC chromatogram of the crude product of the synthesis of V (see Example 6a). Several signals are visible in the UN trace at 220 nm shown above. There is no clear main product. However, the desired target compound N is contained in the analyzed crude product solution. This is clearly demonstrated by the mass spectra of selected HPLC measuring points shown in the lower part of the figure. The mass spectrum belonging to area A of the UN track shows the expected mass for N ([M + l] ⁺ of 522.2 Da) with a retention time of 2.9-3.6 min. The mass spectra belonging to the areas B and C of the UV trace show that these compounds are by-products which have not been elucidated in terms of structure and which have arisen in the synthesis of V.

11 shows the HPLC chromatogram of the modified amino acid sequence VI (cf. Example 6b). Several signals are visible in the UV trace at 220 nm shown above. There is a clear major product with a retention time of 9.8 minutes. The desired target compound VI is contained in the analyzed solution. This is clearly demonstrated by the mass spectra of selected HPLC measuring points shown in the lower part of the figure. The mass spectrum associated with the UV signal at a retention time of 9.8 minutes shows the mass expected for the target compound VI ([M + 1] ⁺ of 800.3 Da (theoretically 800.4)). The comparison of the UV trace in FIG. 10 with that in FIG. 11 clarifies that the target compound has undergone a significant purification through the immobilization and subsequent washing, which can be demonstrated by HPLC-MS after cleavage from the surface.

FIG. 12 A glass surface modified with the peptide N-hydroxy-Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (FIG. 8, IV) (cf. Example 5b) (amino acid sequence was extracted in 200 mM Acetate buffer pH 4.0 dissolved and at RT 1 nL of this solution in an arrangement of 26 rows and 63 columns (1638 spots in total) was applied to the aldehyde-functionalized glass surfaces (Example 1) using a Gesim nano plotter Spot-to-spot distance 0.8 mm. The glass surfaces treated in this way were then incubated for 2 hours at RT. Thereafter, first with 10 ml 100 μM ATP solution in kinase buffer (50 mm Tris-HCL 150 mm NaCl, 30 mm MgCl ₂ , 4 mm DTT, 2mM EGTA, pH 7.5) for 10 minutes, the glass surface was dried and then the Peptide-modified glass surface placed a second, unmodified glass surface of the same dimensions. Then 50 ul of a mixture of protein kinase A (Sigma, P26452, U / ml), 100μM / ml ATP and 100μCi ml γ- ³² P-ATP (Amersham, 9.25mBq / 250μCi / 25μl, activity> 5000Ci / mmol) in Kinase buffer (50mM Tris-HCl, 150mM NaCl, 30mM MgCl ₂ , 4mM DTT, 2mM EGTA, pH 7.5) by capillary forces into the gap, which is formed by the second glass surface resting on the modified glass surface. The mixture was then incubated at RT in an almost water-saturated atmosphere for 30 min and the phosphorylation of the corresponding peptides was detected using a phosphorus leaner from FUJIFILM (Example 7). It becomes clear that protein kinase A tolerates the way in which the immobilized kinase substrate is linked to the glass surface, and that the modification of the glass surfaces proceeds smoothly and without major fluctuations in the immobilization density.

The following examples relate to the functionalization of glass and cellulose, the surface of which is required as a surface for immobilization (Examples 1 to 4), the immobilization of various peptides provided with a reactive group on a surface (Examples 5 and 6) and the analysis of Kinase mediated peptide modification using the peptides immobilized according to the invention (Example 7). The abbreviations listed below were used:

Ala, A L-alanine

Arg, R L-arginine

Asp, D L-aspartic acid

ATP adenosine 5'-triphosphate

Boc tertiary butoxycarbonyl

Cys, CL-cysteine

DCM dichloromethane

DIC NN-diisopropylcarbodiimide

DIPEA N, N-diisopropylethylamine

DMF NN-dimethylformamide

DMSO dimethyl sulfoxide

EGTA ethylene glycol bis (2-aminoethyl) -N, N, N, N'-tetraacetic acid Et ethyl

Fmoc 9-fluorenylmethoxycarbonyl

Gly. G glycine

HBTU O- (benzotriazol-l-yl) -N _J N, N ', N'-tetramethyluronium hexafluorophosphate

HPLC high-performance liquid chromatography

L liter

Leu, L L-leucine

Lys, K L-lysine

M molar

MBHA methylbenzhydrylamine

MeOH methanol mL milliliter mM millimolar mRNA messenger RΝA nL Νanoliter

Phe, F L-phenylalanine

Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl

PTFE polytetrafluoroethylene

PVC polyvinyl chloride

PVDF polyvinyl difluoride

RNA ribonucleic acid

RP reversed phase

RT room temperature

SDS sodium lauryl sulfate

Ser, S 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)

U unit The following reagents and solvents were used in the examples described herein:

tert-butyl methyl ether, 1,3-diisopropylcarbodiimide, N, N-diisopropylethylamine, acetic anhydride, urea, 40% hydroxylamine solution, piperidine, triethylamine, dichloromethane, diethyl ether, N, N-dimethylformamide, ethanol, methanol, and tetrahydrofuran come from Merck Euro lab (Darmstadt, Germany). Trifluoroacetic acid, dimethyl sulfoxide and thiourea were purchased from Fluka (Deisenhofen, Germany). Adenosine 5'-triphosphate, 2-amino-2-hydroxymethyl-l, 3-propanediol hydrochloride, sodium chloride, magnesium chloride, 1,4-dithio-DL-threitol, sodium lauryl sulfate, polyoxyethylene sorbitant monolaurate, and ethylene glycol bis- (2-aminoethyl) -N, N, N ', N'-tetraacetic acid are from Sigma (Taufkirchen, Germany). The Rink amide MBHA resin, (benzotriazol-l-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate and all Fmoc-amino acid pentafluorophenyl esters were obtained from Novabiochem (Bad Soden, Germany).

Cellulose membranes "Whatman 50" (Whatman Maidstone, UK) were used for the SPOT synthesis, on which the Autospot AMS 222 (Abimed, Langenfeld, Germany) and the Autospot XL Ver. Control software were automatically used according to the standard SPOT synthesis method. 2.02 modifications were made. The washing steps were carried out in stainless steel dishes (Merck Eurolab), which were moved on a rocking table.

Chromatography and physical data:

RP-18 HPLC-MS analyzes were performed by chromatography using a Hewlett 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). The separation was carried out on RP-18 column material (Vydac 218 TP5215, 2.1x150 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 (5th -95% B within 17 min, with A: 0.05% TFA in water and B: 0.05% TFA in CH ₃ CN). UV detection was carried out at λ = 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, 10x250 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 synthesized as C-terminal peptide amides using a parallel “Syro” automatic synthesizer (MultiSynTech, Witten, Germany) according to the standard Fmoc protocol on Rink amide MBHA resin. However, an acetylation reaction was also carried out after each coupling step (acetic anhydride / DIPEA / DMF in a volume ratio of 1/2/7, 10 min RT, washing with 3 × DMF). The peptides obtained were analyzed by HPLC-MS after cleavage from the resin and cleavage of all protective groups and showed the desired molecular ion signals. After subsequent HPLC purification, the peptides were lyophilized and stored at -20 ° C.

Examples

Example 1: Aldehyde functionalization of aminopropylsilylated glass surfaces

4-carboxybenzaldehyde (Fluka, 21873) 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 overlaid with the activated carboxybenzaldehyde solution thus obtained and incubated at RT for three hours. The glass surfaces treated in this way were then washed five times with 30 ml of DMF each for 3 minutes at RT. After washing three times for three minutes each with 30 ml of DCM at RT, the glass surfaces were dried and stored at 4 ° C. until further use.

Example 2: Aldehyde functionalization of derivatized cellulose

a) Production of the basic membrane: The production of amino-derivatized cellulose membranes was carried out according to one of Ast et al. (T. Ast, N. Heine, L. Germeroth, J. Schneider-Mergener, H. Wenschuh; efficient assembly of peptomers on continous surfaces, Tetrahedron Lett., 1999, 40, 4317-4318) developed methods (Fig. 7 , I). In these specially derivatized cellulose membranes, the terminal amino group is bound to a photolabile element. The binding of the terminal amino group to the photolabile element can be split by irradiation of UV light (cf. Fig. 7). An array of 20 x 30 amide-linked Fmoc-phenylalanine spots (diameter 6-7 mm, loading approx. 300 nmol / spot) according to standard spot synthesis methods (DE 40 27 675 C 2, and A. Kramer, U. Reineke, L. Dong, B. Hoffmann, U. Hoffmüller, D. Winkler, R. Volkmer-Engert, J. Schneider-Mergener; SPOT synthesis - observations and optimizations, J Pept. Res., 1999, 54, 319-327). After washing with DMF (3 x 2 min), the unreacted amino groups on the cellulose membrane were subjected to a blocking reaction with a solution of 10% acetic anhydride and 20% DEPEA in DMF (10 min) in order to further functionalize them in later steps to prevent. After washing with DMF (3 x 2 min), the Fmoc group was split off by treatment with 20% piperidine in DMF (2 x 10 min) and washed with DMF (3 x 2 min) and with DCM (2 x 2 min). A field of 20 spots was used for further functionalization. The remaining cellulose membrane was stored at -18 ° C and used for further experiments.

b) N-terminal modification of the base membrane with 4-carboxybenzaldehyde (Fig. 7, II): 4-carboxybenzaldehyde (Fluka, 21873) was dissolved in 0.4 M DMF. The resulting mixture was activated by the addition of half an equivalent of DIC for 15 min at RT. 2 μl of the symmetrical anhydride solution thus obtained were pipetted onto each of the 20 spots (2 15 min reaction time) (cf. FIG. 7, b) and then washed with DMF (3 2 min) and with DCM (2 2 min) and dried and stored at 4 ° C until further use.

Example 3: 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. Aminopropylsilylated glass surfaces (2.5 x 7.5 cm; Sigma, Silane-Prep ™, S4651) cleaned with compressed air were covered with a layer of the levulinic anhydride solution thus obtained and at three hours RT incubated. The glass surfaces treated in this way were then washed five times with 30 ml of DMF each for 3 minutes at RT. After three washes for three minutes each with 30 ml of DCM at RT, the glass surfaces were dried and stored at 4 ° C. until further use.

Example 4: Ketone functionalization of the cellulose base membrane

20 spots of the cellulose base membrane described in Example 2a) were modified analogously to Example 2b) with levulinic acid (cf. FIG. 7, c) instead of 4-carboxybenzaldehyde and stored at 4 ° C. until further use.

Example 5: Immobilization of N-hydroxy-glycine-containing peptides on aldehyde-functionalized glass surfaces

a) The peptide used for the immobilization (N-hydroxy-Gly) -Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH2 (Fig. 8, IN) was used as the solid phase according to standard methods of Fmoc-based chemistry C-terminal peptide amide synthesized. Appropriately protected Fmoc amino acids were activated with one equivalent of HBTU and three equivalents of diisopropylethylamine in DMF and coupled to Rink amide MBHA resin in DMF. However, an acetylation reaction was additionally carried out after each coupling step (acetic anhydride / DEPEAJDMF in a volume ratio of 1/2/7, 10 min RT, washing with 3 x DMF). The Fmoc protective group was cleaved using 20% piperidine in DMF for 30 min at RT. After the coupling of the N-terminal leucine, the resin-bound peptide was reacted with 3 equivalents of bromoacetic anhydride in DMF for 40 min at RT. A nucleophilic substitution was then carried out by treatment with 25% solution of hydroxylamine in water / DMF (in a mixing ratio of 1/1), which gave the side-chain-protected, resin-bound target peptide. The permanent protective groups (fßu 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 by adding tert. Butyl methyl ether precipitated. 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. The HPLC retention time is 7.1 min. b) The peptide N-hydroxy-Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (Fig. 8, IV) was dissolved in 200 mM acetate buffer pH 4.0 (final concentration of the peptides 0.5 mM) Contained 20 vol% DMSO. Subsequently, 1 nl of this solution was applied at RT in an arrangement of 26 rows and 63 columns (1638 spots in total) to the aldehyde-functionalized glass surfaces (Example 1) by means of a Gesim nano plotter. The spot-to-spot distance was 0.8 mm. The glass surfaces treated in this way were then incubated at RT for two hours. After washing three times at RT with 100 ml of distilled water in each case, the modified glass surfaces were incubated with 30 ml of a 40% strength aqueous hydroxylamine solution for 30 minutes at RT in order to deactivate the remaining aldehyde functions. The glass surfaces were then washed five times for 3 minutes each with 50 ml of water at RT and then twice each for 3 minutes with 50 ml of methanol at RT. The glass surfaces treated in this way were dried and stored at 4 ° C. until further use.

EXAMPLE 6 Immobilization of N-Hydroxy-Glycine-Containing Peptides on Aldehyde-Functionalized Cellulose Spots and Subsequent Cleavage (FIG. 9)

a) The peptide (N-hydroxy-Gly) -Lys-Phe-Arg-NH ₂ (FIG. 9 V) used for the immobilization was prepared analogously to Example 5a). Of this peptide, however, the crude product precipitated with tert-butyl methyl ether was used for the immobilization experiment described below (cf. FIG. 10, it can be seen that the desired compound V is present in the crude product in addition to some by-products).

b) The peptide (N-hydroxy-Gly) -Lys-Phe-Arg-NH ₂ was dissolved in 200 acetate buffer pH 4.0 (final concentration of the peptides 5 mM). A spot of aldehyde-functionalized cellulose (see Example 2b) was then incubated at RT for 24 hours (cf. FIG. 9). After washing three times with 1 ml of distilled water each time, washing with MeOH (3 x 2 min), DMF (3 x 2 min) and DCM (2 x 2 min), the modified cellulose spot was dried and by cleaving the photolabile linker using UV Split light (4 hours on a UV table (Vilbert Lourmat TFX 20 LC, with 100% corresponding to 7 mW / cm ² intensity). The HPLC diagram of the split product (Fig. 9, VI) together with the mass spectra of the individual compounds - gene is shown in Fig. 11. From the figure it can be seen that in addition to a few by-products, the desired compound VI (retention time 9.8 minutes) is the main product.

Example 7: Analysis of kinase-mediated peptide modifications on modified glass surfaces (FIG. 12)

A glass surface modified with the peptide (N-hydroxy-Gly) -Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH2 (see Example 5) was washed with 10 ml 100 μM ATP in kinase buffer (50 mM Tris-HCl, 150 mM NaCl , 30mM MgCl ₂ , 4mM DTT, 2mM 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, U / ml), lOOμM / ml ATP and lOOμCi / ml γ- ³² P-ATP (Amersham, 9.25mBq / 250μCi / 25μl, activity> 5000Ci / 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, which is formed by the second glass surface resting on the modified glass surface. The mixture was then incubated for 30 min at RT in an almost water-saturated atmosphere. 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 washing buffer (1% SDS and 1% Tween20 in 50mM TRIS buffer, pH 7.5, 200mM NaCl)

- 2 times 3 minutes at RT with IM 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 6M urea, 2M thiourea and 1% SDS

- 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, FUJEFILM) (cf. FIG. 12).

The features of the invention disclosed in the above description, the claims and the drawings can be essential both individually and in any combination for realizing the invention in its various embodiments.

Claims

Expectations

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

React the two reactants

characterized in that when the two reactants react, a nitron grappa is formed and the surface is a planar surface.

2. A method for immobilizing polymeric compounds on a surface, the individual polymeric compounds being composed of several subunits, comprising the steps

a) providing a polymeric compound to be immobilized in solution as the first reaction partner and a surface as the second reaction partner, the first reaction partner comprising a first reactive group and the second reaction partner comprising a first reactive group;

3. The method according to any one of claims 1 to 2, characterized in that the nitron group is formed between the first and the second reaction partner.

4. The method according to any one of claims 1 to 3, characterized in that the nitrone group is formed from parts of the first reactive group of the first reaction partner and the first reactive group of the second reaction partner.

5. The method according to any one of claims 1 to 4, characterized in that the first reactive group of the compound to be immobilized is a reactive group which is selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals.

6. The method according to any one of claims 1 to 5, characterized in that the first reactive Grappe of the surface is a reactive Grappe, which is selected from the Grappe, the hydroxylamines, preferably N-substituted hydroxylamines, aldehydes, ketones, acetals, ketals, Includes thiocarbonyl compounds, thioketals and thioacetals.

7. The method according to any one of claims 1 to 6, characterized in that the reaction is a chemoselective reaction.

8. The method according to any one of claims 1 to 7, characterized in that the immobilization is a directed immobilization.

9. The method according to any one of claims 1 to 8, characterized in that the compound to be immobilized is selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides , Protein domains, proteins, sugars, poly sugars, lipids, polylipids, coenzymes, natural products and low molecular weight compounds as well as derivatives and analogs from the above-mentioned groups.

10. The method according to any one of claims 1 to 9, characterized in that at least one of the subunits is selected from the group consisting of amino acids, nucleotides, carbohydrates, amino alcohols, polyalcohols, diols, terpenes, lipids, olefins, epoxides, haloalkanes, alcohols, Polyamines, diamines, amines, aminothiols, mercapto alcohols, polythiols, dithiols and thiols.

11. The method according to any one of claims 2 to 10, characterized in that the surface is a planar surface.

12. The method according to any one of claims 1 to 11, characterized in that the planar surface is a chip, preferably the surface of a chip.

13. The method according to any one of claims 1 to 12, characterized in that the surface comprises a material which is selected from the group consisting of silicates, ceramics, glass, metals, organic carrier materials, cellulose, nitrocellulose, chitin, PTFE, polypropylene, Polystyrene, copolymers, gold, platinum, silver, copper, silicon, titanium dioxide, magnetic materials, magnetizable materials and silicon oxide.

14. The method according to any one of claims 1 to 13, characterized in that the planar surface is a non-porous surface.

15. The method according to any one of claims 1 to 14, characterized in that the reaction of the compound to be immobilized with the surface takes place under microwave radiation.

16. The method according to any one of claims 1 to 15, characterized in that the nitrone structure has a structure according to formula (I)

in which

the radical X is the surface, in particular a surface of a solid phase support, and at least one of the radicals Y or Z is an immobilized compound; or at least one of the radicals Y or Z is a surface, in particular a surface of a solid phase support, and the radical X is an immobilized compound.

17. The method according to claim 16, characterized in that at least Z or Y or X is a surface and the second reactant comprises the structure FR ¹ -, wherein

R ^{1 is} selected from the Grappe, the hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals and

F is a compound functionalized with R ¹ or one functionalized with R ¹

Material is.

18. The method according spoke 16 or 17, characterized in that

Z or Y or X is an immobilized compound and and the first reactant comprises the structure GR ² -, wherein

R ^{2 is} selected from the Grappe, the hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals and

G is a compound functionalized with R ² .

19. The method according to any one of claims 1 to 18, characterized in that the immobilized compound has a structure G ^! -G ² -, where

G ^{1 is} selected from the group consisting of nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, PNA, peptides, polypeptides, protein domains, proteins, sugars, poly- cker, lipids, polylipids, coenzymes, natural products, and low molecular weight compounds as well as derivatives and analogues each thereof, and

G ^{2 is} a connecting element which connects G ¹ and the nitrone group directly and the connecting element is selectively cleavable at the point of connection to G ¹ or selectively between G ¹ and the nitronic group.

20. The method according spoke 19, characterized in that the connecting element G ^{2 is} selected from the Grappe, which additionally comprises functionalized protective groups, the protective groups preferably being those for amines, alcohols, diols, carboxylic acids, carbonyl groups and thiols.

21. Structure comprising a planar surface, preferably a surface of a solid phase support, with a compound immobilized thereon, the structure comprising a nitrone cluster.

22. Structure according to spoke 21, characterized in that the immobilized compound is a polymeric compound.

23. Structure according to claim 21 or 22, characterized in that the nitrone cluster is arranged between the surface and the immobilized connection.

24. Structure according spoke 23, characterized in that a connecting element is arranged between the Nitronrappe and the immobilized connection.

25. A method for producing an arrangement comprising at least two different compounds, preferably polymeric compounds, at at least two different locations on a planar surface, characterized in that the first connection at a first location on the planar surface and the second connection at a second location on the planar surface Surface is immobilized according to a method according to any one of the preceding claims.

26. Planar surface producible by a method according to one of the preceding claims.

27. Planar surface comprising a compound covalently immobilized thereon, preferably a polymeric compound, a nitron group being arranged between the immobilized compound and the surface.

28. Arrangement producible by a method according to claim 25.

29. Arrangement, in particular according spoke 28, comprising a planar surface according to one of the preceding claims.

30. A method for determining the substrate specificity of an enzymatic activity comprising the following steps:

characterized in that

the planar surface is a planar surface according to one of claims 26 or 27 or a planar surface of an arrangement according to one of claims 28 or 29, and

31. The method according spoke 30, characterized in that the detection of the reaction is carried out on or using the amino acid sequence immobilized on the surface of the carrier material.

32. The method according spoke 30 or 31, characterized in that the change in molecular weight is carried out by forming or cleaving a covalent bond on one of the amino acid sequences, preferably on that amino acid sequence that reacts with the enzymatic activity.

33. The method according to any one of claims 30 to 32, characterized in that the detection of the reaction is carried out by detecting the change in molecular weight.

34. The method according to any one of claims 30 to 33, characterized in that the detection is carried out by a detection method which is selected from Grappe, auto-radiography, plasmon resonance spectroscopy, fluorescence spectroscopy, UV spectroscopy, infrared spectroscopy, electron microscopy, atomic force microscopy and Includes scanning tunneling microscopy.

35. The method according to any one of claims 30 to 34, characterized in that the enzymatic activity is selected from Grappe, the kinases, sulfotransferases, glycosyltransferases, acetyltransferases, farnesyltransferases, palmityltransferases, phosphatases, sulfatases, esterases, lipases, acetylases and proteases.

36. A method for purifying a compound, especially a polymeric compound, comprising the steps

a) providing a mixture of compounds, the mixture being composed such that the mixture has at least one chemical compound to be immobilized which is provided with one or more reactive groups, in particular a polymeric compound, together with one or more these reactive grapples (n) includes non-containing by-products, b) immobilizing the chemical compound containing the reactive grapple (s) on a surface, in particular a solid phase support, by reacting the chemical compound containing the reactive grapple (s) with the surface to form one or more covalent bonds ( en) between the connection and the surface, at least one nitrone layer being formed or at least present between the surface and the immobilized connection by the reaction,

c) removing the non-immobilized chemical compound (s) and the non-immobilized by-products.

37. The method of claim 36 further comprising the step

d) cleaving off the immobilized compound from the surface, preferably by cleaving a covalent bond other than that formed in step b).

38. The method according to claim 36 or 37, characterized in that the surface comprises a reactive group.

39. The method according to any one of claims 36 to 38, characterized in that the one or more covalent bond (s) by reaction of the reactive group (s) on the compound with one or more of the reactive grapple (s) on the Surface is / are being formed.

40. The method according to any one of claims 36 to 39, characterized in that the reactive group is selected from the group consisting of hydroxylamines, preferably N-substituted hydroxylamines; aldehydes; ketones; acetals; ketals; thiocarbonyl; Includes thioketals and thioacetals.

41. The method according to any one of claims 36 to 40, characterized in that the reactive group is contained in the compound several times, preferably 2 times to 50 times, preferably 2 to 10 times.

42. The method according to any one of claims 36 to 41, characterized in that a structure according to one of claims 21 to 24 is formed as a result of the one or more covalent bond (s) between the connection and the surface.

43. The method according to any one of claims 36 to 42, characterized in that the surface is planar.

44. The method according to any one of claims 36 to 43, characterized in that the surface consists of a material which is selected from the group consisting of solid phase supports for synthesis, activated carbon, inorganic particles, magnetic particles and magnetizable particles, materials suitable for chromatography or solid phase extraction , Silicon dioxide, glass, cellulose, chitin, cotton, polyolefins, titanium oxide, gold, platinum, silver, copper, halogenated polyolefins such as PVDF or PVC, and polytetrafluoroethylene and ceramic.

45. The method according to any one of claims 36 to 44, characterized in that the compound is selected from the Grappe, the nucleic acids, genes, chromosomal nucleic acids, cDNA, RNA, oligonucleotides, polynucleotides, primers, probes, PNA, peptides, polypeptides, protein domains , Proteins, sugars, poly sugars, lipids, polylipids, coenzymes, natural products, and low molecular compounds as well as derivatives and analogues thereof.

46. The method according to claim 45, characterized in that the compound is a polypeptide comprising 2 to 1000 amino acids, preferably 3 to 200 amino acids, preferably 4 to 100 amino acids and more preferably 5 to 20 amino acids.

47. Method according to spoke 36 or 46, characterized in that one or more of the reactive groups is / are directly or indirectly connected to the terminal subunit of the compound to be immobilized, in particular the polymeric compound to be immobilized.

48. The method according to any one of claims 36 to 47, characterized in that one or more of the reactive groups is / are directly or indirectly connected to the side chain of the N-terminal amino acid of the polypeptide.

49. The method according to any one of claims 36 to 48, characterized in that; that the compound, in particular the peptide or polypeptide, is a substrate for an enzymatic activity which is selected from the group consisting of kinases, sulfotransferases, glycosyltransferases, acetyltransferases, farnesyltransferases, palmityltransferases, phosphatases, sulfatases, esterases, lipases, acetylases , Includes proteases.

50. The method according to any one of claims 36 to 49, characterized in that the implementation is a chemoselective reaction.

51. The method according to any one of claims 36 to 50, characterized in that the reaction takes place within a period of 0.1 seconds to 2 hours, preferably within a period of 1 second to 1 hour and preferably within less than 10 minutes.