WO1994020521A1 - Process for synthesizing and selecting sequences of covalently bound components - Google Patents

Abstract

A process is disclosed for the solid phase or liquid phase synthesis of selectable sequences of aminoacids of monosaccharides, the coupling sites being predetermined on a substrate. The sequences consist of determined aminoacids or monosaccharides and of undetermined aminoacids or monosaccharides at a defined position in the sequence. According to the process the synthesis of the sequence is carried out in a continuous way and comprises, the following steps: (aa) adding the determined aminoacid or the determined monosaccharide at a defined site on the substrate, and (bb) adding to the substrate a mixture of undetermined aminoacids or monosaccharides equimolar with regard to the coupling sites.

Description

 Process for the synthesis and selection of sequences from covalently linked building blocks.

introduction

The invention relates to a method for the synthesis of peptide libraries (peptide libraries) or saccharide libraries; a method for the synthesis and selection of peptides or saccharides for the determination of sequences; furthermore processes for the preparation of sequences which have been obtained using the synthesis and selection process and peptides or saccharides, the structure of which have been determined using the synthesis and selection process.

State of the art

The development and research of new drugs, which are carried out by organic chemists using the traditional method, are based on the synthesis of a large number of substances. The previously known selection methods are labor-intensive and only have a limited variety of substances per unit of time. Theoretical substance design, in which structures of molecules are determined using computer models, is becoming increasingly important. However, this method is not yet sufficiently mature, so that classic selection methods are still of great value.

Different approaches have been taken to manufacture and select substances. In general, peptide banks (peptide library) are produced, which are then tested.

A major breakthrough in this process was achieved with a peptide library (peptide library) on phage surfaces (J. SCOTT and GP SMITH, (1990) Science 249: 386). The phages which are positive in the selection process are analyzed with regard to their DNA, as a result of which the amino acid structure expressed on the surface is determined. It is disadvantageous that the phages in host cells have to be replicated and that the selection methods must be suitable for recognizing the peptides on the cell surface. The peptide to be selected is a peptide on the cells in addition to many other expressed proteins. This selection therefore has a system-related uncertainty. Furthermore, only the twenty natural amino acids can be used in the process for the production and selection of the desired sequences.

Another known method uses spherical carrier materials on which the peptides are synthesized. (KS LAM et al. (1991) Nature 354: 82. Twenty batches are run, one batch per amino acid. After each synthesis step, the spheres are mixed and again divided into twenty reaction vessels. During the selection, the positive spheres are determined and the sequence The disadvantage of this method is the cumbersome handling after the synthesis. The selection of positive spheres is difficult, a sequence analysis is necessary. The quantity of material present in a sphere is very small.

A variant of the method described above is described in the publication HOUGHTEN et al. (1991) Natur 354: 84. A combinatorial library is created. For example, two positions are set in the amino acid sequence. The rest of the pairing

These approaches should be kept separate from each other. Although these approaches have defined amino acids in certain positions and these amino acids are also known; however, this method requires a large number of reaction vessels to be treated in parallel. Only a certain amino acid is coupled with each reaction. Cysteine is not used as an amino acid to create the sequences.

Another method is that peptide coupling sites are arranged on a solid planar carrier, which can be excited with the aid of light energy to react with another building block. The light energy can come from a laser that shines on points. (F.S. TJOENG et al. (1990) Int. J. Peptide Protein Res. 35: 141-146)

It is also possible to use pinhole diaphragms, which also allow the targeted exposure of individual points. (PA FODOR et al. (1991) Science 251: 767) A disadvantage of this method is that a high level of technical effort is required. For example, a laser or a device for targeted exposure of defined reaction sites must be available. They are too Reaction mechanisms limited, since a photoreaction must absolutely be used with this method.

It is also known to synthesize peptides on a carrier material, for example cellulose. As a result, each peptide has a defined place on the carrier material. (R. FRANK and R. DÖRING (1988) Tetrahedron 44: 6031) Due to the subsequent selection process, the binding molecule can be assigned to a specific amino acid sequence. The disadvantage of this method is that only defined sequences can be synthesized.

Another important prior art is described in the international application WO 86/00991 (H. GEYSEN; publication 13.02.1986). This is a method by means of which peptides can be synthesized and, because of their binding ability, can be selected for the hypervariable range of antibodies. The peptides are composed of amino acids which are either certain amino acids in the respective position or are variable, that is to say indefinite amino acids. A mixture of amino acids is used in the reaction of the variable amino acids. The sum of the amino acids present in the mixture is in excess compared to the coupling sites, that is to say to the growing peptide sequences. The different types of amino acids in the respective mixture should be present in an equimolar amount to one another.

US Pat. No. 5,010,175 (WJ RUTTER and DV SANTI; publication April 23, 1991) also describes a method for producing a peptide bank. Here too, in addition to certain amino acids, variable amino acids should also be arranged in the sequence. In the reaction of the variable amino acid, a mixture of activated amino acids reacts with the acceptor peptide bound to the carrier. The various amino acids are present in the mixture in concentrations which correspond to the relative coupling constants. The concentration of the slow-reacting amino acids is correspondingly higher than the concentration of the fast-reacting amino acids. Since all peptides are located in one reaction vessel, the following selection is used. The mixture of the most varied peptides is passed over a column on which the molecules, for example receptors, are located which are intended to bind the desired peptide. All unbound peptides are eluted. The bound peptides are analyzed to determine the sequence of the amino acids. The analysis of the peptide sequences is cumbersome, it is still fraught with uncertainty, since similar amino acids can sometimes lead to a comparable binding constant.

Once the desired sequence has been determined, the synthesis of peptides is easy to do. A summary of these techniques can be found in J.M. STEWART and J.D. YOUNG, San Francisco, 1969, and J. MEIERHOFER, Hormonal Proteins and Peptides, vol. 2 p. 46 Academic Press (New York), 1973 for the solid phase method and E. SCHRÖDER and K. LUBKE, The Peptides, Vol. 1, Academic Press (New York) 1965 for the liquid phase method. The steps of the synthesis are described in the publication EP 0 097 031. The general process steps from the European publication can be applied analogously to the synthesis of almost all peptide compounds.

task

It is an object of the invention to offer a method with which peptides or saccharides can be specifically produced which are suitable for selection. The sequences should have certain desirable properties. These properties enable a targeted selection process, the desired properties consisting of the specific recognition of predetermined molecules, for example receptor molecules. The sequences obtained by this process are then to be used as templates for molecules (pharmacological lead structures) that are produced in the classic chemical manner.

solution

The object is inventively achieved by a method for producing ^'of peptide libraries (Peptide libraries) of peptides, which process consists of the following features: the peptides have per position in the peptide sequence is either a fixed or an indefinite amino acid on; the peptides are produced on or in a carrier material after the solid phase synthesis or in separate reaction vessels after the liquid phase synthesis; spatially separate reaction sites are located on or in the support material or in the reaction vessels; every reaction site has

(i) during the solid phase synthesis defined number of peptide coupling sites for the binding of those activated for the reaction

Amino acids and

(ii) in liquid phase synthesis, a defined number of start amino acids or start peptides (peptide coupling sites; the method consists of the following steps, each with its own procedure for the synthesis of the particular amino acid and the undetermined amino acid : If an activated certain amino acid is reacted at the intended position of the peptide sequence, this certain amino acid is present in excess compared to the peptide coupling sites; if an activated indefinite amino acid is reacted at the intended position of the peptide sequence, this undetermined amino acid lies in a mixture of amino acids, the sum of all undetermined amino acids in the mixture being at most equimolar to the peptide coupling sites, and after the coupling reaction a further amino acid is added for saturation.

The object is further achieved by a method according to the invention for producing saccharide libraries (saccharide libraries) from saccharides, which method consists of the features: the saccharides have either a specific or an undetermined monosaccharide per position in the saccharide sequence; the saccharides are on or in a carrier material after the solid phase synthesis or produced in separate reaction vessels after liquid phase synthesis; spatially separate reaction sites are located on or in the support material or in the reaction vessels; every reaction site has

(i) during the solid phase synthesis defined number of saccharide coupling sites for the binding of the monosaccharides activated for the reaction and

(ii) in liquid phase synthesis, a defined number of start monosaccharides or start saccharides (saccharide coupling sites); The method consists of the following steps, each with its own procedure for the synthesis of the particular monosaccharide and the indefinite monosaccharide: If an activated particular monosaccharide is used on the intended one

When the position of the saccharide sequence is reacted, this particular monosaccharide is in excess of the saccharide coupling sites; if an activated indefinite monosaccharide is reacted at the intended position of the saccharide sequence, this indefinite monosaccharide is present in a mixture of monosaccharides, the sum of all indefinite monosaccharides in the mixture being at most equimolar to the saccharide coupling sites, and after that Coupling reaction is another monosaccharide

Saturation added.

The mixture of the undetermined amino acids or monosaccharides in the mixture compared to the peptide coupling sites or saccharide coupling sites can be at most in a 0.9-fold excess.

Advantages of the method according to the invention

One of the advantages of this method according to the invention is that structures of, for example, peptide ligands can be determined in a very short time and can bind specifically to, for example, the extracellular domains of receptors. With the help of this random principle, all theoretically conceivable structures are available during the step-by-step selection. The Structures can be determined for each coupling point on the basis of the process protocol.

The results can be influenced differently only by determining which amino acids or which monosaccharides are determined first in the sequence and which ones are undetermined. Otherwise, the process is characterized by the fact that the peptides or saccharides to be obtained can be reworked in spite of a random process in the case of the not indefinite amino acids or monosaccharides by separate process approaches.

This invention is made possible by reaction conditions which are present in the synthesis of the undetermined amino acids or undefined monosaccharides. It is customary in the prior art to offer the mixture of the amino acids or monosaccharides to be synthesized in their sum in a molar excess compared to the coupling sites, since otherwise not all sequence chains come to reaction. A sequence that is not extended by another amino acid or another monosaccharide leads to defects, defects, occurring in the sequence.

Furthermore, according to the prior art, a molar excess of the amino acid to be coupled (acylation reagent) is necessary when coupling the particular amino acid, since the coupling kinetics of the individual amino acids are different, but the coupling yield for a specific coupling step and a specific amino acid never changes lets predict. A clear molar excess circumvents this problem. Two to eight-fold excesses are routine for peptide chemists. A six-fold excess is common. This is the excess when coupling a certain amino acid.

The supply of mixtures has already been mentioned in the literature when indefinite amino acids are to be coupled in one position. In WO 86/00991 a mixture is offered in excess. The mixture mentioned in US Pat. No. 5,010,175 is also present in excess and is adjusted with regard to the concentrations of the individual components so that the concentration is reciprocal to the reaction rate of the amino acid. Since the reaction kinetics and the coupling rates are practically unpredictable, none of the methods mentioned in the prior art can be used Produce peptides or saccharides that have randomly inserted amino acids or monosaccharides in predetermined positions. However, this random principle is important because otherwise an undesired drift occurs during the selection.

By coupling a complex amino acid mixture in molar deficit, the problem of different kinetics for individual amino acids can be avoided. Since these are in deficit in relation to the amine component, all amino acids react. However, some coupling sites remain without a reaction partner. The amine component on the support is then completely converted by at least one further coupling with, for example, alanine in the same, for example, molar ratio to the peptide coupling sites. There is a prejudice among experts that reactions cannot be successful without an excess of the new building blocks to be coupled.

Contrary to the prevailing prejudices, the undetermined amino acids or monosaccharides are offered at most in the equimolar amount in the process according to the invention. As a result, almost all undetermined amino acids or monosaccharides will react equimolar with the sequence. The individual building blocks will have reacted 99% at different times due to their different kinetics, but practically all activated amino acids or monosaccharides react.

Other embodiments

The method according to the invention includes the at most equimolar use of undetermined amino acids or monosaccharides. If the person skilled in the art uses higher concentrations, he will nevertheless obtain results which, however, no longer correspond to the statistical distribution. A worsening of the process is then accepted. In terms of statistical distribution, this method works best when the sum of all amino acids or monosaccharides offered is at most equimolar. If the equimolar range is clearly undershot, only some of the synthesis chains are coupled with the undetermined amino acids or monosaccharides, others remain without reaction partners. A preferred embodiment of the method according to the invention is that the peptide sequence or saccharide sequence (sequence) has three specific amino acids or three specific monosaccharides each at a specific position in the sequence, in that three specific amino acids or three specific monosaccharides are used in the synthesis can be synthesized at a particular position in the sequence.

The affinity of binding molecules increases approximately exponentially to the number of certain amino acids or monosaccharides. Three specific ones

Amino acids or monosaccharides allow a better preselection of the entire sequence than the use of only two specific amino acids or monosaccharides. Three specific amino acids or monosaccharides can also be applied without problems on an essentially plaπar carrier material.

The sequence can also have four or five specific amino acids or monosaccharides at a particular position in the sequence, by synthesizing four or five specific amino acids or monosaccharides at a given position in the synthesis.

Another embodiment of the method according to the invention is that the sequence is branched or unbranched. The method according to the invention is not limited to linear synthesis, branching is easily possible. Appropriate protective groups are to be used. The branched molecules have the advantage that they can also cover flat areas. This opens up a further field of application of the method according to the invention. Cyclic designs are also possible, which increase the conformational stability of the molecule.

Various substances can be used as the carrier material, the cellulose carrier material being particularly suitable. It has the advantage that there is a carrier which is more useful for the synthesis and on which the amino acids or monosaccharides and their mixtures can easily be applied. The flat surface and the high absorbency make handling easier.

The invention further comprises a method wherein the amino acid is (i) a natural amino acid or (ii) a derivative of the natural amino acid. The method according to the invention looks as follows: Synthesis of peptide banks from natural amino acids and / or their derivatives (amino acids) with the coupling of amino acids, the amino acids each occupying a sequence position in the sequence, before the coupling to form the covalent bond on Carboxyl end are activated, and consist of two types of amino acids, on the one hand (i) certain amino acids and on the other (ii) undetermined amino acids, using a solid phase synthesis on or in a carrier material, or using a liquid phase synthesis in solutions, wherein the sequence (i) has at least one specific amino acid, its structure and

Sequence position is determined in the peptide synthesis, and (ii) has at least one undetermined amino acid which can only be determined statistically and which, at a certain sequence position, reacted randomly from an amino acid mixture with structurally known amino acids, whereby on or in the carrier material or in separate reaction vessels for the application of the activated amino acids, the reaction sites have at least in the solid phase synthesis peptide coupling sites for binding the amino acid activated during synthesis in the peptide sequence, which is the first amino acid in the peptide sequence The method consists of the following steps: continuous synthesis of the peptide sequence with coupling of the first amino acid in the peptide sequence to the peptide coupling site or by starting with the first amino acid protected at the carboxyl end and with coupling of the one in the peptide sequence further amino acids at the N-terminal end of the partially synthesized peptide, each with its own procedure for coupling the specific amino acid and the undetermined amino acid, (aa) where, if a specific amino acid is to be coupled at a specific sequence position, the activated specific amino acid is added at at least one specific reaction location on or in the carrier material or in one reaction vessel each, the specific amino acids being opposite the peptide coupling sites or the sequences to be synthesized

Excess, and (bb) whereby if an undetermined amino acid is to be coupled at a certain sequence position, a mixture of activated undetermined amino acid is added to all types of reaction on or in the support material or in one reaction vessel each, the undetermined amino acids being in their Sum compared to the peptide coupling sites or those to be synthesized

Peptide sequences at most equimolar, and further amino acids are added after the coupling reaction.

The mixture of the undetermined amino acids in the mixture compared to the peptide coupling sites or the sequences to be synthesized can be used at most in a 0.9-fold excess.

It is advantageous if the building blocks are D- or L-amino acids and / or their derivatives. In addition to the twenty L-amino acids, there is a large number of amino acid derivatives, which are described, for example, in the catalog of the company BACHEM, Bubendorf, Basellandschaft, Switzerland. Such peptides, which contain modified amino acids, often display different types of pharmacokinetics. They behave differently in the organism than the peptides, which consist exclusively of natural amino acids. They are more stable towards proteases, they penetrate the cell membranes more poorly, making the synthetic peptide used as a drug more suitable for action in the extracellular region than a sequence of L-amino acids.

Suitable carrier material is polystyrene beads, on which the solid phase synthesis of the peptide library (peptide library) can also be carried out by means of simultaneous synthesis using amino acid mixtures. The simultaneous synthesis of individual components (for example in the case of a hexapeptide with the Sequence X- | • X2 ^■ B3 ^• X4 ^• B5 ^• XQ [X = undetermined amino acid with random distribution; B = specific amino acid]) of the peptide library (peptide library) can easily be carried out on a multiple peptide synthesis machine (for example from Abimed AMS 422). For example, 48 approaches can be synthesized simultaneously using a previously mentioned device. With the help of this method, larger amounts of free peptides can be obtained, which can be used, for example, for cell tests.

A further embodiment of the invention is that the peptide coupling sites are β-alanine residues which are covalently linked to the carrier material. β-alanine is coupled to the cellulose material via an ester bond. A defined number of peptide coupling sites is required in order to know what amounts of amino acids have to be offered in each synthesis step. It is also possible to detach the sequences from the support at the peptide coupling sites.

Glycine, other amino carboxylic acids (apart from β-alanine), polyethylene glycol (RAPP et al. In Peptides 1988; Ed. G. JUNG, E. BAYER, Walter deGRUYTER, Berlin 1989, pages 199 are suitable as further pepeptide coupling sites - 201) and all linker groups for peptide synthesis that are commercially available and from which the peptide can be split off again after synthesis (see catalog of NOVABIOCHEM 93).

In a further embodiment of the invention, the mixture can consist of natural amino acids which are present in essentially the same molar proportions.

Furthermore, the amino acids can consist of the D configurations of the natural amino acids, which are essentially in the same molar proportions. The amino acids can also be amino acid derivatives. Such derivatives are listed, for example, in the catalog of BACHEM, Bubendorf, Basellandschaft, Switzerland.

The further embodiment of the invention relates to saccharides. These are described in detail in the literature, (for example RÖMPPS, Chemistry lexicon, ed. NEUMÜLLER, O.-A. (1983) 8th edition, pages 2147 to 2152). It is known that the different sugar units have different reaction kinetics. own techniques (see: PAULSEN, H. (1990) Angew Chem 102: 851; KUNZ, H. (1987) Angew Chem 99: 297; PLEWE, M. et al. (1992) Carbohydr Res 235: 151; and OGAWA, T. et al. (1984) Pur Appl Chem 56: 779) As a result, there is a comparable problem here as with the amino acid sequences. However, there are some differences between the two systems. Due to the high functional density, the saccharides are given a higher information content than the amino acids. In contrast to peptide chemistry, the linkage options for sugars are extremely complex. Theoretically, two monosaccharides can be combined to eleven disaccharides.

A process according to the invention is preferred in which the saccharides are a cyclic pentose or hexose or their derivatives. Typical pentoses are described in the standard work RÖMPPS.

Biologically relevant monosccharides are, for example, glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine or N-acetylneuraminic acid.

The method of synthesis of the particular amino acids or monosaccharides is effective when the particular amino acids or monosaccharides are present in at least a three-fold excess. Good results can be expected in a range from three to eight times the excess.

After the reaction of the undetermined amino acids or monosaccharides, some coupling sites are not occupied with an undetermined amino acid or an undefined monosaccharide. However, in order to achieve the same molecular lengths, after the reaction of the undetermined amino acid or the undefined monosaccharide, the further amino acids or monosaccharides are added at least in a simple excess. A double excess is preferred.

Another embodiment as a selection method

The invention further comprises a method according to the invention for the synthesis and selection of peptides or saccharides, after the synthesis of the sequence, the desired sequences are selected with the aid of binding tests or specific reactions, the selected sequences optionally being represented by indicators.

Advantages and possible uses of the selection process

A simple selection process is used to find, for example, a single peptide from the millions of different molecules that has a certain desired property, such as the effect as a therapeutic substance or the binding and elimination of toxic heavy metals.

This is illustrated using a hexapeptide: The synthesis is carried out in such a way that 400 reaction types (“spots”) are created in an arrangement of 20 × 20 fields. Each of these reaction sites contains a mixture of over 100,000 individual peptides, for example of the composition X-, X ₂ B3B ₄ X ₅ X ₆ . B ₃ and B _{4 are} certain amino acids. In each of the positions X- ^ X ₂ ; Xs _; and X ₆ are simultaneously installed every 20 amino acids with the same probability.

Since there are 20 natural amino acids, this leads to peptide mixtures with two defined positions at the 400 reaction sites. There are 160,000 peptides per reaction site because the positions Xi; X ₂ ; X _5; and X _{6 are occupied} according to the random principle. The 160,000 peptides are represented with the same probability. All reaction sites together contain 64,000,000 different peptides.

When looking for a specific peptide with a specific property (for example binding to a coat protein of the HIV virus), the first step is to find the reaction sites ("spots") that show the best binding. The first two amino acids are thus determined and in the next reaction sequence these two determined amino acids are retained unchanged. In the next step, the next two amino acid positions are determined and the remaining two are varied freely. Again 400 reaction sites are tested for binding and the best ones are identified. Now there are 4 positions. at In the next step, hexapeptides define all peptides in their sequence and identify the peptides with the best binding. This whole process of searching for a specific connection among billions or even billions of connections takes significantly less time than the conventional methods required. The search for peptides with the desired properties can be successfully completed in a few days.

The process is open to many modifications. Natural and non-natural amino acids, D-amino acids, sugar, deaminated amino acids or peptoids can be used. Branches can be introduced via side chains or conformations stabilized via side chain crosslinking or cyclization of oligomers. Longer oligomers can be synthesized, in which several positions remain constant, but which form spatially defined conformations. The method can thus be used to specifically produce active substances and analytical molecules of comparable monoclonal antibodies without the numerous disadvantages of these natural molecules (animal experiments, production costs).

With the process, it is possible in a very small space to produce a variety of connections with minimal material expenditure (mg range) that nature does not even know. This diversity can then be examined for desired properties (binding, enzymatic activity, inhibition of activity). The method thus represents a basic technology in biology, medicine, pharmacy, analysis, sensor technology and microsystem technology.

The potency of the process to produce any variety of molecules and to select such compounds with the desired properties from them is superior to all other processes, in particular with very large numbers of variants (> 10 ⁸ compounds). In recent years there have been various efforts to develop processes which have the advantages of the process according to the invention. However, these attempts failed due to problems in chemical synthesis. Nobody else is able to ensure that, for example, with a supply of 20 amino acids, all are incorporated into the peptide with the same effort, the same reliability and the same probability. Furthermore, this method only makes it possible to use a solid phase that has not previously been used for this purpose. More complex procedures were developed by some groups, which, however, are too high and no longer justifiable from a certain number of variants. real effort. In these methods according to the prior art, small beads are generally used as the solid phase, to which the molecules are covalently bound and the handling of which quickly leads to capacity limits. This problem is solved so successfully by the method according to the invention that use in any chemical laboratory is possible at any time without any problems.

In addition to biotechnology, synthetic biology is becoming increasingly important. The described method enables the targeted search for peptides and other oligomeric chemical compounds with defined properties within a very short time. Up to now, a targeted search for such connections was only possible with a much higher effort, which made this a non-feasible method in most cases. The process is able to significantly accelerate the development of innovative products in the chemical-pharmaceutical industry.

The method according to the invention represents an essential, quickly executable and practicable way in synthetic biology without using genetic engineering methods. The variety of organic compounds known so far only from nature itself can be far exceeded with the present method. From this diversity, the method enables a targeted search for the molecule best suited for a specific application. Biological molecules with completely new properties can be constructed in this way. The biological process of mutation and selection in a certain time interval is exceeded in speed by the present method, since the number of mutants of the different molecules to be tested is significantly higher than in nature.

In the pharmaceutical industry, active ingredient binding has become a more manageable process from the previous random principle (15,000 synthesized molecules to an approved product). Drug development can be accelerated, since drug binding is still one of the most time-consuming phases of development. For example, the method allows the identification of metal-binding peptides that can be used to treat heavy metal poisoning within a few days. The iπ-vitro diagnostics largely based on the use of monoclonal antibodies today will probably be completely replaced by the use of the described method. All analytical and diagnostic methods based on the use of monoclonal antibodies today are replaced by the use of synthetic peptides that are specifically produced and identified using the method described here. No immunization of mice, no month-long production process for antibodies, no bio-technological production of proteins (antibodies), no more 95% unused expensive protein is required. (In the case of antibodies, only the binding sites are of interest, which only make up a few percent of the total mass.)

In environmental analysis, the method opens up the possibility of developing rapid detection systems for environmental pollutants, which enable quantitative determination with minimal expenditure of material and time. For environmental technology, compounds can be shown that selectively bind toxic substances (heavy metals, dioxins, furans) and render them harmless.

Furthermore, the method opens up the possibility of making toxic and ecotoxicological assessments of pollutants using in vitro methods. This is done by identifying the pollutant-binding sequences with the aid of the method and then searching for their occurrence in proteins in protein sequence databases. These proteins can then be specifically examined for receptor-specific toxicity. This is a completely new approach that was previously not possible without the method. The development of ecologically compatible substances is facilitated. Since the investigation can be carried out very early, directly after a first synthesis, the method reduces the number of undesirable developments in new products in the chemical industry.

An application of the method to the development of biosensors for a variety of questions is obvious, especially since the connections are not as sensitive to physical influences, such as high temperatures, which often still limits the use of biosensors today. The use of appropriately selected connections as sensors in microsystem technology is another possible application of the method. The coating of surfaces with correspondingly designed connections in nanotechnology has been made possible. The method works with minimal amounts of substance, in the range of nanograms, so that in the selection phase the user of the method does not incur any noteworthy costs. The reduction in costs for the users, on the other hand, is considerable since it is possible to search specifically for connections.

Further embodiments regarding the selection

An advantage of the synthesis and selection method according to the invention is that after the sequences have been synthesized, known and proven indicator methods can be used to determine which sequences have the properties to be selected.

It is advantageous if the binding test consists in adhering marked test substances and making the marking visible.

Proven and therefore preferred methods are that the label consists of radioactive material or of biotin or of covalently linked enzymes. Other methods consist of measuring fluorescence or luminescence.

When determining formation molecules, the method according to the invention in particular also includes the molecules which trigger a function. This is done by measuring the reactions using, for example, allosteric enzymes. The peptide library (peptide library) can also be used to test the substrate specificity of proteases or to find possible inhibitors. For the substrate specificity, the peptides should e.g. B. be fluorescent labeled. Then the individual coupling sites have to be cut out, treated with protease and the release of fragments measured photometrically. Inhibitors can be found in that certain sequences to which the protease binds but which the protease cannot cleave can be identified at the corresponding coupling sites.

It is also possible to measure the response of a cell system. This also includes all bindings to receptors. A large number of receptors can thus be tested with regard to their ability to bind to the synthesized ligands. In addition to the receptors, metal atoms are also suitable for the selection process, for example technetium, gadolinium, nickel and calcium. Technetium complexing peptides can be used as, for example, tumor diagnostics. Nickel complexing sequences can be used as a tool for protein purification using nickel columns. Peptides should also be able to bind organic chemical molecules. Peptides that bind transition analogues of certain organic chemical reactions may be the precursors for new catalysts.

More complex interactions can also be measured, whereby the reaction can consist of a cell-cell interaction.

Other embodiments

In addition to the method according to the invention, sequences are also claimed, namely unbranched or branched peptides made from D- or L-amino acids and / or amino acid derivatives, which have a structure which uses the previously mentioned synthesis and Selection procedure was obtained. If a sequence has been determined with the aid of the method according to the invention, it is easy for a person skilled in the art to be able, for example, to synthesize the peptide on the basis of this sequence. The technique of peptide synthesis has previously been described in detail. The invention thus provides a technical teaching that reveals the way to the sequence. The sequence thus determined can then be prepared on the basis of the standard knowledge of the person skilled in the art. With the help of this teaching, sequences can be found which have the desired specific functions.

The invention further comprises a method for the peptide synthesis of unbranched or branched peptides from D- or L-amino acids and / or amino acid derivatives, the sequence having the peptide structure being synthesized using the synthesis according to the invention - And selection process was obtained. Definitions

AMINO ACIDS: The amino acids and their derivatives can belong to the group of

L or D amino acids belong. Mixed forms are also possible within a sequence. Derivatives of amino acids are characterized in that the side chains are substituted. Sequence chains over side chains are also possible, e.g. B. γ-glutamine.

All reactive side chains can be used for the synthesis of branched peptide banks (peptide library), such as lysine. Amino groups can be reacted via the reaction of carboxylic acids such as glutamic acid and aspartic acid, for example Arginiπ with dions.

The sequences can have branches, the branch being connected to the main strand of the sequence via a functional group. It is important that the building blocks are all soluble and reactive in at least one reaction medium.

ACTIVATED AMINO ACID OR ACTIVATED MONOSACCHARIDE: A building block in a sequence that has an activated, functional group with which it can bind to existing amino acids / monosaccharides or peptide / saccharide coupling sites. Furthermore, the amino acid or the monosaccharide must have protective groups which permit a reaction of the desired functional groups, but shields those which are not desired.

SOLID PHASE SYNTHESIS: Solid phase synthesis is described in detail in Solid Phase Synthesis, E. ATHERTON and R.C. SHEPPARD (1989) IRL Press, ISBN 1 -85221 -133-4 and Amino Acid and Peptide Syntheses, J. JONES, Oxford Science Publication (1992) ISBN 0-19-855668-3.

LIQUID-PHASE SYNTHESIS: Liquid-phase synthesis or solution technology is described in methods of organic chemistry (HOUBEN / WEYL), vol. 15 / no. 1 and 2, E. WÜNSCH (editor), Thieme Verlag Stuttgart, 1974.

CARRIER MATERIALS: A wide variety of carrier materials are described in the standard literature. All essentially planar supports that are easy to apply are particularly suitable in this process Allows amino acids or monosaccharides. Cellulose is particularly suitable. Here, materials from WHATMAN, type: Whatman paper 540 (hardened cellulose) have proven to be very useful.

In addition, carrier materials are also suitable which are located as spherical structures in reaction vessels. Suitable carrier materials are polystyrene beads, on which the solid phase synthesis of the peptide library (peptide library) can also be carried out by means of simultaneous synthesis using amino acid mixtures. The simultaneous synthesis of individual components (for example in the case of a hexapeptide with the sequence X- | ^• X2 ^• B3 ^• X4 ^• B5 ^■ Xg [X = indefinite amino acid with random distribution; B = specific amino acid]) of the peptide library (peptide library) can can be carried out particularly well on a multiple peptide synthesis machine (for example from Abimed AMS 422). For example, 48 approaches can be synthesized simultaneously using a previously mentioned device. With the help of this method, larger amounts of free peptides can be obtained, which can be used for cell tests, for example.

REACTION LOCATION: Reaction location (also referred to as application location) is to be understood as a reaction space in which identical reaction conditions prevail. It is important that the reaction site makes it possible to understand which reactions have taken place on a support or in a reaction vessel.

A reaction site can thus be part of a cellulose support or else a separate reaction vessel. The same specific amino acids or monosaccharides and the same undetermined amino acids or monosaccharides are reacted on the cellulose support per reaction site.

In order to make the experiment meaningful, the one or two-dimensional or possibly three-dimensional definition of locations on or in the support material is necessary in order to avoid that the amino acids or monosaccharides become uncontrolled at the corresponding reaction sites with the amino acids or monosaccharides of others Can mix reaction sites.

If the reaction sites represent reaction vessels, the reaction can take place as a solid phase synthesis or as a liquid phase synthesis (solution technology). Liquid phase synthesis is currently preferred. PEPTIDE COUPLING OR SACCHARID COUPLING POINTS:

Coupling sites or saccharide coupling sites (coupling sites) are used in solid phase synthesis. Not all carriers are suitable for being able to couple the desired amino acids or saccharides. Furthermore, the amino acids or saccharides must no longer be able to bind to the support material after the second synthesis cycle. It is therefore sensible to attach so-called anchor molecules on or in the carrier material. For this purpose, ß-alanine is suitable for cellulose. The coupling sites define the amount of reactants and the amount of sequences synthesized.

In the solution method (liquid phase synthesis), the coupling site can be formed by a starter to which the first amino acid or the first monosaccharide can couple. This starter is removed again after synthesis. If there is no coupling site in the liquid phase synthesis, the synthesis is started with the first protected amino acid or with the first protected monosaccharide.

Application examples of the inventive method:

The method according to the invention allows new active substances to be found much more quickly and their development to market maturity. Numerous new products can be developed in diagnostics and therapy that could not be developed with the previous methods. Tissue-specific diagnostics in nuclear diagnostics, magnetic resonance imaging, ultrasound diagnostics and X-ray diagnostics have finally moved into the realm of feasibility. New methods, such as the use of lasers for diagnostics (laser-induced fluorescence) and therapy (photodynamic therapy), which have hitherto failed due to a lack of structure, cell- or tissue-specific (target-specific) dyes, are made possible by the present method.

(1) peptides against AIDS

The method according to the invention can also be used to find substances which are suitable for the treatment of AIDS (aquired immune definciency syndrome) (HIV antagonists, human immunodeficiency virus). Two different ways can be labeled here: a) Enzymes essential for virus replication, e.g. the HIV protease or reverse transcriptase can be inhibited by antagonists determined using this method, which would block the spread of the virus. b) the docking of the virus to the T lymphocytes, mediated by the CD4 receptor, can be suppressed either by CD4 antagonists or by molecules which bind the CD4 binding site on the coat protein gp120 of the virus and thus prevent a viral infection.

Example for a:

The method can be used to determine HIV protease antagonists (M. Miller et al. (1989), Science 246: 1149) by adding fluorescent or radioactively labeled protease to the cellulose bank and determining the mixtures which bound by the protease but not hydrolyzed. The binding of the protease to the membrane can be determined by the quantification of the radioactivity. The antagonistic properties of the peptides leave are determined by incubating the peptides in the presence of the HIV protease and natural substrates. If the peptides have an antagonistic effect, the cleavage of the natural substrate is prevented (J. Schneider and SBH Kent (1998), Cell 54: 363). Furthermore, the potential antagonists can be tested by adding to a T cell culture to determine whether they prevent the virus from spreading (S. Seelmeier et al. (1988), Proc. Natl. Acad. Sci. 85: 6616).

Example for b:

The method can be used to determine peptides that may bind the site of the HIV coat protein gp120, which is responsible for the docking to the CD4 receptor of the T lymphocytes (MH Brodsky et al. (1990), J. Immun. 144: 3078). Blocking this site would prevent infection of the T cells by the HIV virus. The method described here can be used to determine both peptides that specifically bind the CD4 receptor binding site on the coat protein gp120 or also the gp120 binding site on CD4 and thus prevent the virus from docking onto the T lymphocyte. So far, either soluble CD4 receptors or recombinant gp120 produced in E. coli (size over 100 amino acids) have been used for such a therapeutic approach (M. Watanabe et al. (1991), Proc. Natl. Acad. Sci. 88: 120; F LaRosa et al. (1990) Science 249: 932). Here too, peptides, because of their smaller size, would have many advantages, such as lower antigenicity, more favorable pharmacokinetics, etc. Peptides which specifically bind these two proteins can be determined with the method via radioactively labeled gp120 or CD4. The sequences determined in this way can now be tested in vitro to determine whether they are able to protect a T-lymphocyte culture from a virus infection (Seelmeir et al., See above).

(II) Pepetide against atherosclerosis:

Furthermore, atherosclerotic processes can be prevented by finding peptides that bind T-lymphocytes and monocytes, which have been transformed in foam cells by too much absorption of oxidized "low density lipoprotein (LDL), to the receptors of the vessel walls (arteries). block (SM Edgiπgton (1993), BIO / TECHNOLOGY 1 1: 676) The foam cells secrete chemokines, cytokines and growth factors which are involved in the formation of atherosclerotic plaques (S. Cushing et al. (1990), Proc. Natl. Acad. Sci. 87: 5134; Schall et al. (1990), Nature 347: 669; Tanaka et al. (1993) Nature 361: 79). The chemokine MCP-1 (monocyte chemoatractant protein 1, Cushing et al., See above) plays a special role in this process, which, among other things, mediates the binding of the monocytes to the vessel walls. A substance that blocks the binding of MCP-1 to its receptor (D. Michiel (1993), BIO / TECHNOLOGY 1 1: 739) could be used as a medicament for the treatment of arthereosclerosis. For this purpose, the receptor is radioactively labeled and the peptide banks are examined for peptides which bind the MCP-1 receptor. The peptides obtained in this way are then examined in an in vitro model (Cushing et al., See above) to determine whether they are able to prevent macrophages from binding to endothelial cells.

(III) Peptides against thrombosis:

The method could also be used to develop a drug for the treatment and prevention of thrombosis. The binding of the von Willebrand factor (vWF) to the "platelet glycoprotein (GP) Ib" receptor (Z. M. Ruggeri and T. Zimmermaπ (1985), Semin. Hematol 22: 230) plays a decisive role in the formation of thrombi. An anti-thrombotic peptide must prevent the interaction of vWF and GPIb. The peptide banks can now be used to determine peptides that specifically bind the receptor. The receptor GPIb (M. Sugimoto et al. (1993), J. Biol. Chem., In press) is again radioactively labeled and incubated with the banks. GPIb-binding peptides obtained in this way can then be examined in an in vitro model to determine whether they prevent the binding of vWF to GPIb and thereby inhibit the fibrinogen binding to the glycoprotein III / Illa complex triggered by the binding. This would prevent platelet aggregation (L. DeMarco et al. (1985), Proc. Natl. Acad. Sci. 82: 7424) and the peptide obtained by the process is suitable for the treatment of thrombosis.

(IV) Pepetides for immune stimulation:

This method can also be used to develop immunostimulants, which are particularly useful for the treatment of mycobacterial (tuberculosis, leprosy), fungal (Cryptococcus, histoplasma), viral (herpes simplex, HIV, cytomegalovirus) and parasitic infections (toxoplasmosis, Leishmania, pneumocystis) ) is suitable. The cytokine interleukin-2 (IL-2) plays a special role here. IL-2 is secreted by T lymphocytes after stimulation of antigens (proteins) (KA Smith (1988), Science 240: 1169). Then IL-2 stimulates itself the proliferation and differentiation of all lymphoid cells by binding to IL-2 specific cell surface receptors p55 and p75 (KA Smith (1989), Annual Review of Cell Biology 5: 397). The method can thus be used to find IL-2 agonists in that peptides which specifically bind p55 and p75 are determined by means of labeled receptors. A possible agonistic effect of the peptides can now be tested in an in vitro experiment. If the peptides are active, NK - ("natural killer"), B and T lymphocytes will be stimulated and quantified using various secretory markers. A variety of different tests have been developed for this purpose (for an overview, see Kaplan et al. (1992), BIO / TECHNOLOGY 10: 157).

(V) Pepetide for immunoscintigraphy:

This method can also be used to determine substances for immunoscintography. Such peptides complexing the radioactive metal technetium 99m (Tc99m) can be genetically fused to an anti-tumor antibody. Such an antibody, e.g. recognizes the tumor cell surface marker protein CEA (Carcinoembryonic antigen, J. Beatty et al. (1979), Arch. Surg. 1 14: 563; TR Barnett et al. (1989), J. Cell Biol. 108: 267) and which is linked to a Tc-binding peptide can generally be used for imaging tumors, since CEA is a general marker protein for tumors. The peptide can be produced using the method described, in that the peptide library is incubated with Tc99m and the peptides which bind Tc99m are determined. Tc-binding peptides can be found and quantified using a phosphoimager from Molecular Dynamics. The functioning of the Tc-complexing peptide in solution can be demonstrated using classic HPLC methods (see above). If the peptide binds technetium, this can be determined by means of HPLC by quantifying a "radioactive peptide peak". In vivo, the functioning of the antibody / Tc-peptide complex in mice that carry tumors can now be demonstrated by applying the antibody and then using a gamma camera to check whether the tumors can be made visible (H. Schrewe et al (1990) Molec. Cell Biol. 10: 2738).

(VI) Peptides for Tumor Diagnostics:

Diagnostic markers for tumor cells can also be found using the method. For this purpose, peptides are determined with the method that the Specifically bind tumor cell surface marker protein CEA (see above). CEA-binding peptides are determined by incubating radioactively labeled CEA with the banks and binding peptides are determined by blackening as described and further approximated. The function of the peptide can be demonstrated in in vivo experiments as described above. Such a CEA-binding peptide can be combined with a Tc-complexing peptide to form a bifunctional peptide for imaging tumors as described above.

(VII) Peptides for nickel binding:

Nickel-binding peptides can also be determined using this method. This is of particular importance for the purification of recombinantly produced proteins. So far, so-called "affinity tags" have been added to the proteins by genetic engineering (Hucholi et al.). These tails consist of 6 histidines, which have various disadvantages. The presence of 6 histidines often leads to solubility and folding problems (J. Hucholi et al. (1987), J. Chrom. 41 1: 177). With this method, sequences can now be found using the peptide banks that bind similarly or even more strongly than 6 histidines. The peptide banks are incubated with a nickel salt, washed and the nickel binding to the peptides is demonstrated by means of a classic nickel detection with dimethylglyoxime as a red color complex (Jander / Blasius, textbook of analytical and preparative inorganic chemistry, S. Hirzel Verlag Stuttgart; U. Reineke and J. Schneider-Mergener et al., Manuscript in preparation). The nickel bond can then be dissolved in solution using HPLC (R. Oliver, HPLC of Macromolecules, IRL Press, Oxford), capillary zone electrophoresis (IS Kreull and JR Mazzeo (1992), Nature 357: 92) and laser desorption mass spectrometry (J. Hodgson (1992 ), BIO / TECHNOLOGY 10: 399).

(VIII) Peptides Against Hemochromatosis:

Furthermore, the process can be found a peptide which binds the metal iron specifically. Such a peptide could be a substitute for the drug desferrioxamine, which is used to treat various diseases of iron metabolism, such as hemochromatosis, thalassemia, but also to inhibit tumor cells (Voest et al. (1993) Cell Prolif. 26: 77) . Desferrioxamine works by binding iron. Serious side effects are common when treating patients with this drug. Effects such as kidney failure (Cianciulli et al. (1992) Haematologica 77: 514). Iron-binding peptides that should not be toxic are now determined by incubating radioactive iron 55 with the peptide bank and determining the best-binding ones using a phosphoimager (Molecular Dynamics) and, as described, continue to iteratively to at least be determined for the hepta peptide. The biological function can, as described in Voest et al. and Cianciulli et al. described. Iron-binding peptides may still be used to treat malaria (Loyevsky (1993) J. Clin. Invest. 91: 218). Iancet 341, 1088 L1 ciba-geigy

(IX) Peptides for tumor treatment:

Furthermore, an anti-colon tumor or anti-pancreatic tumor drug can be developed with the method. The oncogene product ras, a cellular GTP binding protein (M. Barbicid (1987) Ann. Rev Biochm. 56: 779), plays a major role in tumor development. Mutations in the ras protein lead to the transformation of intestinal or pancreatic cells (J. L Bos (1989) Cancer Res. 49: 4682). Potential anti-tumor agents are thus directed against the ras-induced transformation of certain cell types and thus directly against ras. Decisive for the function of ras is a post-translational modification, namely the attachment of a fatty acid (farnesyl) residue to the C-terminal cysteine of ras by a farnesyl transferase (J. B. Gibbs (1991) Cell 65: 1). The peptide banks can now be used to determine peptides that specifically bind the binding site of farnesayl transferase to ras and thus block farnesylation. For this purpose, ras is incubated in labeled form with the banks, and ras-binding peptides determined in this way are examined in an in vitro system for blocking ras (J. F. Hancock et al. (1989) Cell 57: 1167). The inhibition of the growth of L731 rat cells in soft agar in the presence of the peptides is examined (S. Powers et al. (1986) Cell 47, 413). If a peptide found has an antagonistic effect, it prevents the formation of cell clusters.

(X) peptides against lupus erythematosis:

Furthermore, the method can be developed into a medicament which is suitable for the treatment of severe human autoimmune deficiency lupus erythematosus (LE). The cell surface protein MHC I, which occurs on almost all somatic tissue types, appears to be an essential one Role play (Singer and Maguire (1990) Crit. Rev Immun. 10: 235). With this method, a peptide can be found that specifically blocks MHC I and thus prevents the autoimmune response. The radioactive or fluorescence-labeled MHC protein is incubated with the peptide bank and, as described, MHC-binding peptides are selected. In an in vivo model it can now be checked whether the peptides found suppress the autoimmune response. In this model, mice are immunized with an antibody of the idiotype 6/61 d. These then develop symptoms similar to LE (Mozes et al. (1993) Science 261: 91). Peptides determined in this way, which act as LE antagonists, should suppress this reaction.

(XI) peptides against wound infection:

Furthermore, a drug for the treatment of wound infections can be found with this method. Staphylococcus aureus (S. aureus) plays a crucial role here as a pathogenic germ. The partially life-threatening infections are caused by the protein alpha toxin from S. aureus. (Bhakdi and Tranum-Jensen (1987) Rev. Physiol. Biochem. Pharm. 107: 147) Antibodies against α-toxin have already been successfully tested in the treatment of wounds, particularly burns (Dodd and Stutman (1991) Adv. Paed. Inf Disease 6: 137). α-toxin antagonists are determined by incubating labeled α-toxin with the peptide bank and examining binding peptides for their antagonistic properties in an in vitro model. The activity or the inhibition of the activity is detected using a hemolysis test on rabbit erythricytes. (Füssle et al. (1981) J. Cell Biol. 91: 83) Peptides that have an antagonistic effect block the destruction of the cell membrane of the rabbit cell.

(XII) Peptides Against Lung Cell Carcinoma:

Furthermore, the method can be used to develop a medicament for the treatment of small lucent carcinoma (SCLC) (Corps et al. (1985) Biochemistry 213: 781). The proteins bombe and the "gastrine releasing peptide (GRP)" play an important role here. A drug for the treatment of SCLC should now be either a Bombesin or GRP antagonist or a Bombesin or GRP rezeoptor antagonist. The labeled proteins bombesin and GRP or their antagonists described are labeled with the libraries and the best-binding peptides in an in vitro model tested whether they are able to inhibit bobesin-induced growth of fibroblast cell or bronchial epithelium (Cuttitta et al. (1985) Nature 316: 828).

(XIII) peptides against asthma

The method can also be used to develop a medication for the treatment of asthma. The "vaso active intestinal peptide (VIP") plays an important role here. VIP or derivatives are suitable for the treatment of asthma (Bolin et al. (1993) Int. J. Peptide Protein Res. 41: 124). VIP agoists determined by incubating the VIP receptor with the peptide bank as described, identifying VIP receptor-binding peptides and testing their biological effectiveness. VIP agonists should have bronchodilatory properties (O'Doπnell et al. (1991) J. Biol. Chem. 266: 6389).

(XIV) peptides against tumors and infectious diseases:

Furthermore, the method can be developed into a medicament which is suitable for general stimulation of the immune system and thus for the general treatment of tumors and chronic infectious diseases. The lymphocyte protein CD3 plays an important role here. Antikör¬ perfragmente against CD3 are capable of the immune system in vivo ^'be capitalized ren (Wedrychowsky et al (1993) Bio / Technology. 11: 486). The binding of the antibody fragment to CD3 is a prerequisite. With the described method, peptides or derivatives can be found that specifically bind CD3. Labeled CD3 protein is in turn incubated with the peptide bank and the best binding peptides are tested in vitro for their immunostimulatory effect. Peptides that have an immunostimulatory effect should, like the antibody fragments, be able to accelerate the growth of certain cell types (VanWauwe et al. (1980) J. Immuno. 124: 2708).

The method according to the invention can also be used in environmental analysis. Here, the method offers the possibility of decisively improving on-site analysis of environmental pollutants by using peptides to identify multiple substances using multisensors. For every pollutant peptides can be identified as specifically binding substances, which then allow clear detection in suitable biosensors. Rapid analysis in the event of an accident can help to minimize damage to the affected population.

Examples:

The method according to the invention can be used to find substances which are suitable for the treatment of septic shock, cachexia and arthritis. A major mediator of these diseases is the tumor necrosis factor alpha (TNFa, for an overview of the modes of action of the TNF see A.G. Porter (1991), TIBTECH 9: 158). Such a substance, namely a TNFa antagonist, must specifically bind to the site of the molecule which is normally responsible for binding to the two TNF receptors sp55 and sp75 (L.A. Tartaglia and DV Goeddel (1992), Immunology Today, 13: 151). is responsible.

The antagonistic properties of the peptides obtained with this method are determined in the following way: A cell culture system is used to test whether these peptides are able to protect certain cell types from the cytotoxic effect of TNF (CJ Galloway et al. ( 1991), J. Immun. Meth. 140: 37). For this purpose, TNF is added alone and in the presence of the peptides to a cell culture that contains TNF sensitive cells. When TNF is added alone or in the presence of inactive peptides, the cells die within a day. If the peptides act as antagonists, the cells survive. The effect of the peptides can be quantified using calorimetric methods (L.M. Green et al. (1984), J. Immun, Meth. 70: 257), because only living cells can be stained with tetrazolium dye.

The description of the examples clarifies that the process according to the invention makes it possible to find functional TNF antagonists with the aid of the peptide bank according to the invention.

1. Material

1.1. amino acids

There are Fmoc-protected amino acids from Novabiochem, Cambridge

Research Biochemicals and the company BACHEM used. Some of the amino acids are already activated as PfP or Dhbt esters, some are activated in vitro with TBTU or DIC / NMI. The active esters of the amino acids are called Solution in NMP stored at -20 ° C with the exception of the active ester of arginine, which must always be prepared again.

1.2. cellulose

Hardened cellulose (Whatman 540) is used.

1.3. ¹²⁵ l-labeled TNFα

The ¹²⁵ l-labeled TNFα has a specific activity of about 650 Ci / mmol and is obtained from the Amersham company.

2. Methods

2.1. Modification of cellulose

The cellulose membrane is chemically modified in order to enable the subsequent peptide synthesis at the peptide coupling site and to facilitate the subsequent selection process. A spacer is inserted between the carrier material and the peptide sequence to be synthesized. This spacer serves to ensure the free accessibility of the immobilized peptides. In a first step, the entire membrane is incubated with a β-alanine solution, so that an esterification with the OH groups of the cellulose results in a uniform distribution of β-alanine.

The process consists of the following steps:

Activate 0.2 mol / l Fmoc-ß-alanine with 0.24 mol / l DIC and 0.4 mol / l NMI (in DMF) and incubate for 3 hours with the cellulose

Membrane, wash three times with DMF,

Removal of the Fmoc protective group of the esterified β-alanine by incubation with 20% piperidine in DMF for 20 minutes, washing five times with DMF, washing twice with methanol and drying. In a second step, a second β-alanine is dripped onto defined points on the membrane (reaction sites or "spots"), so that a second β-alanine is coupled there. This results in a certain number of peptide coupling sites per reaction site. The points are marked beforehand with a pencil on the back of the membrane. All remaining amino functions of the membrane are blocked by acetylation and Fmoc is cleaved off again with piperidine. The free amino groups are now made visible by staining with BPB and appear as blue distinguishable spots. This procedure has the following steps: - 0.5 μl Fmoc ß-alanine (0.3 mol / l), TBTU (0.3 mol / l 1: 1 in

NMP, incubate twice with 2% acetic anhydride in DMF for 3 minutes, incubate once in 2% acetic anhydride plus 1% disopropylethylamine in DMF for 30 minutes, wash three times with DMF,

Incubate in 20% piperidine in DMF for 15 minutes, wash five times with DMF,

Stain with 0.01% BPB in DMF for five minutes and

dry

2.2 Determination of the functionality of the spots ("spots")

In order to achieve statistical incorporation of all amino acids when coupling with the amino acid mixture, coupling is carried out in a ratio of 0.95 to 1 (sum of all amino acids in the mixture to the peptide coupling sites of the individual spot). Therefore, the number of free amino functions per "spot" must first be determined. For this purpose, a defined area of the membrane coupled twice with β-alanine is cut out and stained with 0.05% BPB in DMF. After washing three times with methanol and drying the membrane piece, it is decolorized again with 20% piperidine in DMF and the colored solution is measured in a photometer at 605 nm. Assuming that a BPB molecule is bound per NH ₂ group and with the extinction coefficient ε = 95,000 mol ^{"1 •} I ^• cm ^-1 , the number of free amino functions can be determined using the Lambert-Beers law per "spot". In general, this results in a value of approximately 60 nmol amino functions per "spot" (reactants per peptide coupling sites).

2.3. Coupling of the amino acids At positions 1, 2, 4 and 6 of the hexapeptide to be synthesized (direction of synthesis from the carboxyl to the amino residue) an equimariamic amino acid mixture of all amino acids except tryptophan, methionine and cysteine is used in a ratio of 0.95 to 1 ( Sum of all amino acids in the mixture to peptide coupling sites at the respective "spot"). D-leucine is used instead of L-leucine. With the above-mentioned functionality of the membrane of about 60 nmol peptide coupling sites per "spot", the concentration of the amino acid solution is 50 nmol / l.

At positions 3 and 5 (direction of synthesis as before), the defined amino acid required to create a combinatorial peptide library (peptide library) is coupled at a concentration of 0.3 mol / l at least twice in succession in order to ensure complete acetylation.

The Fmoc protective group is split off with piperidine between the individual couplings.

The process comprises the following steps:

Drop 1 μl amino acid or amino acid mixture onto each "spot",

Reaction over 15 minutes,

Repeat: once with defined amino acids and twice with the mixture, wash three times with DMF, incubate with 20% piperidine in DMF for 15 minutes, wash five times with DMF, wash twice with methanol and dry and carry out the next couplings analogously for the next building block . 2.4. Acetylation of the N-terminus and cleavage of the side protecting groups

When all couplings have been carried out, the N-terminus is acetylated after the last Fmoc protective group has been split off in order to protect the peptides from protease degradation. Then the side protection groups are split off. The following process steps are available: incubate twice with 2% acetic anhydride in DMF for five minutes, wash three times with DMF, wash twice with methanol,

Dry,

Incubate with 50% TFA, 3% triisobytylsilane, 2% water, 1% phenol in DCM for three hours, wash four times with DCM, wash three times with DMF, wash twice with methanol and dry.

2.5. Block the membrane

Before incubation with TNF, the free binding sites on the membrane are blocked by incubation for 1 hour with 1% BSA, 0.05% TweenδO (detergent) in PBS at room temperature.

2.6. Incubation with TNFα

The membrane is incubated for five hours at room temperature with 20 μCi TNFα in 40 ml 1% BSA, 0.05% TweenδO in PBS. After washing five times with PBS, the dried membrane is exposed to an X-ray film or a coated plate of a phosphoimager is exposed. 3. Results

3.1. First synthesis and selection process of the combinatorial peptide library (peptide library) with TNFα

Among other things, the first synthesis and selection process becomes a well-binding sequence

NH ₂ -Xi ^• X2 ^• ^ 3 ^• 4 ^■ ₅ ^• X ₆ -COOH determined. X stands for a mixture of D-leucine and the natural, randomly coupled amino acids apart from L-leucine, tryptophan, methionine and cysteine. F stands for phenylalanine according to the international code. The index indicates the position in the sequence of the hexapeptide, the counting beginning with the N-terminus.

3.2. Second synthesis and selection process of the combinatorial peptide bank with TNFα

In the second synthesis step, position 4 of the sequence is occupied by a defined amino acid. This results in 20 different approaches with respect to position 4. The aforementioned selection process is also used here.

It is called the best binding sequence

NH ₂ -X1 ^• X2 ^• F3 ^• R ₄ ^• F ₅ ^• X ₆ -COOH found.

3.3. Third method of synthesis and selection of the combinatorial peptide bank with TNFα

The third synthesis proceeds as described under 3.2, but in this case position 6 is occupied by a defined amino acid.

It is ^■ R ₃ ^• R found as best-binding sequence NH ₂ -X 1 X ₂ ^• ₄ ^• R ₅ ^• R ₆ -COOH. 50811 AWOM1 XX00 + P 09.03.94 BR Abbreviations:

BPB bromophenol blue

BSA bovine serum albumine (bovine serum albumine)

DCM dichloromethane DIC diisopropylcarbodiimide

Dhbt 3-hydroxy-2,3-dihydro-4-oxo-bezotriazine

DMF dimethylformamide

Fmoc 9-Fiorenylmethoxycarbonyl

NMI N-methylimidazole PBS phosphate buffered saline

PfP pentafluorophenyl

TFA trifluoroacetic acid

TNFα tumor necrosis factor α

SEQUENCE LOG:

Voluntary, since sequences containing D-leucine

(1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: SCHERING AKTIENGESELLSCHAFT

(B) STREET: MÜLLERSTRASSE 178

(C) LOCATION: 13353 BERLIN

(E) COUNTRY: GERMANY

(F) POSTAL NUMBER: 13353 (ii) DESCRIPTION OF THE INVENTION: Methods of Synthesis and

Selection of sequences from covalently linked building blocks (iii) NUMBER OF SEQUENCES: 3 sequence protocols (iv) COMPUTER READABLE VERSION (A) DATA CARRIER: DISKETTE

(B) COMPUTER: 486 / INTEL

(C) OPERATING SYSTEM: WINDOWS

(D) SOFTWARE: WINWORD; (v) DATE OF REGISTRATION:

(2) INFORMATION ON SEQ ID NO: 1: (i) SEQUENCE IDENTIFIER SEQUENCE LENGTH: 6 amino acids TYPE OF SEQUENCE: amino acid sequence TOPOLOGY: linear

MOLECULE TYPE: synthetic peptide CHARACTERISTIC: binding to TNF

SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Xaa Xaa Phe Xaa Phe Xaa

Xaa = L-Ala; L-Arg; L-Asn; L-Asp; L-Gln; L-Glu; L-Gly; L-His; L-Ile; D-Leu; L-Lys; L-Phe; L-Pro; L-Ser; L-Thr L-Tyr and L-Val. (2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL

SEQUENCE LENGTH: 6 amino acids

TYPE OF SEQUENCE: amino acid sequence TOPOLOGY: linear

MOLECULE TYPE: synthetic peptide

CHARACTERISTIC: Binding to TNF

SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Xaa Xaa Phe Arg Phe Xaa

Xaa = L-Ala; L-Arg; L-Asn; L-Asp; L-Gln; L-Glu; L-Gly; L-His; L-Ile; D-Leu; L-Lys; L-Phe; L-Pro; L-Ser; L-Thr L-Tyr and L-Val.

(2) INFORMATION ON SEQ ID NO: 3: (i) SEQUENCE IDENTIFIER SEQUENCE LENGTH: 6 amino acids TYPE OF SEQUENCE: amino acid sequence TOPOLOGY: linear

MOLECULE TYPE: synthetic peptide CHARACTERISTIC: binding to TNF

SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Xaa Xaa Phe Arg Phe Arg

Xaa = L-Ala; L-Arg; L-Asn; L-Asp; L-Gln; L-Glu; L-Gly; L-His; L-Ile; D-Leu; L-Lys; L-Phe; L-Pro; L-Ser; L-Thr L-Tyr and L-Val.

Claims

Claims

1. Method for producing peptide libraries from peptides consisting of the features: the peptides have either a specific or an undetermined amino acid per position in the peptide sequence; the peptides are produced on or in a carrier material after the solid phase synthesis or in separate reaction vessels according to the liquid phase synthesis; spatially separate reaction sites are located on or in the support material or in the reaction vessels; each reaction site has (i) a defined number of peptide

Coupling sites for the binding of the activated amino acids for the reaction and

(ii) in liquid phase synthesis, a defined number of start amino acids or start peptides (peptide coupling sites); The method consists of the following steps, each with its own procedure for the synthesis of the particular amino acid and the undetermined amino acid: Coupling points in excess; If an activated indefinite amino acid is reacted at the intended position of the peptide sequence, this indefinite amino acid is present in a mixture of amino acids, the sum of all indefinite amino acids in the mixture being at most equimolar to the peptide coupling sites , and after the coupling reaction another amino acid is added for saturation.

2. Process for the production of saccharide libraries from saccharides consisting of the features: the saccharides have either a specific or an undetermined monosaccharide per position in the saccharide sequence; the saccharides are produced on or in a carrier material after the solid phase synthesis 5 or in separate reaction vessels after the liquid phase synthesis; spatially separate reaction sites are located on or in the support material or in the reaction vessels; o each reaction site has

(i) during the solid phase synthesis defined number of saccharide coupling sites for the binding of the monosaccharides activated for the reaction and

(ii) in liquid phase synthesis, a defined number of start 5 monosaccharides or start saccharides (saccharide coupling sites); The method consists of the following steps, each with its own procedure for the synthesis of the particular monosaccharide and the non-specific monosaccharide: 0 an activated particular monosaccharide on the intended

When the position of the saccharide sequence is reacted, this particular monosaccharide is in excess of the saccharide coupling sites; If an activated indefinite monosaccharide is reacted at the intended position of the 5 saccharide sequence, this indefinite monosaccharide is present in a mixture of monosaccharides, the sum of all indefinite monosaccharides in the mixture being at most equimolar to the saccharide coupling sites, and 0 after the coupling reaction another monosaccharide is used

Saturation added.

3. The method according to claim 1 or 2, wherein the peptide sequence or saccharide sequence (sequence) has three specific amino acids or three specific 5 monosaccharides each at a specific position in the sequence, by three specific amino acids or three specific monosaccharides during synthesis can be synthesized at a specific position in the sequence.

4. The method according to any one of the preceding claims, wherein the sequence is branched or unbranched.

5. The method according to any one of the preceding claims, wherein the carrier material consists of cellulose.

6. The method according to claim 1, 3 to 5 (with reference to claim 1), wherein the amino acid (i) is a natural amino acid or (ii) is a derivative of the natural amino acid.

7. The method according to claim 6, wherein the peptide coupling sites consist of alanine residues which are covalently linked to the carrier material.

8. The method according to claim 7, wherein the alanine residues are connected to the cellulose via an ester bond.

9. The method according to any one of claims 2 and 3 to 5 (with reference to claim 2), wherein the monosaccharides are a cyclic pentose or hexose or their derivatives.

10. A method for the synthesis and selection of peptides or saccharides according to one of the preceding claims, wherein after the synthesis of the sequence a selection of the desired sequences is carried out with the aid of binding tests or specific reactions, the selected sequences optionally being represented by indicators .

1 1. The method of claim 10, wherein the binding test consists in adhering labeled test substances and visualization of the label.

12. The method according to claim 11, wherein the label consists of radioactive material or of biotin or of covalently linked enzymes.

13. The method of claim 10, wherein the reactions are measured by allosteric enzymes.

.

14. The method of claim 10, wherein the response of a cell system is measured.

15. The method of claim 10, wherein the reaction consists of a cell-cell interaction.

16. Unbranched or branched peptide from D- or L-amino acids and / or amino acid derivatives, which peptide has a structure using the aforementioned synthesis and selection method according to one of the preceding claims 10 to 15 with reference to claims 6 until 8 is available.

17. A method for peptide synthesis of unbranched or branched peptides from D- or L-amino acids and / or amino acid derivatives, the sequence having the peptide structure being synthesized using the synthesis and selection methods according to one of the previous claims 10 to 15 can be obtained with reference to claims 6 to 8.