DE4308410A1 - Process for the synthesis and selection of sequences composed of covalently linked building blocks - Google Patents

Abstract

The invention relates to a process for the synthesis and selection of defined sequences of building blocks with the aid of a solid-phase synthesis, where building block coupling sites are predefined on support material. The sequences consist of defined building blocks and variable building blocks at defined positions in the sequence. The process comprises the following steps: (i) continuous synthesis of the sequence, (aa) where the defined building block is added at a defined site on the support material, and (bb) where a mixture of variable building blocks is added to the support material, (ii) selection of the desired sequences with the aid of binding assays or specific reactions.

Description

The invention relates to a method for the synthesis and selection of Se sequences from covalently linked building blocks for the determination of sequences Zen; continue to use methods for producing sequences of the synthesis and selection process, and peptides, their structure using the synthesis and selection process rens were determined.

State of the art

The development and research of new drugs by Organi chemists using traditional methods is based on the Synthesis of a large number of substances. The previously known Selek tionation processes are labor-intensive and have only one per time unit limited variety of substances. The theory wins more and more The importance of chemical substance design, when structuring with computer models molecules. However, this method is not yet sufficiently mature, so that classic selection procedures still are always of great value.

Different approaches have been taken to manufacture substances and to select.

A major breakthrough in this process was the peptide Bank (peptide library) achieved on phage surfaces (J. SCOTT and G.P. SMITH, (1990) Science 249: 386). The positi in the selection process Ven phages are analyzed for their DNA, which means that those on the upper Area expressed amino acid structure is determined. The disadvantage is that the  Phages in host cells must be propagated and that the selection must be suitable to detect the peptides on the cell surface nen. The peptide to be selected is a peptide on the cells as well many other expressed proteins. This selection has one systemic uncertainty. Furthermore, only the twenty natural Chen amino acids in the process for the production and selection of desired sequences can be used.

In another known method, spherical carrier materials are used Used on which the peptides are synthesized. (K.S. LAM et al. (1991) Nature 354: 82). Twenty approaches are made, one approach per Amino acid. After each synthesis step, the balls are mixed and again divided into twenty reaction vessels. When selecting the positive spheres are determined and the sequence of the peptides on them certainly. The disadvantage of this method is the cumbersome handling of synthesis. The selection of positive spheres is difficult, one Sequence analysis is necessary. The material present in a sphere is very low in quantity.

A variant of the previously described method is described in the publication HOUGHTEN et al. (1991) Natur 354: 84. A com binatory library. For example, two positions in the amino acid sequence. The rest of the pairing These approaches must be kept separate from each other. Although own these approaches defined building blocks in certain positions and this is also Block known, however, this method requires a large number of parallel to treating reaction vessels. It only becomes one with each reaction certain amino acid coupled. Cysteine is not used as a building block for Er position of the sequences used.

Another method is that on a solid planar support Block coupling points are arranged, which with the help of light energy Reaction can be stimulated with another building block. The light energy can come from a laser that illuminates specific points. Exactly it is possible to use pinholes, which are also targeted Allow exposure of individual points. (P.A. FODOR et al. (1991) Science 251: 767) A disadvantage of this method is that a high techni effort is required. So a laser or device must be targeted  Exposure of some defined reaction sites may be present. Also are the reaction mechanisms are limited because a photoreaction is essential this method must be applied.

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

Once the sequence sought has been determined, the synthesis of peptides is easy to do. A summary of these techniques can be found at J.M. STEWART and J.D. YOUNG, San Francisco, 1969, and J. MEIERHO- FER, Hormonal Proteins and Peptides, vol. 2 p. 46 Academic Press (New York), 1973 for solid phase method and E. SCHRODER and K. LUBKE, The Peptides, Vol. 1, Academic Press (New York) 1965 for the liquid phase me method can be read. The steps of the synthesis are in the publication EP 0 097 031. The general procedural steps from the euro publication can be analogously applied to the synthesis of almost all peptide Transfer connections.

task

It is an object of the invention to offer a method with which Se sequences are to be produced and selected, the sequences being agreed to have desired properties, the desired egg properties from the specific recognition of predetermined molecules, for example receptor molecules. The obtained after this procedure sequences should then serve as a template for molecules (pharmacological Lead structures) are used, which are manufactured in the classic chemical way become.  

solution

The invention is achieved by a process for the synthesis and selection of functionally determined sequences from covalently linked building blocks,
which have different reaction kinetics in the synthesis and which are reactive building blocks before the synthesis,
with the help of a solid phase synthesis,
where on or in the carrier material on locations with building block coupling points are specified,
being the sequence
has at least one certain building block at a certain position in the sequence and
has at least one variable building block, which is randomly selected from a mixture, at a likewise determined position in the sequence,
the process consists of the following steps:

• (i) continuously synthesizing the sequence, each have their own procedure for the synthesis of the specific building blocks and showing the variable building blocks,
• (aa) where,
  if a certain building block is to be synthesized at a certain position,

the reactive, specific building block is added at at least one defined application location on or in the carrier material,
the certain modules are in excess compared to the module coupling points, and

• (bb) where,
  if a variable building block is to be synthesized at a specific position in the sequence,

a mixture of reactive, variable building blocks is added at all application locations on or in the carrier material,
The sum of the variable building blocks is at most a fourfold excess compared to the building block coupling points, and further building blocks are added after the coupling reaction,

• (ii) selection of the desired sequences with the aid of binding tests or specific reactions, giving the functionally determined sequences otherwise represented by indicators.

In the case of the mixture, the total of the variable building blocks is greater than that Block coupling points at most in a 2-fold excess, more be preferably at most in a 1.5-fold excess, more preferably at most at least in a 1.2-fold excess, and most preferably at most in a 0.94-fold excess.

Advantages of the method according to the invention

One of the advantages of this method according to the invention is that in structures of peptide ligands, for example, can be determined in a very short time that are specific to, for example, the extracellular domains of Re can bind receptors. With the help of this random principle, all theories lie conceivable structures during the step-by-step selection. You can use the process protocol for each block coupling point le can be determined.

Simply by determining which building blocks in the sequence order First determine and which are variable, the results can be found under affect differently. Otherwise, the process stands out by from that the peptides to be obtained despite the random process in the certain building blocks by separate process approaches are workable.

This invention has been made possible by reaction conditions which occur in the Synthesis of the variable building blocks is available. It is common to mix the new to be synthesized building blocks in total in molar excess ge compared to the module coupling points, otherwise not all Se quenz chains react. One is not another building block extended sequence leads to defects in the sequence.

Furthermore, there is a molar excess of the amino acid to be coupled (Acylation reagent) necessary because the coupling kinetics of the individual Ami no acids is different, but the coupling yield for one be  agreed coupling step and never predict a particular amino acid leaves. A clear molar excess circumvents this problem. Both Peptide chemists are two routine routine with eight-fold excesses. A is common fourfold surplus. This is the excess of Coupling a specific amino acid. The offer of mixtures is has not been mentioned so far. By coupling a complex Ami nosic acid mixture in molar deficit can solve the problem of under different kinetics for individual amino acids can be avoided. Since this in In relation to the amine component in the deficit, all Ami react essentially to quantitative acids. By at least one further coupling in the same molar ratio becomes the amine component then fully implemented on the carrier. So there is in the specialist circles Prejudice that reactions without excess of the new building blocks to be coupled cannot run successfully.

Contrary to the prevailing prejudices in the inventive Process the variable building blocks not in significant excess, preferred offered at most equimolar. This will make almost all variable construction stones react equimolar with the sequence. The individual Building blocks due to their different kinetics at different times reacted 99%, but practically all activated amino acids react.

Other embodiments

A method according to the invention is preferred in which the variable construction stones in their total compared to the module coupling points at most are equimolar. If the expert uses higher concentrations, he will still get results, but no longer statistical Distribution. A deterioration in the process is then in Taken purchase. This method works on the statistical distribution It is best if the sum of all the variable modules offered is at most equimolar. If the equimolar range is clearly undershot, only some of the synthesis chains are coupled with the variable building blocks, others remain without a reaction partner.

There is a preferred embodiment of the method according to the invention in that the sequence has three specific building blocks at a specific position  tion of the sequence has three specific building blocks in the synthesis can be synthesized in a specific position. The affinity of attachment molecules increase exponentially to the number of defined building blocks. Three defined modules allow a better pre-selection of the whole sequence as the use of only two defined building blocks. Three Defined building blocks can also be on an essentially planar support Apply the germ material without any problems. If for example peptide sequences should be created, then there are 8000 block coupling points, that are still easy to handle on a cellulose carrier. The use of three defined building blocks is a sensible compromise for peptides between the high number of building block coupling sites and binding capacities the peptides.

The sequence can also have four or five specific building blocks on one each possess specific position of the sequence by in the synthesis corresponding to four or five specific building blocks on a specific one Position to be synthesized.

Another embodiment of the method according to the invention exists in that the sequence is branched or unbranched. Natural sequences zen of amino acids are unbranched. Such linear molecules are therefore very well suited to attach to other structures and conformation to effect changes. However, the inventive method is up linear synthesis is not restricted, 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 invention Procedure. Cyclic versions are also possible, which conform mation stability of the molecule increase.

It is advantageous if the building blocks D- or L-amino acids and / or their Are derivatives. In addition to the twenty L-amino acids, there is a large number of amino acid derivatives, which are listed, for example, in the BACHEM catalog, Bubendorf, Basellandschaft, Switzerland. Such peptides, which contain modified amino acids often place a different type Pharmacokinetics to the day. They behave differently in the organism the peptides, which consist exclusively of natural amino acids. they are more stable to proteases, they partially penetrate the cell membrane  worse, which makes the synthetic peptide used as a drug for the action in the extracellular region is more suitable than a sequence from L- Is amino acids.

Various substances can serve as carrier material, particularly suitable net is the carrier material made of cellulose. It has the advantage that one for the Synthesis of more usable supports is available, on which the building blocks and their easily Mixtures can be applied. The flat surface and the high one Absorption makes handling easier.

Also suitable as a 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 ₁ * X ₂ * B ₃ * X ₄ * B ₅ * X ₆ [X = amino acid with random distribution; B = specific amino acid]) of the peptide bank (peptides library) 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.

Another embodiment of the invention consists in that the building block Coupling sites are β-alanine residues that ver covalently ver with the carrier material are bound. β-alanine is bound to the cellulose via an ester bond material coupled. It is a defined number of module coupling points required to know what amounts of building blocks in each synthesis step must be offered. It is also possible to the Kopp to loosen the sequences from the support.

Glycine, other amino acid Carboxylic acids (except β-alanine), polyethylene glycol (RAPP et al. In Peptides 1988; Ed. G. JUNG, E. BAYER, Walter deGRUYTER, Berlin 1989, pages 199 - 201) and all linker groups for peptide synthesis, which he commercially are available and of which even the peptide is recovered after synthesis can be split off (see catalog of NOVABIOCHEM 93).  

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

Furthermore, the mixture of the D configurations of the natural Ami best acids that are present in essentially the same molar proportions hen. The building blocks can also be amino acid derivatives act. Such derivatives are, for example, in the BACHEM catalog, Bubendorf, Basel-Landschaft, Switzerland listed.

Another embodiment of the invention is that the building blocks Sugar building blocks are. Sugar building blocks are extensively in the literature wrote. (for example RÖMPPS, chemistry lexicon, ed. NEUMÜLLER, O.-A. (1983) 8th edition, pages 2147 to 2152) It is known that the various Sugar building blocks have different reaction kinetics. (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) This also makes a comparison problem as with the amino acid sequences. Both systems white but note some differences. Because of the high functional density the saccharides have a higher information content than the amino acids give. In contrast to peptide chemistry, the linkage possibilities are the sugar extremely complex. Theoretically, two monosaccharides can be used combine eleven disaccharides.

A method according to the invention is particularly preferred in which the construction stones are a cyclic pentose or hexose or their derivatives. Typi pentoses are described in the standard work RÖMPPS.

Biologically relevant monosaccharides are, for example, glucose, galactose, Mannose, N-acetylglucosamine, N-acetylgalactosamine or N-acetylneuramine acid.

The synthesis process of the specific building blocks is effective when the certain building blocks are at least in a triple excess. Good results can be expected in a range of three times extends up to eight times the excess.  

After the reaction of the variable blocks, there are some block coupling points len are not occupied by a variable module. But same molecule to achieve lengths, after the reaction of the variable building blocks, the others Blocks at least added in a two-fold excess.

The advantage of the process according to the invention is that after the synthesis of the Se known and proven indicator methods can to determine which sequences the Eigen to be selected own properties.

It is advantageous if the binding test is in a sticky labeled test sub punching and visualization of the marking.

Proven and therefore preferred methods are that the marking from radioactive material or from biotin or from covalently linked ene Zymen exists. Another method is to measure fluorescence or the luminescence.

The method according to the invention includes the detection of binding mo in particular, also read the molecules that trigger a function. This happens that the reaction by, for example, allosteric En zyme is measured. 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 would have to e.g. B. be fluorescent labeled. Then the individual building blocks Coupling sites are cut out, treated with protease and the release of fragments can be measured photometrically. Inhibitors can be found in that certain sequences to which the Protease binds to, but cannot cleave the protease the module coupling points can be identified.

It is also possible to measure the response of a cell system. This also includes all bindings to receptors. So one can Variety of receptors with regard to their binding ability to the Test the synthesized ligand.

In addition to the receptors, there are also metal atoms for the selection process suitable, such as technetium, gadolinium, nickel and calcium.  

Technetium complexing peptides can be used, for example, to diagnose tumors ka can be used. Nickel complexing sequences can work as be used for protein purification over nickel columns. Organic chemical molecules should also be able to be bound by peptides. Pepetide, the transition analogues of certain organic chemical reactions bind, may possibly form the precursors of novel catalysts.

Even more complex interactions have to be measured, the reaction from one Cell-cell interaction can exist.

In addition to the method according to the invention, sequences are also used speaks, namely unbranched or branched peptides from D- or L-Ami noacids and / or amino acid derivatives, which peptide has a structure, those using the aforementioned synthesis and Selection procedure was obtained. If using the fiction According to the procedure, a sequence has been determined, so it is for the subject a light one, due to this sequence for example the peptide synthe to be able to produce a table. The technique of peptide synthesis is already before have been described in detail. The invention thus provides a technical Teaching that reveals the way to sequence. The sequence determined in this way can then prepared on the basis of the standard knowledge of the person skilled in the art become. With the help of this teaching, sequences can be found, the desired one have specific functions.

The invention further comprises a method for peptide synthesis of un branched or branched peptides from D- or L-amino acids and / or Ami nosacid derivatives, the sequence being synthesized that the peptide structure has that using the synthesis and selection according to the invention nation process was obtained.

Definitions

BUILDING BLOCKS: The building blocks are chemical compounds that form chains or branching are suitable.

The building blocks preferably belong to a group of chemical sub punch, for example to the amino acids and their derivatives. The Ami  noacids 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 are. Sequence chains over side chains are also possible, e.g. B. y-glutamine.

All reactive side chains can be used for the synthesis of branched peptide banks (peptide library) can be used, such as lysine. About the Implementation of carboxylic acids such as glutamic acid and aspartic acid can Amino groups are implemented, such as argingine with dions.

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

Sugar or sugar derivatives are also conceivable as building blocks.

REAGIBLE BLOCK: A brick that is an activated functional group with whom he sits on existing modules or module coupling points len can bind. Furthermore, he must have such protective groups that one Reaction of the desired functional groups allowed, but not the ge wished shields.

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.

CARRIER MATERIALS: Various carrier materials are in the Standard literature described. Are particularly suitable in this process all essentially planar carriers that are easy to apply to the reactive Blocks enabled. Cellulose is particularly suitable. Have here WHATMAN materials, type: Whatman paper 540 (hardened Cellulose) was found to be very useful.

LOCATION: To make the experiment meaningful, the one or two-dimensional or possibly three-dimensional definition of Places on or in the carrier material required to avoid that the  Building blocks at the corresponding application locations become uncontrolled with the Can mix blocks of other application locations.

MODULE COUPLING POINTS: Not all carriers are suitable, the desired ones to be able to couple th building blocks. Furthermore, the building blocks after the second synthesis cycle can no longer bind to the support material. It is therefore useful to have so-called anchor moles on or in the carrier material to fix the cooler. In cellulose, for example, β-alanine is suitable for this.

Examples 1. Material 1.1. amino acids

There are Fmoc-protected amino acids from Novabiochem, Cambridge Research Biochemicals and the company BACHEM used. The amino acids some are already activated as PfP or Dhbt esters, some are in vitro activated with TBTU or DIC / NMI. The active esters of the amino acids are called Solution in NMP at -20 ° C with the exception of the arginine active ester maintains that always has to be manufactured 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 to link to the building block to enable the subsequent peptide synthesis and that to facilitate closing selection procedures. It is between a distance between the carrier material and the peptide sequence to be synthesized Holder (spacer) inserted. This spacer is used to free the To ensure the mobility 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 cellulose is the same moderate distribution of β-alanine results.  

The process consists of the following steps:

• - Activation of 0.2 mol / l Fmoc-β-alanine with 0.24 mol / l DIC and 0.4 mol / l NMI (in DMF) and 3 hours incubation with the cellulose Membrane,
• - washing three times with DMF,
• - Cleavage 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,
• - Wash twice with methanol and
• - Dry.

In a second step, a second β-alanine is added to defined points on the Dripped onto the membrane so that a second β-alanine is coupled there. The Points are marked beforehand with a pencil on the back of the membrane kiert. All remaining amino functions of the membrane are by acetylation blocked and Fmoc is split off again with piperidine. The free amino Groups are now made visible and appear by dyeing with BPB as blue distinguishable spots.

This procedure has the following steps:

• - 0.5 µl Fmoc β-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% disopropylethyla min in DMF for 30 minutes,
• - washing three times with DMF,
• Incubate in 20% piperidine in DMF for 15 minutes,
• - washing five times with DMF,
• - Staining 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 a statistical incorporation of all amino acids when coupling with the amino acid mixture, coupling is carried out at least twice, usually three times in a ratio of 0.8 to 1 (sum of all amino acids in the mixture to building block coupling points 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 saturated 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 · | · 1 cm ^-1 , the number of free amino functions per “spot” can be determined using the Lambert-Beer law will. In general, there is a value of about 60 nmol amino functions per "spot" (reactants per module 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 carboxyl to amino radical) becomes an equimolar amino mixture of all amino acids except tryptophan, methionine and Cysteine used in a ratio of 0.8 to 1 (sum of all amino acids in Ge mix to block coupling points at the respective "spot"). In the above ge called functionality of the membrane of about 60 nmol building block coupling len per "spot" is the concentration of the amino acid solution 50 nmol / l. To one To ensure complete acylation must be coupled at least twice become.

At positions 3 and 5 (direction of synthesis as before) is used for creation a combinatorial peptide library (peptide library) necessary defi nated amino acid in a concentration of 0.3 mol / l at least twice sequentially coupled to ensure complete acetylation.

Between the individual couplings the Fmoc protecting group with piperidine cleaved.

The process comprises the following steps:

• - Drop 1 µl of amino acid or amino acid mixture on every "spot",
• Reaction over 15 minutes,
• - Repeat: once for defined amino acids and twice with the mixture,
• - washing three times with DMF, Incubate with 20% piperidine in DMF for 15 minutes,  
• - washing five times with DMF,
• - Wash twice with methanol and
• - drying and
• - for the next block Perform the next pairings in the same way.

2.4. Acetylation of the N-terminus and cleavage of the side protection groups

If all couplings have been made, after splitting off the last Fmoc Protecting group of the N-terminus acetylated to protect the peptides from protease degradation protect. 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,
• - washing three times with DMF,
• - washing twice with methanol,
• - Dry,
• - Incubate with 50% TFA, 3% triisobytylsilane, 2% water, 1% Phenol in DCM for three hours,
• - washing four times with DCM,
• - washing 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 mem bran by 1 hour incubation with 1% BSA, 0.05% Tween80 (Detergent) blocked in PBS at room temperature.

2.6. Incubation with TNFα

The membrane is five hours at room temperature with 20 uCi TNFα in 40 ml 1% BSA, 0.05% Tween80 incubated in PBS. After washing five times with PBS, the dried membrane is exposed to an X-ray film, or it becomes exposed a coated plate of a phosphoimager.  

3. Results 3.1. First combinatori synthesis and selection process peptide library with TNFα

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

NH ₂ -X ₁ · X ₂ · F ₃ · X ₄ · F ₅ · X ₆ -COOH

determined. X stands for one of the natural, randomly ge coupled amino acids other than cysteine. F stands according to the international Code for phenylalanine. The index gives the position in the sequence of the Hexapeptides, where the counting begins with the N-terminus.

3.2. Second synthesis and selection process of the combination Roman peptide library with TNFα

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

It is called the best binding sequence

NH ₂ -X ₁ · X ₂ · F · ₃ R ₄ · F ₅ · X ₆ -COOH

found.

3.3. Third synthesis and selection process of the combinatorial peptide bank with TNF _α

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

It is called the best binding sequence

NH ₂ -X ₁ · X ₂ · F · ₃ R ₄ · F · ₅ R ₆ -COOH

found.  

Abbreviations

BPB bromophenol blue
BSA bovine serum albumin (bovine serum albumine)
DCM dichloromethane
DIC diisopropyicarbodiimide
Dhbt 3-hydroxy-2,3-dihydro-4-oxo-bezotriazine
DMF dimethylformamide
Fmoc 9-fluorenylmethoxycarbonyl
NMI N-methylimidazole
PBS phosphate buffered saline
PfP pentafluorophenyl
TFA trifluoroacetic acid
TNFα tumor necrosis factor α

SEQUENCE LOG

SEQ.ID.NO: 1
Sequence type: amino acid sequence
Sequence length: 6 amino acids
Type of molecule: synthetic pepeptide

XaaXaaPheXaaPheXaa

SEQ.ID.NO: 2
Sequence type: amino acid sequence
Sequence length: 6 amino acids
Type of molecule: synthetic pepeptide

XaaXaaPheArgPheXaa

SEQ.ID.NO: 3
Sequence type: amino acid sequence
Sequence length: 6 amino acids
Type of molecule: synthetic pepeptide

XaaXaaPheArgPheArg

SEQUENCE LISTING IN ENGLISH LANGUAGE

SEQ.ID.NO: 1
Sequence Type: Aminoacid Sequence
Sequence length: 6 aminoacids
Molecule Type: Synthetically manufactured pepetide

XaaXaaPheXaaPheXaa

SEQ.ID.NO: 2
Sequence Type: Aminoacid Sequence
Sequence length: 6 aminoacids
Molecule Type: Synthetically manufactured pepetide

XaaXaaPheArgPheXaa

SEQ. ID. NO: 3
Sequence Type: Aminoacid Sequence
Sequence length: 6 aminoacids
Molecule Type: Synthetically manufactured pepetide

XaaXaaPheArgPheArg

Claims (23)

1. Process for the synthesis and selection of functionally determined sequences from covalently linked building blocks,
which have different reaction kinetics in the synthesis and which are reactive building blocks before the synthesis,
with the help of a solid phase synthesis,
where on or in the carrier material on locations with building block coupling points are specified,
being the sequence
has at least one certain building block at a certain position in the sequence and
has at least one variable building block, which is randomly selected from a mixture, at a likewise determined position in the sequence,
the process consists of the following steps:

• (i) continuous synthesis of the sequence, each with its own procedure for the synthesis of the specific building blocks and the variable building blocks,
• (aa) where,
  if a certain building block is to be synthesized at a certain position,

the reactive, specific building block is added at at least one defined application location on or in the carrier material,
the certain modules are in excess compared to the module coupling points, and

• (bb) where,
  if a variable building block is to be synthesized at a specific position in the sequence,

a mixture of reactive, variable building blocks is added at all application locations on or in the carrier material,
The sum of the variable building blocks is at most a fourfold excess compared to the building block coupling points, and further building blocks are added after the coupling reaction,

• (ii) Selection of the desired sequences with the aid of binding tests or specific reactions, the functionally determined sequences optionally being represented by indicators.

2. The method according to claim 1, wherein the variable building blocks in their Sum compared to the module coupling points are at most equimolar. 3. The method of claim 1 or 2, wherein the sequence determined three Has blocks at a specific position in the sequence by using the Synthesis three specific building blocks at a specific position be settled. 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 building blocks D- or L-amino acids and / or their derivatives. 6. The method according to any one of the preceding claims, wherein the Trägerma material consists of cellulose. 7. The method according to any one of the preceding claims, wherein the building block Coupling sites are alanine residues that are covalently bonded to the carrier material they are. 8. The method of claim 7, wherein the alanine residue via an ester bond associated with the cellulose. 9. The method according to any one of the preceding claims, wherein the mixture from natural amino acids, essentially in equal molar proportions exist. 10. The method according to any one of the preceding claims 1 to 8, wherein the Mixture of the D configurations of the natural amino acids, which we are present in equal molar proportions. 11. The method according to any one of the preceding claims, wherein the building blocks Sugar building blocks are.   12. The method of claim 11, wherein the building blocks a cyclic pen are tose or hexose or their derivatives. 13. The method according to any one of the preceding claims, wherein the specific There are at least three times the excess of building blocks. 14. The method according to any one of the preceding claims, wherein after reaction of the variable building blocks, the other building blocks at least in two Excess is added. 15. The method according to any one of the preceding claims, wherein the binding test in an adherence of marked test substances and visualization of the Mark exists. 16. The method of claim 15, wherein the radioactive label Material or consists of biotin or covalently linked enzymes. 17. The method according to any one of the preceding claims 1 to 14, wherein the Re action is measured by allosteric enzymes. 18. The method according to any one of the preceding claims 1 to 14, wherein the Re action of a cell system is measured. 19. The method according to any one of the preceding claims 1 to 14, wherein the Re action consists of a cell-cell interaction. 20. Unbranched or branched peptides from D or L amino acids and / or amino acid derivatives, which peptide has a structure which under Use of the aforementioned synthesis and selection methods was obtained according to one of the preceding claims 1 to 10 and 13 to 19. 21. Method for peptide synthesis of unbranched or branched pep tiden from D- or L-amino acids and / or amino acid derivatives, the Sequence is synthesized, which has the peptide structure, which use the synthesis and selection methods according to one of the previous ones Claims 1 to 10 and 13 to 19 was obtained.