WO1994020521A1 - Elements de synthese et de selection de sequences d'elements constitutifs lies par covalence - Google Patents

Elements de synthese et de selection de sequences d'elements constitutifs lies par covalence Download PDF

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Publication number
WO1994020521A1
WO1994020521A1 PCT/DE1994/000281 DE9400281W WO9420521A1 WO 1994020521 A1 WO1994020521 A1 WO 1994020521A1 DE 9400281 W DE9400281 W DE 9400281W WO 9420521 A1 WO9420521 A1 WO 9420521A1
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Prior art keywords
peptide
sequence
amino acids
synthesis
peptides
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PCT/DE1994/000281
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German (de)
English (en)
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Jens Schneider-Mergener
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Jerini Bio Chemicals Gmbh
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Priority claimed from DE19934308410 external-priority patent/DE4308410A1/de
Priority claimed from DE19934328332 external-priority patent/DE4328332A1/de
Priority claimed from DE19934328637 external-priority patent/DE4328637A1/de
Application filed by Jerini Bio Chemicals Gmbh filed Critical Jerini Bio Chemicals Gmbh
Priority to AU62815/94A priority Critical patent/AU6281594A/en
Publication of WO1994020521A1 publication Critical patent/WO1994020521A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00731—Saccharides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/12—Libraries containing saccharides or polysaccharides, or derivatives thereof

Definitions

  • 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.
  • peptide banks (peptide library) are produced, which are then tested.
  • 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.
  • 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.
  • 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.
  • variable amino acids should also be arranged in the sequence.
  • 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.
  • 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.
  • 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
  • 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.
  • saccharide libraries saccharide libraries
  • 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
  • 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
  • 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.
  • 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.
  • acylation reagent 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 undetermined amino acids or monosaccharides are offered at most in the equimolar amount in the process according to the invention.
  • 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.
  • 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 are possible.
  • 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 pla ⁇ ar 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.
  • the carrier material 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
  • 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.
  • the building blocks are D- or L-amino acids and / or their derivatives.
  • 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, Baselischen, 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-
  • 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 ⁇ -alanine residues which are covalently linked to the carrier material. ⁇ -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 ⁇ -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).
  • the mixture can consist of natural amino acids which are present in essentially the same molar proportions.
  • 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, Basel Liste, Switzerland.
  • the further embodiment of the invention relates to saccharides. These are described in detail in the literature, (for example R ⁇ MPPS, 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.
  • 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 R ⁇ MPPS.
  • 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.
  • 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.
  • 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.
  • reaction types 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.
  • X- ⁇ X 2 ; Xs ; and X 6 are simultaneously installed every 20 amino acids with the same probability.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • mice 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.)
  • 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.
  • compounds can be shown that selectively bind toxic substances (heavy metals, dioxins, furans) and render them harmless.
  • 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 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.
  • the binding test consists in adhering marked test substances and making the marking visible.
  • the label consists of radioactive material or of biotin or of covalently linked enzymes.
  • Other methods consist of measuring fluorescence or luminescence.
  • 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.
  • 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.
  • 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.
  • 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. ⁇ -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 Argini ⁇ 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.
  • 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.
  • 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-
  • 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 (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.
  • 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.
  • 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.
  • Coupling sites or saccharide 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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 Edgi ⁇ gton (1993), BIO / TECHNOLOGY 1 1: 676)
  • LDL low density lipoprotein
  • 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.
  • 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 could be used as a medicament for the treatment of arthereosclerosis.
  • 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.
  • 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 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • Diagnostic markers for tumor cells can also be found using the method.
  • 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.
  • 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).
  • a peptide which binds the metal iron specifically can be found a peptide which binds the metal iron specifically.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Antibodies against ⁇ -toxin have already been successfully tested in the treatment of wounds, particularly burns (Dodd and Stutman (1991) Adv. Paed. Inf Disease 6: 137).
  • ⁇ -toxin antagonists are determined by incubating labeled ⁇ -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.
  • the method can be used to develop a medicament for the treatment of small lucent carcinoma (SCLC) (Corps et al. (1985) Biochemistry 213: 781).
  • SCLC small lucent carcinoma
  • GRP gastrine releasing peptide
  • 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).
  • 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'Do ⁇ nell et al. (1991) J. Biol. Chem. 266: 6389).
  • 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.
  • 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.
  • 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).
  • TNFa tumor necrosis factor alpha
  • 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.
  • Hardened cellulose (Whatman 540) is used.
  • the 125 l-labeled TNF ⁇ has a specific activity of about 650 Ci / mmol and is obtained from the Amersham company.
  • 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.
  • the entire membrane is incubated with a ⁇ -alanine solution, so that an esterification with the OH groups of the cellulose results in a uniform distribution of ⁇ -alanine.
  • the process consists of the following steps:
  • spots 2.2 Determination of the functionality of the spots (spots).
  • the defined amino acid required to create a combinatorial 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:
  • 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,
  • the membrane is incubated for five hours at room temperature with 20 ⁇ Ci TNF ⁇ in 40 ml 1% BSA, 0.05% Tween ⁇ O 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
  • the first synthesis and selection process becomes a well-binding sequence
  • 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.
  • 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.
  • the third synthesis proceeds as described under 3.2, but in this case position 6 is occupied by a defined amino acid.
  • BSA bovine serum albumine (bovine serum albumine)
  • MOLECULE TYPE synthetic peptide
  • CHARACTERISTIC binding to TNF
  • 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.
  • MOLECULE TYPE synthetic peptide
  • 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.
  • MOLECULE TYPE synthetic peptide
  • CHARACTERISTIC binding to TNF
  • 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.

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Abstract

Un procédé permet de synthétiser en phase solide ou en phase liquide des séquences sélectionnables d'acides aminés ou de monosaccharides, les sites de couplage étant prédéterminés sur un substrat. Les séquences sont constituées d'acides aminés ou de monosaccharides déterminés et d'acides aminés ou de monosaccharides indéterminés à une position définie de la séquence. Selon ledit procédé, la synthèse de la séquence se fait en continu et comprend les étapes suivantes: (aa) addition de l'acide aminé déterminé ou du monosaccharide déterminé à un site défini du substrat, et (bb) addition au substrat d'un mélange d'acides aminés ou de monosaccharides indéterminés équimolaire par rapport aux sites de couplage.
PCT/DE1994/000281 1993-03-12 1994-03-09 Elements de synthese et de selection de sequences d'elements constitutifs lies par covalence WO1994020521A1 (fr)

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DE19934308410 DE4308410A1 (de) 1993-03-12 1993-03-12 Verfahren zur Synthese und Selektionierung von Sequenzen aus kovalent verbundenen Bausteinen
DEP4308410.9 1993-03-12
DE19934328332 DE4328332A1 (de) 1993-08-17 1993-08-17 Verfahren zur Synthese und Selektionierung von Sequenzen aus kovalent verbundenen Bausteinen
DEP4328332.2 1993-08-17
DE19934328637 DE4328637A1 (de) 1993-08-23 1993-08-23 Verfahren zur Synthese und Selektionierung von Sequenzen aus kovalent verbundenen Bausteinen
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US10100131B2 (en) 2014-08-27 2018-10-16 The Board Of Trustees Of The University Of Alabama Chemical pulping of chitinous biomass for chitin
US10927191B2 (en) 2017-01-06 2021-02-23 The Board Of Trustees Of The University Of Alabama Coagulation of chitin from ionic liquid solutions using kosmotropic salts
US10941258B2 (en) 2017-03-24 2021-03-09 The Board Of Trustees Of The University Of Alabama Metal particle-chitin composite materials and methods of making thereof
US10982381B2 (en) 2014-10-06 2021-04-20 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing welded substrates
US11085133B2 (en) 2016-05-03 2021-08-10 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing dyed and welded substrates
US11766835B2 (en) 2016-03-25 2023-09-26 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing welded substrates

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US8668807B2 (en) 2008-02-19 2014-03-11 Board Of Trustees Of The University Of Alabama Ionic liquid systems for the processing of biomass, their components and/or derivatives, and mixtures thereof
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US8784691B2 (en) 2009-07-24 2014-07-22 Board Of Trustees Of The University Of Alabama Conductive composites prepared using ionic liquids
US9394375B2 (en) 2011-03-25 2016-07-19 Board Of Trustees Of The University Of Alabama Compositions containing recyclable ionic liquids for use in biomass processing
US10100131B2 (en) 2014-08-27 2018-10-16 The Board Of Trustees Of The University Of Alabama Chemical pulping of chitinous biomass for chitin
US10011931B2 (en) 2014-10-06 2018-07-03 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing dyed and welded substrates
US10982381B2 (en) 2014-10-06 2021-04-20 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing welded substrates
US11555263B2 (en) 2014-10-06 2023-01-17 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing dyed and welded substrates
US11766835B2 (en) 2016-03-25 2023-09-26 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing welded substrates
US11085133B2 (en) 2016-05-03 2021-08-10 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing dyed and welded substrates
US11920263B2 (en) 2016-05-03 2024-03-05 Natural Fiber Welding, Inc. Methods, processes, and apparatuses for producing dyed and welded substrates
US10927191B2 (en) 2017-01-06 2021-02-23 The Board Of Trustees Of The University Of Alabama Coagulation of chitin from ionic liquid solutions using kosmotropic salts
US10941258B2 (en) 2017-03-24 2021-03-09 The Board Of Trustees Of The University Of Alabama Metal particle-chitin composite materials and methods of making thereof

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