RU2145233C1 - Unordered peptide library, method of preparation thereof, and method of identifying peptide prepared by solid-phase synthesis - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to library of peptides immobilized on solid-phase carrier which represents unordered library of unprotected peptides containing numerous solid-phase carriers wherein one unprotected peptide type is bound to each solid-phase carrier and all statistically possible subunit combinations are included. Preparation of library involves introduction of subunit combination into solid-phase carrier aliquot in such a way as to introduce only one protected subunit into each aliquot, after which subunit is covalently bound to all reactive groups of solid-phase carrier and blocking and protective are removed. For identification of peptide, an active molecule specifically linking to library peptides is added to form complex, which is separated and peptide sequence therein is determined. EFFECT: expanded properties in preparing a variety of amino acid combinations of unordered peptide library. 15 cl, 12 dwg, 4 tbl, 14 ex

Description

1 Field of Invention
The invention relates to a library of bio-oligomers immobilized on solid-phase substrates, where each solid-phase substrate is associated with only one bio-oligomeric form, and all possible combinations of monomeric subunits making up these bio-oligomers are included in this library. As the bio-oligomer of the invention, a peptide, oligonucleotide or chimeric peptide-oligonucleotide construct can be used. The invention also relates to a method for synthesizing said library.

 The invention also relates to the use of bio-oligomers of the proposed library for the identification and characterization of ligands having the ability to bind an acceptor molecule or to transfer the studied biological activity. The bio-oligomers of the library may also accelerate a chemical reaction.

2. Background of the invention
In the recognition and binding of ligands, almost all biological processes are regulated, such as immune recognition, signal transmission to cellular receptors, transcription and translation, intracellular signal transmission and catalysis, i.e. enzymatic reactions. For a long time in this field of knowledge, the problem of recognizing acceptor molecules acting as agonists or capable of suppressing and inhibiting the biological activity of ligands, for example, as hormones, growth factors, and neurotransmitters, has been of interest; inducing B-cell (antibody-mediated) or T-cell (cell-mediated) immunity; with the ability to catalyze chemical reactions; and regulating gene expression at the level of transcription or translation.

 Of particular interest are protein and peptide ligands. These include most hormones, growth factors, neuro-reactive molecules, and immunodominant determinants. Moreover, as mentioned earlier, the main efforts to create antagonists or agonists of receptor-mediated biological activity, or antibodies or T-cell antigenic determinants, are concentrated on peptides. The creation of drugs recognizing receptor binding sites, however, was hindered to a large extent by the difficulty in determining the sequence of peptide ligands. The number of variations and the multitude of such peptide sequences made it impossible to achieve this goal by any methods, except for the use of a laborious technique, namely, that a specific complex was isolated, the location of the epitope was identified, and the indicated epitope was sequenced. The solution to this problem is also hampered by the fact that the specified antigenic determinant consists of amino acid residues that do not match in the primary sequence.

 Some researchers in this field have attempted to overcome this laborious process by determining the amino acid sequence based on the nucleotide sequence of its complement. Protein is a high molecular weight peptide consisting of an amino acid; each amino acid is copied by one or more codons consisting of three nucleic acid residues. So, for example, peptide A containing glutamine encodes a codon of three nucleic acid residues: cytosine, adenine and guanine. The complement to the indicated codon is guanine (bound to cytosine), thymine (bound to adenine) and cytosine encoding the amino acid in peptide B. According to the complementarity theory, said peptide B is linked to peptide A. In particular, Bost and Blalock (1989, Hethods in Enzymology, issue 168, pages 16-28), it is believed that any given peptide binds to a peptide encoding a complementary sequence of nucleic acid residues, and based on this information they predicted the amino acid sequence of any complementary peptide. These authors used this sequence in peptide synthesis and in studies of the ability of the peptide to bind.

This method, however, does not provide a solution to this problem due to the fact that the degree of binding affinity between complementary peptides is usually low and the presence of complementary peptides containing more than 15 nucleic acid residues is necessary. Moreover, this method requires information on either the amino acid sequence or the sequence of the binding partner of the analyzed protein. This method, in addition, is not effective for antigenic determinants consisting of amino acid residues that do not match in the primary sequence. Recently, there have been several reports of obtaining peptide libraries and their use for identifying peptide ligands that can bind to acceptors. In one method, a recombinant bacteriophage is used to create large libraries. Using the phage method (Scott and Smith, 1990, Science, issue 249, pp. 386-390; Cwirla et al., 1990, Proc Natl. Acad Sci., Issue 87, pp. 6378-6382; Devlin et al., 1990, Science, issue 249, pp. 404-406) it is possible to construct very large libraries (10 ⁶ -10 ⁸ chemical units), but the genetic code and biosystem impose severe restrictions on the versatility and diversity of their design. In the second methodology, mainly chemical synthesis methods are used, as examples of which we can mention the Geisen method (Geysen et al., 1986, Molecular Immunology, no. 23, pp. 709-715; Geysen et al., 1987, J. Immunologic Method, issue 102, pp. 259-274) and the most modern method of Fodor and others, Science, issue. 251, pp. 767-772, 1991). The methodology of Geysen et al. Provides for the possibility of synthesizing a limited number of peptides on polyethylene pins in a few days. Fodor et al. Uses the technology of “light-inducible spatially addressable parallel chemical synthesis. This technology also has limited capabilities due to the relative lack of development of photochemical methods for peptide synthesis.

 Technologies have also been developed for large-scale parallel competitive peptide synthesis. A Houghton publication reports the synthesis of hundreds of similar peptides simultaneously in polypropylene mesh bags (tea bag method (Houghton, 1985, Proc Natl. Acad. Sci. USA, 82, pp. 5131-1535; Berg et al., 1989, J Am. Chem. Soc., Issue 111, pp. 8024-8026) announces a new substrate based on a polystyrene-grafted polyethylene film suitable for parallel peptide synthesis, using standard Boc amino acid resin and traditional operations in both technologies. deprotection, neutralization, binding and washing according to the original met Odice solid-phase synthesis Merrifield (1963, J. Am. Chem. Soc., issue 85, pp. 2149-2154).

 Furka et al. (1988, 14th International Congress of Biochemistry, x. 5, Abstract FR: OB) describe a method for preparing a peptide mixture by individually binding each of three different amino acids, followed by mixing all three different amino acids containing the resin. In the disclosed technique, Glysen et al. Do not propose any effective method for isolating a test peptide from a plurality of obtained peptides.

Despite their practical suitability, chemical synthesis methods of Glysen, Fodor, Houghton, Berg, Furka et al. Allow the synthesis and analysis of only peptides containing from several hundred to less than a thousand amino acid sequences at a time. These methods are very limited in the light of millions of possible peptide sequences, one or more of which must correspond to the binding sites between the analyzed structural units. In the case of peptides of 20 amino acids, in any sequence of five amino acids, there are 20 ⁵ or about 3.2 • 10 ⁶ possible amino acid combinations. None of these methods allows the synthesis of such a large number of peptides at a time. Similarly, well-known peptide synthesis technologies such as those described by Stewart and Goung (1984, Solid Phase Synthesis, Second Edition, Pierce chemical Co. Rock ford, IN) do not offer a synthesis method from several thousand to several million peptides at a time.

 Moreover, none of the other known methods of peptide synthesis offers a library of peptides immobilized on a solid-phase carrier, which is completely randomized. An absolutely randomized peptide library is a bank with a good static distribution of all molecular structures, as a result of which the indicated library contains approximately equimolar ratios of all individual types of peptides.

 Synthesis of a completely randomized peptide usually cannot be ensured under the conditions of simultaneous introduction of various amino acids into a one-stage reactor due to the fact that the binding rate of various amino acids differs significantly during the process of solid-phase peptide synthesis (TFPS) (Ragnarsoon et al., Acta Chem. Scand. 25, pp. 1487, 1489; Ragnarsson et al., 1974, J. Org. Chem, issue 39, pp. 3837-2842). For example, the binding rate of Fmoc-glycine to the growing petid chain is much faster than Fmos-valine, possibly due to steric hindrance caused by the voluminous side chain of valine. In the case when all 20 activated eukaryotic L-amino acids had to be mixed with the polymer during each binding cycle, the most rapidly reacting amino acids were included in the indicated peptide, and it was not possible to obtain the equimolar ratio of each peptide form. In addition, each of the possible nucleophiles usually has a different reactivity.

Moreover, none of the known methods of peptide synthesis provides the creation of a library of more than 10 ⁵ peptides, where only one type of peptide is immobilized on one solid phase carrier. The immobilization of only one type of peptide on a carrier usually significantly increases the effectiveness of current peptide isolation techniques.

 Thus, there is a need in this field of knowledge to create a library consisting of absolutely disordered peptide and oligonucleotide sequences, that is, sequences of bio-oligomers, where only one oligomeric form can be easily and quickly isolated from the remaining components of this library. In addition, there is a willingness in this field of knowledge to create a method for carrying out fast and cheap synthesis from several thousand to several million of these absolutely randomized bio-oligomeric sequences.

3. Summary of the invention
The invention relates to a library of bio-oligomers containing all possible combinations of subunits, by the method of synthesis of this library, which consists in the fact that repeatedly carry out operations to obtain at least two aliquots of a solid phase carrier; to these aliquots of the solid-phase carrier, a separate set of subunits is added individually; completely bind the subunit to all sites of the solid-phase carrier with the formation of a combination of a new subunit and a solid-phase carrier, assess the completeness of binding, and if necessary continue the reaction until it is complete; aliquots of the resulting combination from the new subunit and the solid-phase carrier are thoroughly mixed and, after repeating the above operations, the protective groups are removed as many times as necessary, as a result of which the resulting bio-oligomer remains immobilized on the solid-phase carrier. In another embodiment, an amino acid can be used as the corresponding subunit, and a peptide as a bio-oligomer. In another embodiment, a nucleoside can be used as a subunit, and the bio-oligomer can be represented by an oligonucleoside. In yet another embodiment, DNA is used as the nucleoside, and in yet another aspect, RNA is used as the nucleoside. In another embodiment, the amino acid or nucleoside may be used as a subunit, and the chimeric peptide-oligonucleotide complex as a bio-oligomer.

 The invention provides a method for determining the sequence of a bio-oligomeric ligand of an acceptor molecule, which consists in the synthesis of a randomized library of bio-oligomers immobilized on solid-phase carriers, in which each solid-phase carrier is associated with only one bio-oligomeric form, and all possible combinations of monomeric subunits of which these bio-oligomers consist are part of this library; the required acceptor or substrate molecule is introduced into said randomized library, as a result of which one or several bio-oligomeric subunits are recognized and immobilized on a solid-phase substrate within the specified library, or the indicated substrate molecule undergoes a chemical reaction catalyzed by one or more combinations of a bio-oligomer and a solid phase; isolating a combination of a bio-oligomer and a solid phase carrier exhibiting the desired ability, and sequencing a bio-oligomer of an isolated combination of a bio-oligomer and a solid phase carrier. In another embodiment, a bio-oligomeric fragment of a bio-oligomer is isolated from a combination of a bio-oligomer and a solid phase carrier in situ, followed by in situ detection of the biological activity being studied. In another embodiment, the peptide is a bio oligomer. In yet another embodiment, an oligonucleotide, in particular DNA or RNA, is used as a bio oligomer. In yet another embodiment, a chimeric peptide-oligonucleotide complex is used as a bio oligomer.

 The invention also provides drugs and diagnostics containing bio-oligomeric sequences established in accordance with the above methods.

4. Brief Description of the Drawings
In FIG. Figure 1 shows a synthesis scheme for peptides consisting of randomized sequences using the split method for the synthesis of a disordered tripeptide with the insertion of tryptophan at the end of the chain: XXX W (where X is S, A or Y; there may be 3 ³ or 27 combinations).

 In FIG. 2 shows in a schematic form cyclic peptides, n = 0, 1, 2, 3, ... and m = 1, 2, 3, ...; n and m may have, although not necessarily, the same values. The solid lines show the linear peptide bonds; dashed lines indicate cross-linking. Pairs of specifically crosslinkable subunits are designated A and B. A has crosslinking only with A, and B crosslinks only with B.

(a) a “basket-shaped” structure;
(b) a ladder shape;
(c) “lariate” form.

 In FIG. 3 - chromatograms (reverse phase HPLC, Wilak system) of disordered tetrapeptides (X-X-X-W, where X is S, A or Y), synthesized: (A) using the new method (see description text) and (B) using the standard method of solid-phase peptide synthesis. A chromatogram was obtained by eluting the column in a linear gradient of acetonitrile. Solvent A: 0.1% trifluoroacetic acid and 5% acetonitrile; solvent B: 0.1% trifluoroacetic acid and 100% acetonitrile.

 In FIG. 4 is a snapshot of a peptide / sycrosphere with a "long V-mos sequence" labeled with an anti-V-mos antibody and a second antibody.

 In FIG. 5 is a snapshot of a mixture of microspheres with a "long V-mos sequence" and a "short V-mos sequence" labeled with an anti-V-mos antibody and a second antibody.

 In FIG. 6 is a microsphere image of a "long V-mos sequence" and a "short V-mos sequence" labeled with an anti-V-mos antibody and a second antibody.

 In FIG. 7 is a micrograph of a screening of a library of a typical peptide ligand where a positive microsphere (dark blue) can easily be identified against the background of many thousands of negative (colorless) microspheres.

 In FIG. 8 is a micrograph showing the concentration-dependent inhibitory effect of biotin when streptavidin-alkaline phosphatase stained with LHPQF polymer coated mimeotope microspheres. A: 100 nmol; B: 10 nmol; C: 1 nmol; and D: 0.1 nmol of biotin. Control microspheres (polymer composition based on beta-Ala-aminocaproic acid) are mixed in a 1: 1 ratio with LHPQF-resin before incubation with streptavidin-alkaline phosphatase to use them as an internal control of the negative.

5. Detailed description of the invention
In the following, preferred embodiments of the invention will be described in detail.

 As used herein, the term “library” means a bank of virtually disordered bio-oligomers. As used herein, the term “bio-oligomer” means a polymer containing less than about 100 subunits. As the bio-oligomer of the invention, any peptide consisting of amino acid residues, or an oligonucleotide consisting of nucleovides, or a chimeric peptide-oligonucleotide complex can be provided.

 5.1. Methods for obtaining a disordered library of bio-oligomers.

 As indicated above, the invention relates to a method for producing a bio-oligomeric library by synthesizing bio-oligomers consisting of disordered sequences of monomeric subunits. As used herein, the term “disordered sequence of monomeric subunits” means a sequence in which any monomeric subunit precedes or follows any other monomeric subunit.

In one embodiment of the invention, an amino acid, any amino acid analog, or any peptide mineralotope can be used as a monomeric subunit. Used in this description, the term "peptide mineralotope" means a molecule that is similar in structure and chemical composition to a peptide consisting of two or more amino acids. In another embodiment, any nucleoside can be used as the monomeric subunit; DNA or RNA can be used as nucleoside. In yet another embodiment of the invention, amino acids and nucleosides can be used as monomeric subunits. As a bio-oligomer, you can use a peptide (consisting of amino acids), an oligoribonucleotide (containing deoxyribonucleosides), a chimeric DNA-RNA oligonucleotide complex, or any peptide-oligonucleotide chimeric structure. A library containing peptides, oligonucleotides or peptide-oligonucleotide chimeric structures can be obtained in accordance with the method, including multiple stages from the operations consisting in the fact that:
(i) receive at least two aliquots of a solid phase carrier for randomized sequences of subunits;
(ii) introducing into said aliquots of a solid phase carrier a set of subunits individually;
(iii) bind the completely indicated subunits to virtually all sites of the solid phase carrier, as a result of which a new combination of subunits immobilized on the solid phase carrier is formed;
(iv) evaluate the degree of completeness of binding and, if necessary, continue the specified reaction until it is complete;
(v) carefully mix aliquots of a new combination of subunits immobilized on a solid phase carrier; and after repeating operations
(I) - (V) the required number of times carry out the final operation (VI), removing the protective group, as a result of which the resulting bio-oligomer remains immobilized on a solid phase carrier. In another embodiment of the invention, an unordered bio-logical library can be obtained in such a way that during at least one synthesis step, the same subunit binds to all regions of the solid-phase substrates and for at least one other stage of at least two subunits are immobilized on a solid phase substrate. The disordered bio-oligomeric library can be synthesized by a single implementation of the above operations (i) - (V); in yet another embodiment of the invention, a disordered bio-oligomeric library can be obtained by repeatedly performing the above operations (i) to (V). Any solid phase support can be obtained in combination with one or more pre-linked subunits.

 A bio-oligomeric library may consist of a given, limited number of subunits. In another embodiment, the disordered bio-oligomeric library may be composed of all available subunits.

 In yet another embodiment of the present invention, the analyzed bio-oligomer can be identified in processing by creating an initial library and subsequent identification of the bio-oligomeric sequence exhibiting the test ability. As a result, a solid phase support is obtained containing the thus identified bio-oligomeric sequence. A new segment of the monomeric subunit is introduced into the previously identified sequence, after which the resulting fragment is recognized, consisting of a known sequence and an disordered sequence exhibiting the test ability. Such a consistent optimization-randomization strategy enables rapid identification of the bio-oligomer being analyzed.

 The bio-oligomers of the proposed library may, although not necessarily, be contained in equimolar amounts. It is well known to those skilled in the art that the molar amount means a concentration at which one gram molecule of any substance is dissolved in a solvent sufficient to produce one liter of solution. As used herein, the term “substantially equimolar amounts” of bio-oligomers refers to individual species of monomeric subunits that are contained in approximately the same concentration. So, for example, if the content of bio-oligomer A in a set containing 150,000 bio-oligomers is 200 nmol / L, then all remaining of the 150,000 bio-oligomeric forms will be present in it at a concentration of about 200 pmol / L. However, the term “substantially equimolar amount”, as used herein, is used to evaluate the surface heterogeneity of solid-phase carriers. The heterogeneity of the solid-phase carrier leads to a change in the amount of bio-oligomer that can be immobilized on this carrier.

 In the proposed method, at least two aliquots of a solid phase carrier are used, where the amount of solid phase carriers in said aliquots preferably corresponds to at least the amount of bio-oligomers synthesized. This allows you to create a library where each solid-phase carrier contains only one bio-oligomeric species, that is, one bio-oligomer - one microsphere. Used in this description, the term "aliquot" means a part representing a certain fraction of the total amount of solid phase carriers.

5.2. Randomized Peptide Libraries
In a specific embodiment of the present invention, the proposed randomized bio-oligomeric library may include peptides. The term "peptide" in this description in its broadest sense to mean a compound consisting of two or more amino acids, amino acid analogues or peptidomimetics as subunits. These subunits may be linked by peptide bonds. In another embodiment, the subunit used may be linked by other bonds, for example, ester, ether, etc. As used herein, the term “amino acid” means both natural and / or non-natural and synthetic amino acids, including glycine or both D- or L-forms of optical isomers, as well as amino acid analogs and peptidomimetics. Any peptide consisting of three or more amino acids is usually called an oligopeptide in the case of a short peptide chain. In the presence of a long peptide chain, such a peptide is commonly referred to as a polypeptide or protein.

The present invention is basically based on the chemistry of synthetic peptides and does not rely on any systems of living organisms for amplification or screening. The peptide libraries of the present invention may include organic amino acids. So, for example, the proposed peptides may contain D-amino acids, a mixture of D- and L-amino acids and various amino acid derivatives (for example, beta-methyl amino acids, CL-methyl amino acids, N ^α- methyl amino acids, etc.) to transfer the specific properties of the peptides in the proposed library. In addition, when transferring the specific ability of amino acids at the site-specific binding stages, peptide libraries containing peptides of alpha-helical, beta-curved, beta-folded, gamma-curved and cyclic conformation can be obtained.

 The composition of the proposed library of peptides includes any possible combination of amino acids that make up these peptides. Using as an example a peptide formed from two amino acids, glycine and proline, four combinations are possible: glycine-glycine, glycine-proline, proline-glycine and proline-proline and, therefore, all four combinations will be part of the proposed disordered library .

The set of first amino acids is administered individually in each aliquot. Typically, for peptide synthesis, basic mobile amino acids with a protective 9-fluorenylmethoxycarbonyl (Fmoc) N ^α -amino group, first described by Carpino and Han (1972, J.org Chem, 37, p. 3403-3409), are used as amino acids. In the proposed method, you can also use Boc amino acids (with a protected N ^α -tertbutyloxycarbonyl N ^α -amino group). Both Fmos and Boc amino acids can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambredge Research Biochemical, Bachem or Peninsula Labs, or other chemical firms known to those skilled in the art. In addition, in the proposed method, you can use other well-known specialists N ^α -protective groups.

Returning to the dipeptide example disclosed above, the first set of introduced amino acids typically comprises glycine and proline; each aliquot takes either N ^α -Fmos-glycine or N ^α -Fmos-proline.

 After administration, the specified set of first amino acids is fully associated with all sites of solid-phase carriers. Used in this description, the sign of "complete binding" means that the binding reaction proceeds before it is completed, regardless of differences in the binding rate of individual amino acids. Moreover, these amino acids bind to almost all binding sites on a solid-phase carrier, as a result of which each solid-phase carrier contains only one type of peptide. The complete binding reaction leads to the formation of combinations from a set of first amino acids and solid phase carriers. Using the above dipeptide as an example, after binding is completed, a combination of glycine immobilized on a microsphere and a combination of proline immobilized on a microsphere are obtained.

The binding reaction of these amino acids can be carried out by methods known to those skilled in the art, for example, Sfewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemicol Co, Kockfd, IL. As is known to those skilled in the art, the technology of peptide synthesis on solid phase carriers usually involves constructing a peptide from a carboxyl or the end of a C-terminal region, where a C-terminal amino acid with a protected alpha amino group is immobilized on a solid phase polymer. The protective group is then removed, and the next amino acid, also with protection, is bound via a peptide bond to the alpha-amino group of the amino acid immobilized on a solid phase carrier. The deprotection cycle of the previous amino acid and the binding of the next amino acid is repeated until the synthesis of the peptide is complete. Any reactive side chains are protected by chemical groups that can withstand the binding and N ^α -blocking operations, but which can be removed at the end of the synthesis.

 To bind any amino acid to a growing synthetic chain, it is necessary to activate the carboxyl group of the blocked amino acid. Various activation methods can be used in the practice of the invention, which include, for example, pre-prepared symmetric in-hydrides (in situ), pre-prepared anhydride mixtures (PMA), acid chlorides, reactive esters and carboxylic acid activation according to the method described by Fields and Noble, 1990, "Solid Phese Peptide Syntbesis utilizing 9-fluorenylmethoxycarbonyl amino acid" (solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl amino acids) Int. J. Pept. Protech. Res, 35, 161-214.

 It is necessary to evaluate the completeness of binding. Specialists in this field are well aware of the known reactions when conducting quantitative analysis using, for example, ninhydrin (Kaiser test), picric acid, 2,4,6-trinitrobenzenesulfonic acid (TNBS), fluoroesamine and chloranil, which are based on the interaction of reagents with free amino groups with the formation of a chromophore. In the case of imino acids (e.g. Pro and Hyp), quantitative control using the isatin reaction is the preferred analysis method (see Fields and Noble above). Quantitative analysis or assessment of the degree of completion of the binding reaction can be carried out during the entire synthesis time, for example, as described by Salisbury et al. (Patent Application Agreement PCT No. 91/03485).

 When conducting Fmos-amino acid synthesis, it is preferable to use a Kaiser test. In the Kaiser text, a sample from each tube can be analyzed using the ninhydrin reagent supplied by Pierce Chemicol according to the method of Savine et al. (1981, Anal. Biochem, 117, pp. 147-157).

If the binding reaction was incomplete, as was established as a result of this test, this reaction can be brought to its completion by several methods known to specialists, including (a) conducting a second binding reaction using a five-fold excess of the protected amino acid, (b) additive binding using other or additional solvents (e.g. trifluoroethane) or (c) adding chaotropic salts, e.g. NaClO ₄ or hcBr (Klis and Stewart, 1990, "Peptides: Chemistry, Structure and Biology", Rivier and Harshall, eds., ESCOM Publ., P. 90 4-906).

 After complete binding, aliquots of the combinations of the first amino acids of the solid phase carrier are thoroughly mixed. Thorough mixing is achieved provided that a homogeneous mixture of aliquots is obtained, preferably by stirring said aliquots in a one-stage reactor. Although any method of thorough mixing is included in the scope of the present invention, and specialists are well aware of many other means, it may be preferred, for example, vortex or rotational mixing in any commercially available rotary shuttle apparatus or sparging with an inert gas, for example, nitrogen or argon .

 The resulting mixture is divided into at least two aliquots. These aliquots are equal in volume and, if sufficient homogeneity is achieved, should contain almost equal amounts of combinations of the first amino acids on solid phase carriers. In the case of the dipeptide used as an example, each aliquot contains virtually equal amounts of microsphere-glycine and microsphere-proline combinations.

 A second set of amino acids is introduced individually into each aliquot. This second set may consist of (a) the same amino acids that were introduced in the first set, i.e. glycine or proline; (b) another set of amino acids, for example tryptophan or leucine; (c) only one type of amino acid, for example, isoleucine.

As in the case of the first amino acid set, the second set of amino acids is fully associated individually with a combination of the first amino acids immobilized on solid phase carriers from each aliquot to form peptides containing the first amino acid and the second amino acid. As in the case of the previous binding reaction, the following binding can be performed by any method used in this field to carry out such reactions. Using as an example the discussed dipeptide:
(a) upon administration of the same amino acids, the resulting peptides are combinations of both glycine-glycine, glycine-proline, proline-glycine, and proline-proline;
(b) in the collection of other amino acids, the resulting peptides are combinations of either Gly-Trp, Gly-Leu, Pro-Trp, or Pro-Leu;
(c) upon administration of one type of amino acid, the resulting peptides are Gly-ILe or Pro-ILe.

This binding technique can be repeated as many times as amino acids have to be administered. If the peptide of interest is a tetrapeptide XXXT, where X is either valine, serine, or alanine, for example, then the above binding procedure can be carried out three times to obtain a tetrapeptide with the XXX-Trp structural chain. In the first, second, and third administrations of amino acids, either N ^α -Fmos-valine, N ^α -Fmos-serine (O-Bu '), or N ^α -Fmos-alanine is added to aliquots of a solid-phase carrier with a yield of 27 different peptides in virtually equimolar amounts (Fig. 1). If it is necessary to obtain a hexapeptide, the specified binding procedure is repeated six times. If the hexapeptide must include 5 different amino acids, then this method can be carried out using 5 aliquots, each of which contains a different acid at each stage of binding. If, however, the target hexapeptide must contain any combination of the amino acid backbone, this method can be performed using 20 aliquots at each binding step.

 The proposed peptide synthesis method can be used with solid-phase carriers on which any amino acid is either immobilized or not yet. In addition, it is possible to use a linker that is already connected to a solid phase carrier. As one common substrate on which any amino acid has already been immobilized, beta-alanine-polyacrylamide resin (available from Bachem Biochemical) can be used. Such resins can be obtained from numerous commercial sources or obtained in the laboratory by any specialist in the field of peptide synthesis.

 If a mixture of amino acids immobilized on a solid-phase support or a linker on a solid-phase support is used as a starting reagent, then it is divided into at least two aliquots, each of which contains any amino acid from the first set of amino acids. As mentioned above, the first set of amino acids completely binds to almost all binding sites of a mixture of amino acids immobilized on a solid-phase carrier or on a solid-phase linker, after which aliquots containing these newly introduced amino acids are thoroughly mixed. As indicated previously, the resulting mixture is divided into at least two aliquots, with each aliquot receiving one amino acid from the second set of amino acids, and the binding reaction is carried out again with the formation of a growing peptide. As mentioned earlier, this process can be repeated as many times as necessary to obtain the desired peptides.

The specified method can be used for the synthesis of disordered peptides, as well as for the synthesis of a peptide library containing predefined sequences. The synthesis of predefined sequences involves the use of specific N ^α -Boc, N ^α -Fmos or other properly protected amino acids during site-specific binding operations. For example, anyone can select amino acids at the site-specific binding stages so that the target peptides have the likelihood and preference of obtaining a special secondary structure, for example, beta-fold, alpha-helix, beta-bend, etc. For example, alpha -helix is preferred if Glu, Ala, Leu, His, Trp are used as preferred amino acids; on the other hand, beta folds are usually preferred when using Val, ILe, Tyr and Met. Alternatively, if Gly, Asp, Ser, Pro, Asp are used, a beta curved structure is preferred. Other examples include, for example, acidic amino acids at the N-terminal region and basic amino acids at the C-terminal region that stabilize the alpha helix. D-amino acids can stabilize some curved structures, and numerous other structural forms can be included in the proposed library (see Sections 5.2.1 and 5.2.2 below). A synthesis of a library of cyclic peptides with disulfide, lactam, lactone or other cycle-closing residues is quite likely (see Section 5.2.1 below).

 It should be emphasized that the proposed method allows the synthesis of peptides so that each solid-phase substrate, for example, as a polymer microsphere, contains only one type of peptide. The specified method provides the contact of a single polymer microsphere with only one Fmos amino acid during each binding cycle and the course of this reaction until its completion. The specified synthesis method is one microsphere - one peptide allows to increase the sensitivity and efficiency of the allocation of the peptide, which is specific to the subunit with which it binds.

The proposed method can be easily implemented for the synthesis of a randomized peptide pool containing from 10 ⁵ to 10 ⁷ different peptide forms.

 In one aspect of the invention, the peptides included in the library may contain a special amino acid residue at the C-terminal region with either the COO H or CONH side chain, designed to mimic the free glycine or glycine amide group. Moreover, this special amino acid residue is usually used in the form of a D- or L-amino acid analog with a side chain containing a linker or bond to the microsphere. In another embodiment, the indicated pseudo-free amino acid residue at the C-terminal portion can be used in the form of an optical isomer of the D- or L-configuration; in yet another embodiment, in the form of a racemic mixture containing the D and L isomers.

 In yet another embodiment, the peptides of the library may contain pyroglutamate as the N-terminal amino acid residue. Although pyroglutamate cannot be sequenced by the Edman degradation method, however, when peptides with the N-terminal pyroglutamate are limited to only 50% on any given microsphere, a sufficient amount of pyroglutamate-free peptide remains on the indicated microsphere for sequencing. Any specialist should be clear that this method can be used to sequence any peptide that contains an amino acid residue in the N-terminal region that is resistant to Edman degradation. Other methods for characterizing individual peptides exhibiting the desired activity are described in detail below in the description. The specific activity of a peptide containing a protected N-terminal group, for example, pyroglutamate, if such an N-terminal group is present in 50% of these peptides, can be easily demonstrated by comparing the activity of a fully protected (100%) peptide with an unprotected (0%) peptide.

In another embodiment of the invention, subunits of peptides carrying useful chemical and structural properties are selected. For example, peptides containing L-amino acids are resistant to L-amino acid-specific proteases in vivo. In addition, the present invention addresses the problem of obtaining libraries with more clearly defined structural characteristics, as well as the use of peptidomimetics and peptidomimeotic bonds, for example, as ester bonds to obtain peptide libraries with new properties. In another embodiment of the invention, a peptide library can be created comprising a reduced peptide bond, i.e., Ri-CH ₂ -NH-R ₂ , where R ₁ and R _{2 are} amino acid residues or sequences. The reduced peptide bond may be administered as a dipeptide subuniticin. Such a molecule is usually resistant to cleavage of the peptide bond, for example, protease activity. Such libraries create ligands with unique functional ability and activity, for example, as a prolonged half-life in vivo, due to resistance to metabolic decay or protease activity. Moreover, it is well known that in some biological systems, conformationally restricted peptides exhibit increased functional activity (Hruby, 1982, Kife Sciences vol. 31, p. 189 -; Hruby et al., 1990, Biochem J., 268, p. 249- 262) the present invention provides a method for producing a conformationally restricted peptide containing disordered sequences at all other positions.

 Limited and cyclic peptides.

 Any limited, cyclic or randomized peptide can be obtained in accordance with the above method, provided that at least two positions in the sequence of any of the peptides of the library are inserted amino acid or amino acid analogue, which gives a chemical functional group with the ability to cross-link, leading to to conformational restriction, cyclization and a rigid framework of the peptide after processing with the formation of cross-links. The cyclization reaction will proceed favorably with the introduction of any bending-inducing amino acid. Examples of amino acids that can crosslink a peptide include cysteine to form disulfides, aspartic acid that forms lactone or lactam, and any halting agent, for example, gamma-carboxyl-glutamic acid (61a) (manufactured by Bachem), giving a transition metal chelate complex with the formation of bridging. Protected gamma-carboxyl-glutamic acid can be obtained by modifying the synthesis technology described by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun., Issue 1128, pp. 1128-1132). Any peptide library in which the indicated peptide sequence contains at least two amino acids with the ability to crosslink can be processed, for example, by oxidation of cysteine residues with the formation of disulfide, or by the introduction of a metal ion with the formation of a chelate complex, resulting in crosslinking of the indicated peptide while forming a conformationally limited, cyclic peptide or peptide with a rigid skeleton.

 The present invention provides several basic rigid skeletons for use in the synthesis of the proposed libraries. In one embodiment of the invention shown in FIG. 2, two pairs of crosslinking residues are arranged in such a way that a basket-like framework is formed. Such a “basket-like” skeleton can be used, in particular, as a catalytic pocket. In another embodiment of the invention, a “ladder” skeleton, which is shown in FIG. 2. With the alternate use of D- and L-amino acids in the "ladder" basis, it is possible to obtain a peptide, where each of the side chains is oriented on the same surface similarly to the beta-folded conformation established in gramicidin. Such a surface can potentially form a unique catalytic center. If in one embodiment of the invention it is possible to create a simple “rope” skeleton in which two residues form a bridge, as shown in FIG. 2. In addition to the obtained peptide loop, a shortened “rope” skeleton usually leads to the formation of a conformationally limited linear peptide and, therefore, stabilization of the secondary structure, for example, the alpha helix.

 The present invention also found that interpeptide crosslinking can be obtained with the formation of a rigid peptide frame.

 The present invention provides strategies for systemic bridging. For example, when cysteine residues are introduced into the sequence of such a peptide, various protective groups can be used (HisKey, 1981, In The Peptides: Analysis, Synthesis, Biology, v. 3, Gross and Meienhofer, from Academic Press: New York, p. 137-167; Ponsanti et al., 1990, Tetrahedron, issue 46, pp. 8255-8266). The first pair of cysteine residues can be deprotected and oxidized, and then the second pair can be unlocked and oxidized. As a result of this technique, several disulfide bridges can be formed. Alternatively, a pair of cysteines and a pair of chelating amino acid analogues can be introduced into such a peptide sequence, resulting in the formation of bridging bonds of different chemical nature.

5.2.2. Unconventional amino acids causing conformational limitations
The following non-traditional amino acids can be included in a randomized peptide library to introduce characteristic conformational frameworks: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991, J. Am. Chem. Soc., Issue 113 pg. 2275-2283); (2S, 3S) methylphenylalanine, (2S, 3R) methylphenylalanine, (2R, 3S) methylphenylalanine and (2R, 3R) methylphenylalanine (Kazmiercki and Hruby, 1991, Tetrahedron Lett). 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Nhesis, University of Arisona), hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., 1989, J. Takeda Res. Labs, issue 43, pp. 53-76); beta-carboline (D- and L-forms) (Kazmierski, 1988, Ph. D.Thesis, University of Arisona); HIC (histidine-isokolin-carboxylic acid) (Zechel et al., 1991, Int. J. Pep. Perotein Res. 43) and HIC (histidine-cyclourea) (Dharanipragada).

The following amino acid analogues and peptidomimetics that lead to the formation or beneficial organization of specific secondary structures can be included in such a randomized library: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), beta-bending-inducing dipeptide analogue (Kemp et al., 1985, J. Org. Chem., issue 50, pp. 5384-5838); beta-folding conformation inducing analogues (Kemp et al., 1988, Tetrahedron Lett, vol. 29, pp. 5081-5082); beta-bent conformation inducing analogues (Kemp et al., 1988, Tetrahedron Lett, vol. 29, p. 5057-5060); alpha-helix-inducing analogues (Kemp et al., 1988, Tetrahedron Lett, 29, pp. 4935-4938); gamma-bending inducing analogues (Kemp et al., 1989, J. Org. Chem. no. 54, pp. 109-115); and analogues disclosed in the following references: Nagai and Sato, 1985, Tetrahedron Lett, 26, pp. 647-650; DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p. 1687; in addition, an analog of the Gly-Ala- curved conformation (Kahn et al., 1989, Tetrahedron Lett, vol. 30, p. 2317); amide-bonded isosteres (Jones et al., 1988, Tetrahedron Lett., issue 29, pp. 3853-3856); tertrazole (Zabrocki et al., 1988, J. Am. Chem. Soc., issue 58, pp. 5875-5880); DTC (Samanen et al., 1990, Int. J. Protein Pep. Res., Issue 35, pp. 501-509); and analogues disclosed by Olson et al., 1990, J. Am. Chem. Sci, no. 112, pp. 323-333 and
Garvey et al., 1990, J. Org. Chem., Vol. 56, p. 436. Although the aforementioned unconventional peptides and peptidomimetics may not be capable of sequencing by the conventional Edman degradation method, a combination of the initial Edman degradation followed by analysis of the remaining amino acids can be used to determine the structure of the peptide with the activity being studied. Alternatively, mass spectral analysis can be used.

5.2.3. Derivatized and modified peptides
The present invention also provides for the modification of peptides or the preparation of their derivatives in a library. Those skilled in the art are familiar with the techniques for modifying peptides, including phosphorylation, carboxymethylation and acylation.

In another embodiment, glycosylated or fatty acylated peptide derivatives can be prepared. It is known to obtain glycosylated or fatty acylated peptides, which are given in the following links:
1. Garg and Jeanloz, 1985, in Advances in Cerbohydrate Chemistry and Biochemistry, Academic Press.

 2. Kunz, 1987, in Ang. Chem. Int. Ed. English 26: 294-308.

 3. Horvat et al., 1988, Int. J. Pept. Protein Res. 31: 499-507.

 4. Bardaji et al., 1990, Ang. Chem. Int. Ed. English, 23: 231.

 5. Toth et al. , 1990, in Peptides: Chemistry, Structure and Biology, Rivier and Marshal. eds., ESCOM Publ., Leiden, pp. 1078-1079.

 6. Torres et al., 1989, Experientia 45: 574-576.

 7. Torres et al., 1989, EMBO J. 8: 2925-2932.

 8. Hordever and Musiol, 1990, in Peptides: Chemistry, Structure and Biology, loc. cit., pp. 811-812.

 9. Zee-Cheng and Olson, 1989, Biochem, Biophys, Res. Commun. 94: 1128-1132.

 10. Merki et al., 1977, Helv. Chem. Acta., 60: 807.

 11. Fuji et al. 1987, J. Chem. Soc. Chem. Commun., Pp. 163-164.

 12. Ponsati et al., 1990, Peptides 1990, Giralt and Andreu, eds., ESCOM Publ., Pp. 238-240.

 13. Fuji et al., 1987, 1988, Peptides: Chemistry and Biology, Merahall, ed., ESCOM Publ., Leiden. pp. 217-219.

 There are two main classes of peptide-carbohydrate bonds. First, ester groups combine the hydroxyl of serine or threonine with the hydroxyl group of a carbohydrate. Secondly, amide bonds connect the carboxyl groups of glutamate or aspartate with the amino group of a carbohydrate. In particular, the above references 1 and 2 describe methods for producing peptide-carbohydrate ethers and amides. Acetal and ketal bonds can also bind a carbohydrate to a peptide.

Moreover, fatty acylated peptides can also be prepared. So, for example, but not in any way limiting the volume, an acyl group, for example, niristoyl, can be introduced into the free amino group (at the N-terminus or lysyl). Alternatively, an amino acid containing an aliphatic side chain of the structure (CH ₂ ) _n (CH ₃ ) can be introduced into the peptides of the proposed library. This and other conjugates of peptides and fatty acids suitable for use in the present invention are disclosed in GB Patent GB-8809162.4; PCT / AU 89/00166 and contrasted source 5, see above.

5.3. Randomized Oligonucleotide Libraries
The method of obtaining a selective library consisting of nucleic acids can be based on solid-phase synthesis of DNA by the phosphoramidate mechanism proposed for the first time by Caruthers (1985, Science, issue 230, p. 281; Caruthers et al., 1987, Methods in Ensymology, no. 154, pp. 287-313).

 Both insoluble silica-based polymer number and protective deoxynucleosides are commercially available (e.g., Penin Sula Laboratories, Jnc., California, Applied Biosystems. Jnc.). As protected deoxynucleosides, 5'-0-dimethoxytrityl deoxythymidine, 5'-0-dimethoxytrityl-4-N-benzoseldeoxyadenosine and 5'-0-dimethoxytrityl-N-isobutyldeoxyguanosine can be used. Other specific protecting groups may be used depending on the application. The corresponding deoxynucleoside-3-phosphoramidites can be synthesized with their subsequent binding to a solid-phase carrier according to the method of Caruthers et al., 1987, see above. The first deoxynucleoside can be immobilized, for example, in the form of deoxyadenosine. After removal of the trityl group and washing first with dichloromethane, then with acetonitrile, the resulting carrier product is divided into four equal aliquots and transferred to four separate reactors. Then, four deoxynucleoside-3-phosphoramides are separately introduced into these four reactors. After the completion of the coupling reaction, these carrier products, recovered from the four reactors, are mixed together, washed thoroughly, and then oxidized using a mixture of iodine, water, lutidine and tetrahydrofuran. After oxidation, the resulting carrier product is washed thoroughly with acetonitrile and the above cycle is repeated. After the synthesis of the disordered polydeoxynucleotide chain (for example, after 11 binding cycles) is completed, the residues of the methyl ester are cleaved with thiophenol and dimethoxytrityl with trichloroacetic acid. Unblocked polynucleotide chains can remain covalently attached to a solid phase carrier (when selecting the appropriate linkers), ready for use in the selective screening strategy below.

 The present invention provides for the use of oligonucleotides with other than phosphodiester bonds. Thus, for example, an oligonucleotide may include a phosphorothionate bond. Modified phosphodiester bonds or their analogues are known. Such modified bonds are known to be resistant to exonuclease and endonuclease activity.

Since there are only four deoxyribo- or ribonucleosides per binding cycle, a library with 12 nucleoside bases will contain 412 possible polynucleotide sequences, which will total 1.68 • 10 ⁷ possible sequences. The oligonucleotide can also be synthesized using both deoxyribo and ribonucleosides. Any specialist should also know that, in addition to major nucleosides, unusual (minor) and modified nucleosides can be used. As minor and modified nucleosides, you can use nucleosides containing methylated purines, derivatives of uridine and 2-methylribose, which can be formed in combination with any ribonucleoside.

 5.4. Solid-state carriers and liquors intended for use in a randomized bio-oligomeric library.

 The solid-phase carrier intended for use in the present invention should be inert to the reaction conditions of bio-oligomeric synthesis, for example, peptide or oligonucleotide synthesis, or to each of them. The solid phase carrier of the invention should have reactive groups for attaching a monomeric subunit or for attaching a linker or spacer, which can serve as a starting point for the binding of the monomeric subunit. In another embodiment of the invention, the solid-phase carrier can be suitable for in vivo use, that is, it can be used as a carrier for direct use, or as a substrate when used directly in a bio-oligomeric library (for example, Tentagel, Rapp. Polymerg, Tubingen, Germany : see section 5.8 below). In a specific embodiment, said solid phase carrier can be delivered through the mucous membrane of the oral cavity or can be administered orally. In another embodiment of the present invention, the solid phase carrier of the invention can be used as a chromatographic support.

 The solid phase carrier as used in the invention does not limit the volume of any particular type of carrier. There are a fairly large number of different carriers, and they are known to any person skilled in the art. Silica gels, polymer resins, plastic-based films, glass beads, cotton, polymer microspheres, aluminum gels can be used as solid-phase carriers. A suitable solid phase carrier can be selected taking into account the intended purpose and its suitability for various synthesis modes. For example, resins such as polystyrene (e.g., polyacrylamide resin supplied by Bachem Inc., Peninsula Laboratories, etc.), Polyhipe® resin (supplied by Aminotech, Canada), polyamide can be used as a solid phase carrier for peptide synthesis. resin (supplied by Peninsula Laboratories), polyethylene glycol is a grafted polystyrene resin (Tentagel®, Rapp. Polymex, Tubingen, Germany) or polydimethyl acrylamide resin (supplied by Milligene / Biosecarch, California). In a preferred embodiment, for peptide synthesis, a polydimethyl acrylamide resin is used as a solid phase carrier.

The solid phase carriers of the invention may also contain a linker. As used in this description, the term linker means any molecule that provides a spatial gap between the carrier and the synthesized peptide. Linkers can be covalently attached to a solid phase carrier before binding to it with N ^α -Boc or N ^α -Fmoc amino acids, or other properly protected amino acids. Various linkers can be used to immobilize the obtained oligomer on it. Examples of linkers of the present invention include aminocaproic acid, aminobutyric acid, 7-aminoheptanoic acid and 8-aminocaprylic acid. Fmos - aminocaproic acid is marketed by Bachem Biochem and is most preferred for use. In another embodiment, the linkers may further include one or more beta-alanines as spacers. Moreover, the specified solid-phase carrier can be modified to meet the special requirements of biological analysis or recognition of a specific object. Modification of the solid-phase carrier can be carried out with the introduction of a specific linker. So, for example, the modified solid-phase carrier can be made in the form of a carrier that is sensitive to acids, bases, nucleophiles, electrophiles, light, oxidation or reduction reactions.

 In addition to the above linkers, selectively cleavable linkers can be used. The use of a UV-sensitive linker, namely infra, is described below in section 12 of the present description (see Barany and Albenicia, 1985, J. Am. Chem. Soc., Issue 107, pp. 4936-4942). For other cleavable linkers, hydrogenolysis and photolysis are necessary. Examples of photosensitive (photolysis cleavable) linkers can be found in Wang (1976, J. Org. Chem., Issue 41, p. 32-58), Hammer et al. (1990, Int.J. Pept. Protein, issue 36 , pp. 31-45) and Kreib Cordonier et al. (1990, Peptides-Chemistry, Structure and biology, River and Marshall, second edition, pp. 895-897). (1977, Meth. Enzym., Issue 47, pp. 145-149) used an aqueous solution of formic acid to cleave the Asp-Pro linker group: this method was used to characterize T-cell epitons in combination with hairpin synthesis according to the Van method der Zee et al. (1989, Eur. J. Jmmunol, no. 191, pp. 43-47). Other potential linker groups cleavable when bases are used include linker groups based on para- (hydroxymethyl) benzoic acid (Athirton et al., 1981, J. Chem. Soc. Perkin, part 1, pp. 538-546) and hydroxy acetic acid acids (Baleaux et al., 1986, Int. J. Pept. Protein Res, vol. 28, p. 22-28). Geysen et al. (1990, J. Immunol. Methods, issue 134, pp. 23-33) report peptide cleavage by the diketopiperazine mechanism. Any enzyme can specifically cleave any linker containing a sequence that is sensitive to or is a substrate for enzymatic hydrolysis, for example, protease cleavage of a peptide; endonuclease cleavage of the oligonucleotide. In some cases, it is possible to obtain a polymer mixture, 10-50% of which is a cleavable linker, and the remaining 50-90% is a non-cleavable linker, which provides a sufficient amount of peptide remaining for sequencing after cleavage of the linker. Mixtures of cleavable linkers can also be used, which provides sequential cleavage from only one microsphere.

 The solid phase carrier for use in the present invention may further comprise an assayable bio-oligomer into which a randomized sequence of subunits can be introduced. The pre-mobilized bio-oligomer can be selected according to the methods described in this description, or it may contain a known sequence with the desired properties.

 In the synthesis of oligonucleotides, it is preferable to use a solid phase carrier on silica. As already mentioned in Section 5.3, such solid phase carriers can be obtained commercially (for example, from Peninsula, Laboratories Inc. and Applied Biosystems Inc.).

5.5. Methods of detection and identification of the studied bio-oligomers
In addition to creating authentically randomized libraries of bio-oligomers and methods for their synthesis, the present invention also provides methods for screening any bio-oligomeric library with identification of bio-oligomers within the specified library, which exhibit biological activity under study, for example, binding, inhibitory, stimulating, cytotoxic, etc. Other bio-oligomeric libraries can be screened according to the methods described below for enzymatic activity, enzyme-inhibitory activity and physicochemical Kie properties that are of interest.

 For the studied bio-oligomers established in the first screening, their presentation in the form of ligands at the final stage is not required. In fact, it is preferable to synthesize a second library based on the usual ligand sequences selected in the first screening. Following this technique, ligands with even higher activity can be identified, provided that the second screening is carried out under more stringent conditions.

5.5.1. Methods for determining binding ability
The present invention allows the identification of bio-oligomeric ligands that bind acceptor molecules. The term “acceptor molecule”, as used herein, means any substance that binds to a bio-oligomeric ligand. As the acceptor molecules, you can use a biomacromolecule, for example, but without limiting the volume, antibodies, formulations or viruses. In addition, any chemical compound can be used as acceptor molecules, for example, but not limited to, proteins, carbohydrates, nucleic acids, lipids, drugs, metals or small molecules
The proposed bio-oligomeric library can effectively interact with many different acceptor molecules. As a result of identification of a specific bio-oligomeric ligand with which a specific acceptor molecule binds, it becomes possible to physically isolate the bio-oligomeric ligand under study.

 Since it is possible to extract only a small number of microspheres during each screening-detection-isolation cycle, most of the microspheres remain in the indicated pool. Thus, the specified randomized bioolegomeric library can be screened repeatedly. In the case of using individual staining or identification schemes for different acceptor molecules (for example, when using fluorogens, for example, like fluorescein (green), Texas red (red) and DAPI (blue) deposited on these acceptors) and with the corresponding excitation filters in With a fluorescence microscope or fluorescence detector, various acceptors (receptors) can be introduced into any peptide library and simultaneously evaluated, thereby facilitating the rapid screening of specific ligands. This methodology not only reduces the cost, but also increases the number of acceptor molecules that can be screened.

 In the proposed method, the analyzed acceptor molecule is introduced into a bio-oligomeric library, in which it recognizes and binds to one or more bio-oligomeric ligands within the specified library. Each bio-oligomeric ligand with which the acceptor molecule binds is recognized on only one solid-phase carrier, as a result of which the indicated carrier, and therefore the desired bio-oligomer, can be easily identified and isolated.

The specified bio-oligomer can be distinguished by any well-known specialist method, however, the invention does not limit the scope to the allocation method. So, for example, but in no way limiting the volume, it is possible to physically isolate a bio-oligomer immobilized on a solid-phase carrier, exhibiting the strongest physicochemical interaction with a specific acceptor molecule. In one embodiment of the invention based on physicochemical interaction, a solution containing a specific acceptor is added to a disordered peptide library that is equivalent to about 10 ⁵ to 10 ⁷ solid phase carriers. The acceptor molecule is then incubated with the specified resin for a period of time sufficient to bind the peptide to the antibody, for example, for one hour at a temperature of 22 ^° C. After this, the acceptor molecule coated with a solid phase carrier is isolated with a bio oligomer. More specific embodiments of the present invention are disclosed in the following methods, which describe the use of a monoclonal antibody in the form of a soluble acceptor molecule. It will become apparent to those skilled in the art that these methods can be easily adapted to determine the binding capacity of any acceptor molecule. Moreover, although the following refers to the peptide library, it should be understood that it is also possible to analyze oligonuleotide libraries and peptide-oligonucleotide chimeric complexes.

(i) The monoclonal body is first labeled with fluorogen or a fluorescein tag is introduced according to the techniques that comprise the routine of a person skilled in the art. The antibody at a concentration of 1 μg / ml is introduced into the library of peptides, and after careful mixing at a temperature of 22 ^o C for one hour, the solid-phase carriers are washed. The resulting complexes of peptides with fluorescent antibodies, immobilized on solid phase carriers, are identified and separated using a cell sorter with fluorescence excitation. In an alternative embodiment, the obtained complexes of peptides with fluorescent antibodies immobilized on solid phase carriers are identified and physically separated under a preparative microscope with a fluorescent attachment using a micromanipulator. The relative fluorescence intensity is usually proportional to the affinity of the peptide ligand for the antibody being analyzed.

(ii) The monoclonal antibody is first conjugated to microspheres based on ferromagnetic material according to routine procedures. The resulting antibody conjugate at a concentration of 1 μg / ml is then incubated with the peptide library for one hour at a temperature of 22 ^o C. Magnetic microspheres form a socket around the analyzed peptide on a solid-phase carrier, which can then be highlighted with a strong magnet.

(iii) The monoclonal antibody is first conjugated to an enzyme, for example, alkaline phosphatase according to routine procedures. The resulting antibody-enzyme conjugate is then incubated with a randomized peptide library for a period of time from 30 minutes to 1 hour at a temperature of 22 ^o C. After washing, the entire contents of the library is poured into a Petri dish containing a substrate on alkaline phosphatase, for example, 5-atom 4-chloro-3-indoyl phosphate (BCIP) and nitrosine tetrazolium (NBT). After incubation for several minutes, the antibody-loaded peptide complex on the solid-phase support changes color (turns blue) due to the deposition of the transformed substrate on the solid-phase support, which makes it easy to identify and physically isolated under a preparative microscope using a micromanipulator. The relative intensity of the color reaction is usually proportional to the affinity of the peptide for the antibody being analyzed.

(iv) The monoclonal antibody is first conjugated to an enzyme, for example, as horseradish peroxidase according to routine procedures. The obtained antibody-enzyme conjugate is then incubated in a randomized peptide library for a period of time from 30 minutes to 1 hour at a temperature of 22 ^o C. After washing, the entire contents of the library are poured into a Petri dish containing a substrate for peroxylase, for example, 3.3 ', 4 4'-diaminobenzidine (DAB); 3.3 ', 5.5-tetramethylbenzidine (TMB) or 4-chloro-1-naphthol (4ON). After incubation for several minutes, the obtained antibody-peptide complex on a solid-phase carrier changes color and can be identified and isolated physically under a preparative microscope using a micromanipulator. The relative intensity of the color reaction is usually proportional to the affinity of the peptide for the monoclonal antibody being analyzed.

(v) The monoclonal antibody is first labeled with biotin, that is, a biotin label is introduced according to routine procedures. After that, the biotinylated antibody is incubated together with a randomized peptide library for a period of time from 30 minutes to 1 hour at a temperature of 22 ^o C. After washing, streptazidine-alkaline phosphatase complex or a complex of streptazidine with horseradish peroxidase are added and incubated for 30 minutes. The resulting support is then washed and a color reaction is created as described in the above (iii) in combination with an enzyme-linked immunosorbent assay. The analyzed carrier with the immobilized peptide is isolated according to the procedure described previously.

 In addition to using soluble acceptor molecules, in another embodiment of the invention, bio oligomers that bind to cell surface receptors using intact cells can be detected. Intact cells are preferably used with multivalent or mobile receptors, or with receptors that require a functional lipid domain of the cell membrane. The cells used in this technique can be both viable and immobilized. Typically, these cells are incubated together with a peptide library, which, when bound to certain peptides, form a “socket” between the target cells and the relevant peptide immobilized on a solid support. The outlet can then be isolated by differential centrifugation, or physically removed using a preparative microscope.

Alternatively, the library can be screened using the panning method with cell lines, for example, as (i) the parent cell line, where the analyzed receptor is absent on its cell surface and (ii) the receptor-positive cell line, for example, the cell line formed by transfection the parent line by the genome encoding the receptor under study. After that, you can screen the library using the following strategy:
(i) first, non-specific microspheres binding to cells lacking the indicated receptor are removed from the library by introducing a monolayer of the parent cell line according to the standard panning method, leaving receptor-specific unbound microspheres or irrelevant unbound microspheres;
(ii) remove unbound microspheres, which include both receptor-specific and irrelevant microspheres, and apply them to a monolayer of a receptor-positive cell line, where the specified receptor-specific microsphere binds to a receptor-positive cell line;
(iii) remove the remaining irrelevant unbound microspheres by gentle washing and decanting and
(iv) remove the receptor-specific microsphere (s) using a micromanipulator. Alternatively, the intact cell is analyzed for the presence of receptors and receptors immobilized on the membrane that require a functional lipid domain of the cell membrane, the receptor molecules can be enclosed in liposomes to which a signal group or enzyme can be attached. Although the above examples relate to peptide ligands, any of the bio-oligomers described previously in sections 5.1, 5.2 and 5.3 can be used in the practice of the present invention. Thus, the acceptor molecule can bind to non-classical, cyclic, conformationally mobile or structurally restricted peptides, oligonucleotypes or peptide-oligonucleotide chimeric complexes.

 In yet another embodiment of the invention, a label may be introduced into the acceptor molecule. In yet another embodiment of the invention, a labeled second reagent can be used to detect the site of attachment of the acceptor molecule to a solid phase carrier containing the bio-oligomer under study. The binding site can be determined by in situ chromophore formation using an enzyme tag. Suitable enzymes for use, but not limiting their scope, include alkaline phosphatase and horseradish peroxidase. In yet another embodiment of the invention, a two-color reaction method using two chromogenic substrates with two enzyme labels on different acceptor molecules to be analyzed can be used. Using this two-color sample, cross-reactive and monoreactive ligands can be identified.

 As other methods, color latex microspheres, magnetic microspheres, fluorescent labels (e.g., fluorescent isocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelating salts of lanthanides, especially Eus, some fluorophores), chemiluminescent molecules, radioisotopes and labels based on magnetograms. Two-color samples can be performed using two or more colored latex microspheres or a fluorophore that emit at different wavelengths. Labeled microspheres can be isolated manually or by mechanical means. As mechanical means, one can use sorting with activation of fluorescence, for example, similarly to cell sorters with activation of fluorescence, and micromanipulators for isolating biomaterials.

 Specific exemplary embodiments of the invention, further described, use an enzyme-chromogenic tag and a fluorescent isothiocyanate (FITC) tag.

 Reactive microspheres can be distinguished based on the intensity of the label, for example, brightness, fluorescence intensity, magnetic field strength or radioactivity. The most intensely labeled microspheres can be selected and sequenced, or else analysis can be performed to determine the structure, for example, mass spectral analysis. In another embodiment of the invention, with random selection of microspheres with increased label intensity, any arbitrary fragment can be selected and sequenced. It is possible to effectively use the most advanced methods of microscopy with visual detection to quantify the intensity of the color reaction, and, therefore, to more correctly determine the relative affinity of the ligand to the acceptor molecule before sequencing the microsphere. Similarly, a quantitative analysis can use the method of immunofluorescence luminescence, if the acceptor molecule is equipped with a fluorescent label. In yet another embodiment of the present invention, microspheres showing some label strength are selected from a library for component analysis, for example, determination of amino acid composition. You can get an audited library containing a limited set of monomeric subunits, which, according to the analysis of the chemical composition, are identified as important, and then screened.

 In another embodiment of the present invention, the bio-oligomer (s) with the highest binding affinity, then the affinity constant, can be identified by serial dilution of the solution of the studied acceptor molecule to the point of detection of its binding to only a few solid-phase carriers, alternatively, more stringent conditions of the binding solution can be provided or in the case of nucleic acids hybridization with the target nucleic acid, that is, an acceptor molecule. Anyone skilled in the art generally knows that more stringent binding conditions can be achieved by (i) increasing the ionic strength of the solution; (ii) increasing the concentration of denaturing compounds, for example, urea; (iii) raising or lowering the pH to a neutral state (pH 7.0); in the case of nucleic acids, bring to the melting point (Tp). Other means are known for changing the titer of a solution to limit binding by a high affinity mechanism. A high tetra of an acceptor molecule solution and its binding under strict conditions with a bio-oligomer immobilized on a solid-phase support can be used to detect the analyzed ligand in a randomized library containing all or almost all possible combinations of monomeric subunits, or in an audited library with a limited set of monomeric subunits.

 In another embodiment, low affinity bio-oligomers may also be of interest. They can be selected from the library, initially extracting all bio-oligomers with high binding affinity, followed by detection under conditions of reduced stiffness and lower titer of the solution.

 In a preferred embodiment, a double-labeled sample can be used. The first label can be used to detect non-specific binding of the studied acceptor molecule to microspheres in the presence of a soluble ligand. Labeled microspheres are then removed from the library, and then the soluble ligand is removed. After that, an acceptor molecule is detected showing specific binding to the remaining microspheres of the library. Bio-oligomers immobilized on such microspheres can be assumed to bind an acceptor molecule at the same binding site as the ligand under study and, therefore, mimic this ligand. Double-label analysis has the advantage that it eliminates the need for purification of the studied acceptor molecule, since the first stage of the analysis allows the isolation of non-specific positively responding microspheres.

5.5.2. Methods for determining biological activity
The present invention also provides methods for determining the biological activity of any bio-oligomer from a library by processing it, which removes any toxic molecules remaining from the synthesis, for example, by neutralization with thorough washing with a solvent, sterile water and culture medium. Tests to determine the biological activity can be carried out on toxicity, killer activity, stimulating and growth-stimulating activity and the effect of causing physiological rearrangement of the body.

 In a preferred embodiment of the present invention, the bio-oligomers of the proposed library are capable of being selectively released from a solid phase carrier, also referred to herein as a “microsphere”. In one embodiment, the microspheres are prepared in such a way that only one fraction of bio-oligomers can be selectively released. Selectively released bio-oligomers, linkers and microbuses are discussed above in section 5.4. The library of bio-oligomers is treated with cleavage agents, resulting in the release of one fraction of bio-oligomers. As disintegrating agents, it is possible, but not limiting their scope, to use, for example, UV radiation, an acid, a base, an enzyme or a catalyst. In one embodiment of the invention, the library is processed in such a way that 10 to 90% of bio-oligomers are released. In a more preferred embodiment, 25 to 50% of bio-oligomers are released. In the case when all bio-oligomers are capable of release, non-selective cleavage can be carried out by limiting the action of the cleaving agent. In one embodiment, the exposure time and the intensity of the UV radiation can be limited. In another embodiment, the concentration of the cleavage agent is limited. After processing to ensure cleavage, the library can be reprocessed, for example, by a neutralization reaction to ensure biocompatibility with the desired plug. In practical implementation, any specialist can easily determine the appropriate hydrolysis conditions for partial cleavage, if all the bio-oligomers of the library are immobilized on a solid-phase carrier by means of cleavable linkers or bridging groups. Any specialist should also be aware that the relative concentration of the released bio-oligomer is affected by a change in hydrolysis conditions.

 The immobilization of microspheres on the solid phase creates a concentration gradient of a specific bio-oligomer. High concentrations of bio-oligomer are noted, as a rule, near the location of the microsphere from which it is released. Thus, the establishment of biological activity near the location of the analyzed microspheres allows the identification and isolation of the specified microspheres and, consequently, sequencing and determination of other characteristics of the bio-oligomer. Identification of the bio-oligomer is possible due to the fact that a sufficient amount of it remains on the microsphere after partial hydrolysis for sequencing and determination of other characteristics. In another embodiment, the microspheres can be divided into microtiter plate points (for example, 10 microspheres per well), and a portion of the bio-oligomer is isolated and analyzed for biological activity, thereby eliminating possible diffusion-related difficulties. As will be described below, various fractions of the bio-oligomer can be immobilized on a solid-phase support or on a microsphere using various cleavable linkers for sequencing. In the scope of these examples, the term "microsphere" means a solid phase carrier. The following examples are intended to illustrate methods for determining biological activity, but without limiting the scope.

 (i) A population of cells in a monoclonal suspension culture is applied in layers to a liquid medium or semi-solid matrix containing a randomized bio-oligomeric library. In one embodiment of the present invention, this procedure is performed in 96-well microcultural plates with one or more microspheres per well together with a cell suspension. In another embodiment of the present invention, a barrier matrix or cookie-cutter is applied to the cell suspension and library microspheres to create separate chambers. An aliquot of the peptide on each microsphere is covalently linked to a water-cleavable (e.g., diketopiperazine) or light-cleavable linker. As a result, a peptide fraction sufficient to provide a biological effect can be isolated, while a sufficient amount of the peptide remains immobilized on the microsphere for sequencing. The cell suspension can be obtained in the form of a solution, or a semi-solid matrix. After an appropriate incubation period, the cell population is analyzed for growth and proliferative ability, for example, by identification of colonies. In another embodiment, the tetrazolium salt of MMT (3- (4,5-dimethyltriazol-2-yl) -2,5-diphenyltetrazolium bromide) can be added to the cell culture (Mossor, 1983, J. Jmmunol. Methods, 65: 55- 63; Niks and Otto, 1990, J. Jmmunol Methods, 130: 140-151). Succinate dehydrogenase, found in the mitochondria of living cells, turns MMT into formazan blue. Thus, a saturated blue color indicates the presence of metabolically active cells. In another embodiment of the present invention, a radioactive label, for example, tritium-labeled thymidine, can be quantified to determine the proliferating activity of cells. Similarly, peptide synthesis can be visualized with the introduction of 35S-methionine. A peptide released from microspheres exhibiting both promoter and inhibitory activity against cell growth is isolated and sequenced, while the identified peptides of the sequence are reanalyzed in solution with test cultures against indicator cells.

 (ii) In another embodiment of the invention resulting from the above (i), the microspheres of the library are distributed into microtiter plate counts at a rate of about 10 microspheres per well. The microspheres are suspended in the liquid phase. Sufficient peptide is released from each microsphere to exhibit biological activity, although a sufficient amount of peptide for sequencing remains on the microsphere. The supernatant containing the released peptide is transferred to a microplate for cell replication or left in the wells with microspheres. Then determine the biological activity, for example, the ability to grow or proliferate the cell line. The microspheres from the wells showing biological activity are sequenced, followed by preparation and analysis of each fragment to identify fragments exhibiting the studied biological activity.

 iii 3 it is held on the microsphere. The sequential release of the bio-oligomer is due to the use of two different cleavable linkers, or is the result of limiting the action of the cleaving agent, which leads to the release of only part of the bio-oligomer at each stage. To limit this effect, it is preferable to use controlled irradiation of the light-cleavable linker, although partial hydrolysis can be achieved with a carefully defined duration of irradiation with respect to the cleaving agent by chemical and enzymatic methods. A library of sequentially cleaved bio-oligomers is synthesized and distributed into the wells of microtiter plates so that each well contains more than 50, more preferably about 50 to 250 microspheres / well. The microspheres are then digested with about 1/3 of these bio-oligomers. The resulting supernatant is then analyzed for biological activity by replication. Microspheres isolated from the wells, showing biological activity, are then suspended and distributed into the wells of a microtiter plate, assuming that each well contains about 1 to 10 microspheres. The resulting microspheres are then treated to release another 1/3 of the bio-oligomer, and the supernatant is analyzed for biological activity. The microspheres from the wells exhibiting biological activity are isolated and the immobilized bio-oligomer is sequenced. In the case where more than 1 microsphere is detected, all identified sequences are obtained, which are analyzed individually for biological activity. Such a two-stage processing of biological samples leads to the creation of a highly effective method for screening very large libraries of bio-oligomers with specific biological activity.

 (iv) The stimulating activity of cytokine release can be determined by the addition of a suspension cell culture immobilized on a semi-solid matrix, for example, an agarose gel. If the bio-oligomer of the present invention induces the release of a cytokine, for example, lymphokine, growth factor, hormone, etc., then the presence of a cytokine can be detected by the activity of the indicator cell line. Specific tests with an indicator cell line can be performed according to the technique disclosed in paragraph (i) above. In another embodiment of the present invention, the cytokine released during cell stimulation can be blotted onto a membrane, for example, a nitrocellulose membrane, and the presence of the cytokine is determined by immunoassay or receptor-binding analysis.

 (v) In another embodiment of the present invention, the cytotoxic activity of the bio-oligomer can be observed. Zones or colonies containing a non-growing, for example, transformed or cancerous cell line covering the bio-oligomeric library, indicate cytotoxic activity. In a specific embodiment of the invention, in a semi-solid matrix, two populations of cells can cover one another. In this case, it is possible to identify a cytotoxic bio-oligomer to target cells and not showing cytotoxicity to any cell with an antigenic phenotype. This analysis method allows you to quickly identify bio-oligomers that can be used as chemotherapeutic agents. Cytotoxic bio-oligomers include toxic peptides and anti-sense oligonucleotides.

 (vi) Physiological changes can also be detected. In one embodiment of the present invention, a myocardial cell suspension culture is applied to a bio-oligomeric library. One can observe the "beating" of cells stimulated by a bio-oligomer. In another embodiment of the present invention, it is possible to determine the activation of a particular enzyme by detecting an increase in the activity of a specific enzyme in the presence of an appropriate substrate, for example, a chromogen (e.g. MMT, see above (i)), a fluorophore, or a chemiluminescent agent. Alternatively, enzyme activation can be determined by immunological analysis. In yet another embodiment of the present invention, histological examinations may also indicate physiological and morphological changes due to the bio-oligomer of the library.

 (vii) The present invention provides a method for determining the activity of any bio-oligomer in a library on polarized cells, for example, cells with a basal and inertial membrane surface. Polar cell cultures can be prepared on a semipermeable membrane corresponding to the indicated interstitium. The library is introduced into a semi-solid matrix on an interstitial or basement membrane surface. Various effects of the bio-oligomer of the present invention can be determined, such as polar transport, proliferation, intercellular interaction, etc. In particular, by introducing a label into a bio-oligomer, for example, a radioactive label or fluorophore, the transport ability of bio-oligomers can be identified. For a long time there is the problem of creating specifically absorbable molecules. In particular, such molecules could be used for oral and nasal administration of drugs, in the case where transport of molecules from the interstitial to the basal surface of the epithelium is necessary.

 The biological activity of non-degradable bio-oligomers can also be determined. After that, the biological activity of all bio-oligomer-coated microspheres can be screened. In one embodiment, any library of the invention may be isolated from a specific tissue. Microspheres that are specifically absorbed after oral, nasal or subcutaneous administration can be distinguished. In a preferred embodiment of the present invention, they are magnetized or have some other identifying label, and thus they are easily isolated from the tissue.

 Any specialist should be clear that all of the above methods for determining biological activity are applicable to bio-oligomers, including peptides, oligonucleotides or peptide-nucleotide chimeras. The use of peptides or peptide analogs as promoters of growth factor, growth factor inhibitors and regulatory molecules is known. Peptides can act as gene regulators by binding them to the regulatory region on any gene, for example, by enhancing or inhibiting the effects of promoter, enhancer and regulatory proteins. Similarly, nucleic acids on gene expression inducers at the transcription level can act as inhibitors (for example, by binding or blocking promoters, enhancers, transcription termination sites, etc.), processing and translation. It is well known to use an oligonucleotide or oligonucleotide analog to inhibit the translation of specific nRNAs. Any and each of the libraries described earlier in sections 5.1 - 5.3 can be analyzed for biological activity.

 The specialist should also understand that any cell supported in tissue culture, for both long and short periods of time can be used in the analysis of biological activity. The term "cell", as used herein, should mean prokaryotic (eg, bacterial) and eukaryotic cells, yeast, mold, or fungus. Culture-preserved primary cells or cell lines may be used. Moreover, the applicant contemplates that bioassays for viruses can be carried out by infection or transformation of cells with a virus. So, for example, but without limiting the volume, it is possible to determine the ability of a bio-oligomer to inhibit the ligogenic activity of the bacteriophage lambda by identifying infected E. coli colonies that do not form clear plaques upon infection.

 The proposed methods for determining the biological activity of any bio-oligomer library are not limited to the above examples; Applicants believe that any analysis system can be modified and included in the invention disclosed herein, and such modifications should be within the scope of this invention.

5.5.2.1. Bioassay for an erythropoietin agonist
In a specific embodiment, the present invention provides a method for determining the bio-oligomeric erythropoietin agonist. It should be borne in mind that the method disclosed in this invention offers an effective strategy for identifying any agonist, for example, an agonist of growth factors, hormones, cytokines, lymphokines and other intercellular transporters, which are described earlier in section 5.5.2.

 In this example, the bio-oligomeric library may consist of pentapeptides obtained with 19 common amino acids (except cysteine). The estimated number of distinct peptides is 2,476,099. The library can be synthesized from 19 cycles, which facilitates screening. Such a library is screened by selecting only one amino acid of the C-terminus at each stage, which results in ordering only the positions of the other four amino acids. Thus, the sequence of each step corresponds to a XXXXY linker resin, where Y is selected for this step, and X stands for arbitrary administration of amino acids. This method reduces the number of possible peptides of each stage to 130,321. If you add a 96 well microtiter plate of 10 microspheres (peptides) to each well, 136 microtiter plates are required (one additional tablet is required as standards and controls for bioassay, so their total number is 137). The specified method allows you to distribute the full cycle of creating the library for the specified number of micronization tablets in one working day.

Most (50-80%) of the peptides synthesized on the microspheres can be cleaved for use in the biological activity assay, resulting in at least 50 pmol being constantly released from each such microsphere. As a cleavable linker, it is preferable to use diketopiperazine
a bridging group, although a light cleavable linker can also be used. This group is quite stable under mild acidic conditions (for example, 0.1 - 1.0 mmol HCl), which allows you to distribute the microspheres in 6-8 hours. Cleavage is accelerated (pH 8.5). Cleavage takes place overnight (12-18 hours) while maintaining the final volume of the hole. The degree of evaporation can be adjusted by keeping the tablet in a humidified chamber.

 The peptide solution obtained by hydrolysis of the microspheres of the library must meet the requirements of asepsis if there is no sterility. Compliance with aseptic requirements is necessary, since the solution is 25% of the volume of the final product, while the cultivation time is at least 24 hours. The aseptic regimen can be achieved (1) by hydrating the microspheres in sterile water after completion of the synthesis, (2) diluting the initial suspension culture of the microspheres in acidified sterile water, and (3) using a sterile technique to distribute these microspheres. In the final suspension culture of the microspheres, at least 20% DMSO is present to facilitate the solubilization of hydrophobic peptides. DMSO (DMSO) should be introduced, if necessary, at the beginning of hydration to facilitate the solubilization of peptides. The final concentration of the suspension culture of the microspheres is 10 microspheres / 50 μl. Retention of the microspheres in suspension during pipetting can add about 0.8 to 3.2% methylcellulose to it. The use of methyl cellulose can reduce the effect of DMSO, which is used to accelerate solubilization. The final concentration of methylcellulose in the cultures is less than 0.8, so as not to affect the bioanalysis.

 The released peptides can be transferred to microbiological plates in the form of 50 μl of samples, providing an exact match between sample plates and culture plates. During this transfer, sterile conditions must be ensured. Recombinant human EPO can be added to selected wells of plates for use as a positive control (each tablet) and for constructing calibration curves (first and last plate). Control wells may contain 0.1 M units. EPO, with calibration curves obtained from six sets of duplicate wells, containing from 1.0 to 100 milli units of EPO (D 'Andrea et al., 1991, Moll.Cell.Biol., 11: 1980-1087).

A bioassay can be obtained with a Ba / F3-T recombinant cell line expressing an erythropoietin receptor (EPO receptor). These cells depend on the presence of either interleukin-3 (IL-3) or EPO. Cultures of these cells containing IL-3 (supplied as 10% (v / v) WEHI -conditioned culture medium) prevents the possible effects of EPO in a bioassay environment. As a basal culture medium, RPMI 1640 medium containing 2.0 g / l NaHCO ₃ , 10% fetal bovine serum (v / v), 1 x penicillin-streptomycin, 5 μl beta-mercaptoethanol and / or 10 mM HEPES buffer is used (final concentration 7.0). It is necessary to add 10% WEHI - conditioned medium (v / v) to the indicated medium, added to IL-3 WEHI - conditioned medium is obtained by culturing WEHI cells with their lysis in the same medium. After this, the conditioned medium is centrifuged to remove cells, passed through a 22 μm filter and stored in a frozen state. Ba / F3-T cells are cultured with a yield of 1.31 and 10 ⁷ cells / well, which are distributed in points in the amount of 1 • 10 ³ cells / well in a volume of 150 μm according to the method described in (Joshimura et al., 1990, Proc. Natl. Acad. Sei USA, 87: 4139-4143).

Ba / F3-T cells are transferred from roller bottles to large (250 ml) sterile roller flasks. Cells are harvested by centrifugation at a speed of about 500 • r / 5 min. After this, the cells are resuspended in 200 ml of fresh basal medium without the presence of JL-3 for cell counting. The final volume was adjusted to a titer of 6.67 x 10 ³ cells / ml (1 x 10 ³ cells / 150 μl) with the addition of the indicated medium. After this, it is necessary to divide the resulting suspension cell culture into 4-8 aliquots for storage in an incubator during a long cell distribution process. The number of cells and their viability should be determined by samples taken at the beginning and at the end of the distribution process to ensure the presence of the same number of viable cells in the first and last culture plate. The obtained cells are distributed on culture plates containing the supernatant from the released peptides, followed by their incubation for 3 days (Joshimura et al., See above).

 The endpoint bioassay titer corresponds to the number of living cells present in each cell culture well. It can be determined using a Mosmann test on MTT (1983, J. Jmmunol. Methods 65: 55-63), modified by Nixon and Otto (1990, J. Jmmunol. Methods, 130: 140-151). The specified modified technique allows you to determine the number of viable cells without removing them from the culture medium.

MTT (3 (4,5-dimethyltriazol-2-yl) -2,5-diphenyltetrazolium bromide is obtained as a 5 mg / ml solution in PBS (about 270 μl). 20 μl of this solution is pipetted into each well of a bioassay plate, followed by incubation for 4 hours at a temperature of 37 ^o C. After that, 100 μl of extraction solution is added to each well and the plate is placed in an ultrasonic homogenizer for 120 minutes.The extraction solution contains 50% (v / v) N, N-dimethylformamide in 20% ( w / v) a solution containing sodium dodecyl sulfate (SDS), adjusted to a pH of 4.7 with the introduction of the mixture acetic and hydrochloric acid according to the method described by Mansen et al. (1989, J. Jmmunol. Methods, 119: 203). With this treatment, the formazan metabolic product of MTT is solubilized to determine the optical density at a wavelength of 570 nm using a plate reader.

 The obtained optical density data of the bioassay is averaged over all wells and a 95% confidence interval of the averaged value is determined. The optical density values of each well beyond this interval use the determination of the degree of reliability by the Student method. Reliability indicators are compared with the EPO calibration curve and calculated data are obtained in the form of the dependence of M. units-EPO. The calibration curve is determined by non-linear regression analysis using a logical equation. The data and obtained from the two calibration curves are analyzed separately, to determine, if there is a significant difference, the reaction rates taken at both ends of the bioassay (test to determine the F-index). A comparative analysis of the control values obtained for each tablet for each sample is used to determine if there is a constant change in the bioassay reaction.

 It must be pointed out that two growth factors receptors (IL-3 receptor and EPO receptor) are present on Ba / F3-T cells, and that activation of any of them produces a positive bioassay reaction. Thus, the biological activity assay described previously is selected for both the JL-3 receptor agonist and the EPO receptor agonist. There are two ways to solve this problem. The first is to use a strain of different cells, or possibly spleen cells, taken from mice injected with phenylhydrazine (Krystal, 1983, Exp. Hematol., II, 649-660). The second solution to the problem is to study synthetic peptides for biological activity against EPO and IL-3 by conducting a second bioassay, or using methods of binding to a radiolabeled ligand.

5.5.3 Enzyme mimetics and enzyme inhibitors
The present invention also includes within its scope bio-oligomers that catalyze reactions, in particular an enzyme library, as well as acting as coenzymes or inhibiting enzymatic reactions. Accordingly, the present invention provides methods for determining enzyme and coenzyme activity or inhibition of enzyme activity.

 Enzyme activity can be observed in the formation of a detectable reaction product. In a specific embodiment of the present invention, the bio-oligomer of the library catalyzes an enzymatic reaction of an alkaline phosphatase-based substrate, for example 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), and forms a blue, unsolubilized solid-phase reaction product (see Example 13 below) )

 In another embodiment of the present invention, a zone of a visible product, for example, dyed or fluorescent, can be obtained on a semi-solid matrix. The library is obtained in layers in a semi-solid matrix, for example, an agarose gel, and then a chromophore or other indicator substrate is added. In the area where the bio-oligomer immobilized on a solid-phase substrate exhibits the analyzed enzymatic activity, a zone of the visible product is formed. For example, but in no way limiting the volume, the bio-oligomeric horseradish peroxidase analogue can be identified by adding a solution containing aminoantipyrene (0.25 mg / ml; Kodac), phenol (8 mg / ml) and water (0.005%) to 0. 1 M phosphate buffer, pH 7.07. Microspheres with enzymatic activity form a purple color zone. In another embodiment of the present invention, protease activity bio-oligomers / microspheres can be identified by adding colorimetric protease substrates well known to those skilled in the art.

 Coenzyme activity can be observed on a sample analyzed for coenzyme-mediated enzyme activity, where there is no natural or linked coenzyme.

 Enzyme-inhibiting activity can be detected in the case of a partially-released bio-oligomer. The release of bio-oligomers is discussed previously in sections 5.4 and 5.5.2. In one embodiment of the present invention, but not limiting the scope, a library is prepared in layers in a semi-solid matrix that contains any enzyme. The library is treated with partial release of the bio-oligomer. In the area where the bio-oligomer inhibits enzymatic activity, an invisible product zone can be identified. In one embodiment of the present invention, where a chromogenic substrate is used as the enzyme substrate, a colored product is formed. Thus, the presence of an enzyme inhibitor leads to the formation of an invisible zone. In another embodiment of the present invention, inhibition of the proteolysis of hemoglobin or an indicator enzyme, for example, alkaline phosphatase, can be determined by the presence of an opaque zone in the semi-solid matrix. In other words, the presence of a protease inhibitor prevents the degradation of hemoglobin and the indicator enzyme.

 Any specialist knows well that any bio-oligomer exhibiting enzyme and coenzyme activity or inhibiting the activity of enzymes may refer to a peptide, oligonucleotide, or peptide-oligonucleotide chimeric complexes. Of particular interest are limited and cyclic peptides that produce a unique catalytic center or binding surface (see section 5.2.1 above). Moreover, peptide-oligonucleotide chimeric complexes are thought to be capable of exhibiting a specific chemical ability, for example, enzyme or coenzyme activity, due to the unique arrangement of the corresponding functional groups in close proximity to each other. In addition, a solid phase carrier with a bio-oligomer immobilized therein is believed to be able to exhibit enzyme or coenzyme activity, while an unbound bio-oligomer may have no activity. Such a high concentration of bio-oligomer near the location of the corresponding functional groups can transfer the unique chemical properties of the bio-oligomeric product on a solid-phase carrier. It is also believed that any bio-oligomer may exhibit enzymatic or coenzyme activity upon release from the microsphere. Well-known coenzymes (cofactors) are believed to be chemically incorporated into a conformationally restricted bio-oligomer to stimulate a simple or complex enzyme, including, for example, an electron transport chain.

5.6 Methods of characterization of bio-oligomers
The present invention provides a means for determining the structure and sequence of a bio-oligomer immediately after isolation from the library of a microsphere containing the bio-oligomer under study by any of the methods described previously in section 5.5.

 In the case where the bio-oligomer relates to a peptide, the Edman degradation method is used as the preferred method for sequencing. An especially preferred sequencing method uses the Applied Biosystem Model 477 A protein sequencer. The amino acid sequence of the peptides can also be determined either by fast atomic bombardment mass spectroscopy (PAB-MS) or other analytical methods.

 The resulting peptides can be sequenced by immobilizing them on a solid phase carrier, or by cleaving them from a solid phase carrier. To peptide off the peptide, the isolated microspheres with the peptide are treated with conventional cleaving agents, which are used by specialists, to separate the polymer from the solid-phase substrates. The selection of suitable cleavage agents depends on the solid phase carrier used. So, for example, to remove peptides from Wung resin, it is preferable to use 50% trifluoroacetic acid (TPA) in dichloromethane.

 In an alternative embodiment of the present invention, it is possible to isolate only one solid-phase carrier, for example, a microsphere, with bio-oligomers attached to it and then transferring it to a sequencer without first cleaving the bio-oligomers from the microsphere. So, for example, if the studied bio-oligomers belong to a peptide, approximately 200 pmol of peptide is contained in a separate microsphere based on a polymer resin with a diameter of 100 μm, which is equivalent to 0.5 meq / gram of resin. In one 250 μm microsphere based on polyacrylamide resin at the rate of 0.5 meq / gram of resin contains 3125 pmol of peptide. When using a traditional peptide sequencer, only 5-10 pmoles of peptide are necessary for correct sequencing. Thus, one microsphere based on a PAM resin with a diameter of 100 μm of a standard size contains a peptide in an amount greater than is necessary for sequencing.

 In the case where the amino acid peptides or initiating peptides (pseudopeptides) that cannot be analyzed by the Edman degradation method, it is possible to obtain such a microsphere provided that 10-50% of its peptides should not contain a sequence with an unsequenced amino acid residue. You can determine the structure of the remaining amino acid sequence, and then on its basis to extrapolate the sequence containing non-sequencing residue.

 Another method for determining the structure of non-sequencing residues is to temporarily cap a portion of the indicated peptide before introducing a non-sequencing residue during library synthesis. In the process of subsequent structural identification, Edman degradation can be used up to the non-sequencing residue, followed by removing the temporary “cap” and resuming sequencing of a distant region (for example, the C-terminal region) in the direction of the non-sequencing residue.

 In the case of oligonucleotides, sequencing can be performed on an automatic oligonucleotide sequencer (e.g., Applied Biosystems). Alternatively, it is preferable to use the method of Maxam and Hilbert (1977, Proc. Natl. Acad. Sci USA, 75: 560-564). Other oligonucleotide sequencing methods known in the art may also be used.

 Mass spectroscopy with fast ion bombardment provides the most effective structural analysis possible. When detecting fragments, as well as bio-oligomeric forms themselves, any sequence can be reconstructed. Data on the structure and sequence of oligonucleotides can also be obtained using high resolution electrostatic spray mass spectrometry (Finnigan, MAT).

 Immediately after determining the sequence of the selected bio-oligomer, a large number of peptides can be chemically synthesized using an automatic peptide synthesizer or other means used in bio- or chemical synthesis. Moreover, as soon as the bio-oligomeric sequence is established, subunit analogues can be replaced to enhance the activity of a specific bio-oligomer.

5.7. Therapeutic diagnostic tools based on randomized bio-oligomeric libraries
Once the test bio-oligomeric sequence has been established, the present invention provides the use of molecules containing the specified bio-oligomeric sequence for therapeutic or diagnostic purposes. The indicated bio-oligomer sequence may itself provide a diagnostic or therapeutic agent, or it may be incorporated into a larger molecule. Any molecule containing any sequence of bio-oligomer with biological or binding activity may be called an "effector molecule. The invention also provides libraries for various applications. The effector function of such an effector molecule may be any of those described or known in the art.

 The method disclosed in this description offers not only a new tool for searching for specific ligands with potential diagnostic and therapeutic effects, but also provides important information on a number of ligands that differ greatly in their primary structure and chemical composition, but nevertheless can physically interact with one and the same acceptor molecule. The integration of such information with molecular modeling methods and modern computer technology will probably lead to the creation of a new fundamental concept of ligand-receptor interactions.

 The drugs of the present invention contain effector molecules that bind to the biologically active center of cytokines, growth factors or hormones, and thereby enhance or neutralize their action, as well as inhibiting or promoting transcription and / or translation.

 Medicines of the present invention include, for example, effector molecules that bind to a receptor of pharmacological interest of interest, for example, as growth factor receptors, neurotransmitter receptors or hormone receptors. These effector molecules can be used as either agonists or antagonists of the ligand-receptor interaction.

 Another use of effector molecules that bind to receptors is to use binding to block the attachment of viruses or microbes that gain access to the cell when attached to the normal cellular receptor and invade it. As an example of such a phenomenon, one can mention the binding of HIV to the CD4 receptor, as well as the herpes virus vulgar with the receptor of fibroblast growth factor. Effector molecules that invade the receptor could be used as drugs to inhibit viral infection of target cells. Spurious cell invasion can be likewise inhibited after identification of the corresponding effector molecules of the present invention.

 In another embodiment of the present invention, an effector molecule containing a sequence of bio-oligomers that binds to any acceptor molecule of interest can be used for targeted delivery of a drug or toxin. In a preferred embodiment, the indicated acceptor molecule may be a receptor or antigen identified on the surface of a tumor cell, an animal parasite or a microbe, for example, a bacterium, a virus, a single cell parasite or pathogen, fungus or mold.

 It is also possible that several of the millions of bio-oligomers in a pool can represent sequences that have biological activity. For example, bio-oligomers that have antitumor, antiparasitic or antimicrobial activity, for example, antifungicidal, antibacterial activity against a monocellular parasite or pathogen, or antiviral activity, can be distinguished. Moreover, some of these bio-oligomers can act as agonists or antagonists of growth factors, for example, erythropoietin, epidermal growth factor, fibroblast growth factor, tumor growth factors, as well as hormones, neurotransmitters, or other regulatory molecules. In one embodiment, peptides are used as such bio oligomers.

 The therapeutic agents of the present invention also include within their scope effector molecules containing a bio-oligomeric sequence with high affinity for drugs, for example, digoxin, heroin, cocaine or theophylline. Such peptides can be used as antidotes for overdosing of such indicated drugs. Similarly, therapeutic agents of the present invention include effector molecules conjugated to small molecules or metal ions, including heavy metal ions. Bio-oligomeric sequences with high affinity for bilirubin can be used in the treatment of newborns with high bilirubin in the blood.

 Moreover, the present invention provides methods for identifying bio-oligomeric sequences used for the treatment of diseases or pathologies, for example, which are listed in the Index of Categories of Organic Foods of the Doctor's Handbook (PDR, 1991, 45th edition, Medical Economics Data: Oradell N 1, p. 201 - 202). For example, one can identify the effector molecule with the activity of any of the antiparasitic, anticoagulant antagonist, antidiabetic, anticonvulsant, antidepressant, antidiarrheal, antipotnogo, antigonadotropnym, antihistamine, antihypertensive, antiinflammatory, antipruritic, antimigraine, protivoparkinsonnogo, antiplatelet, an anti-inflammatory, antipyretic, toksinneytralizuyuschego, bronchodilatory , vasodilating, chelating, contraceptive , Miorelaksantnogo, antiglaucoma or sedative.

 The medicaments of the invention may also contain appropriate pharmaceutically acceptable carriers, diluents and adjuvants. As such pharmaceutical carriers, sterile liquids, for example, water and oils, including petroleum, animal, vegetable or synthetic oils, for example, peanut, soy, mineral, sesame oil and the like, can be used. Water is used as the preferred carrier if the drug is administered intravenously. As liquid carriers, physiological solutions and aqueous solutions of pextrose and glycerol, especially infusion solutions, can also be used. As pharmaceutically acceptable excipients, starch, glucose, lactose, sucrose, gelatin, maltose, rice flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene can be used. water, ethanol and the like. These pharmaceutical compositions can be prepared in the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release dosage forms and the like. Pharmaceutically acceptable carriers are described by E.W. Martin in the journal Renington's Pharmaceutical Sciences. Such compositions typically contain an effective amount of the active compound in admixture with an appropriate amount of a carrier in a form suitable for administration to a patient. Although intravenous infusion is a very effective form for administering the drug, other forms can be used, for example, by injection, or by oral, nasal or parenteral administration.

 A molecule containing a bio-oligomeric sequence identified according to the present invention can also be used to obtain diagnostic tools. A diagnostic preparation can be prepared on the basis of one or more bio-oligomer sequences of the invention, for example, more than one peptide or oligonucleotide sequence. In addition, the diagnostic tool may contain any of the above pharmaceutically acceptable carriers.

 Used in this description, the term "diagnostic tool" means any substance that can be used to detect diseases, for example, but without limiting the volume of cancer, for example, as T- or B-cell-mediated lymphoma and the infectious diseases mentioned above . The term "detection" is used in this description in the broadest sense, covering confirmation of the presence of the disease, the location of the affected organ or an indicator of the severity of the disease. For example, a peptide-immunoperoxidase complex or similar immunohistochemical agent can be used to detect and analyze specific receptors or antibody molecules in tissues, serum or total water in the body. Diagnostic agents can be used in vitro or in vivo. More specifically, the present invention provides effective diagnostic reagents for use in immunoassays, southern or Northern blot hybridization, and in situ assays.

 Moreover, the proposed diagnostic tool may contain one or more markers, for example, like, but not limiting their scope, radioisotope, fluorescent labels, markers based on paramagnets or other substances that enhance the image. Specialists in this field usually know a lot of markers and methods of their introduction to obtain diagnostic agents.

 The drugs and diagnostic tools of the present invention can be used to treat and / or diagnose diseases in animals, more preferably mammals, including humans, as well as experimental mammals, for example, dogs, cats, horses, cows, pigs, guinea pigs, mice and rats. Medicines and diagnostics can also be used to treat and diagnose plant diseases.

 Diseases and syndromes that can be treated or diagnosed with the bio-oligomers found according to the present invention are also numerous and vary over a wide range, as well as structural variations in a randomized bio-oligomeric library. The following examples are intended to be illustrative only and not to limit the scope of the invention.

5.7.1. Cytotoxic compositions
A molecule containing a bio-oligomeric sequence may exhibit specific cytotoxic activity in relation to itself. Moreover, the bio-oligomer binding the studied acceptor can be modified by routine methods used in this field, for example, by conjugation with cytotoxic substances, for example, drugs or radionuclides to form a cytotoxic carrier molecule. Such a conjugate of a cytotoxic substance with a bio-oligomer, for example, a peptide, can provide a targeted supply of this substance and specifically destroy cells representing a specific acceptor molecule. For example, such cytotoxic peptide conjugates can directly destroy unwanted B-cell populations, B-cell lymphomas, T-cell populations, or T-cell lymphomas in a patient. As a possible clinical use of such conjugates, one can indicate their use for the treatment of autoimmune diseases, lymphomas and in immunosuppressive therapy for organ transplantation. Other cancer forms in which cancer cells exhibit receptor-mediated ligand binding, such as breast or uterine cancer, where epidermal growth factor receptors (EGPs) are thought to play an important role, can also be treated using this technique.
Cytotoxic preparations specific for the target cell, for example, a cancer or virus-infected cell, but not leading to the death of immunophenotypic cells, tissues or organs, can be obtained by the methods of the present invention. In addition to the targeted delivery of cytotoxic drugs to target cells, for example, cancerous, these specific toxins can be used as antimicrobial agents. In particular, these drugs may exhibit bacteriostatic, bactericidal, antiviral, antiparasitic or fungicidal activity. Similarly, toxins exhibiting insecticidal or herbicidal activity can be identified.

In one embodiment of the present invention, a peptide can be used as a bio oligomer. Such a peptide can act as a carrier with any toxin immobilized on it. The specified peptide can itself infect the cell and act as a toxin. In another embodiment of the present invention, an oligonucleotide may be used as such a bio oligomer. The specified oligonucleotide can transmit its toxic effect at the level of transcription and translation of the gene responsible for cell viability
5.7.2. Immunomodulators
The present invention provides molecules and compositions for use as immunomodulators. The term “immunomodulator” is a molecule or compound capable of effecting changes in the immune system. In particular, immunomodulators can stimulate or inhibit T-cell immune responses, a humoral immune response, and non-specific immune responses, for example, which are mediated by macrophages, neutrophils, polymorphic nuclear phagocytes, granulocytes and other cells of the myeloid line of differentiation. Effective molecules can be used to treat the following diseases associated with impaired immune systems: (1) various autoimmune diseases, including myastenin gravis, multiple sclerosis, Graves disease, rheumatoid arthritis, systemic lupus erythematosus (SLF), pemphigus vulgaris, autoimmune hemolytic anemia , immune thrombocytosis, (2) Hodgkin's disease and various other cancer forms, (3) allergies, (4) immune symptom complexes, (5) conditions associated with rejection of transplanted organs, (6) infectious pain znei and (7) diabetes.

 Immunomodulators can stimulate the immune effect by mimicking the activity of a stimulating lymphokine, for example, (IL) -1, (IL) -2, (IL) -3, (IL) -4, (IL) -6, granulocyte colony stimulating factor (CSF) , macrophage-colony-stimulating factor; and granulocyte-macrophage-colony-stimulating factor. Such stimulation can be provided by a peptide that binds a ligand to a lymphokine receptor, or an oligonucleotide that acts as a activator of activation of cell regulation at the level of transcription. An immunomodulator can be active in binding to a white blood cell or lymphocyte, for example, as the Gc receptor, LAP-1, LAP-2, etc., including phagocytic activity and cytoxin release activity. The effector molecule of the invention may act as chemotaxin.

 In a preferred embodiment of the present invention, a molecule containing a bio-oligomer can mimic an antigen. In such a form, said bio-oligomer can be used as a vaccine for the manifestation of a T-cell or B-cell reaction specific for a particular pathogen.

 The proposed effector molecules are believed to have an effective effect in released form. However, a separate bio-oligomer may show a greater effect if it remains immobilized on a solid-phase carrier. In particular, a simulator with a high density of antigenic determinants can more effectively stimulate a humoral immune response by binding and capping a membrane immunoglobulin or other receptor-mediated immune response.

 In addition, a limited library of the invention is believed to be useful as a vaccine against a pathogen represented by a plurality of epitopes. It is known, for example, that the primary structure (sequence) of VSG (variable membrane glycoprotein) trypanosomes changes continuously during infection. Pori changes the VSG trypanosome epitope loses immunological recognition. Similarly, blood-sucking parasites have been found to express multiple antigenic epitopes between species at different stages of the life cycle, and within subspecies. Thus, a peptide library with limited variability could be used to immunize against the caribel antigenic diversity represented by trypanosomal or blood-sucking parasites. A limited library can be used as a vaccine in any case where immunity to a number of antigens is required.

 The effector molecules of the invention are also believed to be able to inhibit the immune response by (i) blocking specific immunological recognition at the antibody or T cell antigen receptor level; (ii) blocking the Fc fragment or other immune receptors; or (iii) binding to lymphokines and cytokines and inhibiting their activity; (iv) creating negative feedback signals to immunocompetent cells. Effector molecules can be used for tolerization of the immune system, in particular, for autoantigens. So, for example, immune tolerance against DNA can be provided by any oligonucleotide or oligonucleotide library, and in the treatment of systemic lupus erythematosus. Moreover, inhibition of the immune response can be achieved by the mechanisms described above in section 5.7.1.

 The drugs of the present invention may also include selected synthetic antigenic peptides, with which you can manipulate the immune system so as to deprive this antigen of immunogenicity and, therefore, to suppress or cure the corresponding autoimmune disease. Similarly, the use of specific synthetic antigenic peptides can inhibit the formation of multimeric immune complexes and, therefore, prevent the occurrence of specific immunocomplex diseases.

 Peptides that bind to tumor-specific monoclonal antibodies can be isolated, sequenced and synthesized for use as an immunogen that induces an active immune response against this neoplasm.

 Specific peptides exhibiting high affinity for Fc fragment receptors can be used as drugs for inhibiting the Fc receptors of the reticuloendothelial system, which should be especially effective for patients with autoimmune diseases, for example, idiopathic thrombocytopenia and autoimmune hemolytic anemia.

 The potential for the use of such peptides may be even greater. Peptides that are similar to the epitopes of invasive organisms can be used to block infection with such an organism. For example, recent studies of diseases associated with acquired immunodeficiency (AIDS) have shown that infection with the AIDS virus begins with recognition and binding between a specific glycoprotein (dr 120) on the shell of the AIDS virus - CD4 - a surface receptor on the cell. The introduction of a peptide that mimics protein dr 120, can significantly inhibit the CD4 receptor, as a result of which the AIDS virus loses its ability to bind to and infect this cell. Similarly, parasitic infestation and infection can be suppressed.

5.7.3. Neuroreactive Agonists and Antagonists
The effector molecules of the invention are believed to act as agonists (mimics) or antagonists (inhibitors) of the effects of hormones, neurotransmitters, analgesics, antipsychotics, antidepressants or drugs. Such effector molecules should be used in the search for regulators of appetite, psychiatric drugs, regulators of attention and absorption, and anamnestic drugs. The present invention also provides substitutes for flavoring and aromatic additives, for example, substitutes for sweets, salt and aromatic additives.

5.8. Limited libraries
In addition, a limited library of the present invention can be used as a mixed aromatic additive, for example, as a spice, at its low cost and without possible allergic reactions from such aromatics. In this case, you can get inexpensive substitutes for aromatic additives, such as saffron. In another embodiment of the present invention, a new class of aromatic substances can be obtained.

 In yet another embodiment of the present invention, a limited library can be used as a carrier for chromatography. A library of bio-oligomers, for example, peptides that exhibit the same chemical properties but have a wide variety of sequences, is believed to be a more efficient chromatographic support compared to current carriers. Such a chromatographic carrier has greater selectivity than an ion exchange or reverse phase carrier. In yet another aspect of the present invention, the purified acceptor molecule can be easily eluted from said carrier under milder conditions than, for example, a high affinity carrier can be used, which reduces the likelihood of denaturation. In one embodiment of the present invention, said carrier is prepared based on the composition or structure of bio-oligomers that have been found to either exhibit the affinity of the intermediate, for example, the labeling intensity of the intermediate form, or only bind at a high concentration of a specific acceptor molecule. Moreover, a highly selective carrier used under reduced stiffness conditions can be quickly identified without cleaning it (see section 5.5.1 above).

 In another embodiment of the present invention, low affinity microspheres can be selected and a limited library can be obtained based on the composition of the selected microspheres. In yet another embodiment of the present invention, a conventional low affinity or high affinity carrier comprising one or more bio-oligomeric sequences identified from millions of sequences obtained according to the present invention can be used for chromatography.

 The following examples further illustrate the invention and are intended for illustrative purposes only.

6. Example: Synthesis of a tetrapeptide library
The proposed method is used for the synthesis of a tetrapeptide family of the formula XXX-Trp, where X can mean either valine, serine, or alanine, provided that tryptophan is always used as the first amino acid. Tryptophan is introduced at the C-terminus to facilitate spectrophotometric analysis at OD 280.

 Na-Fmos-tryptophan-alkoxymethyl-polystyrene, which is described by Wang (1973, J. Amer. Chem., 95: 1328-1333), obtained from Bachem. Inc. Torrence, Ca, and placed in a conventional solid phase peptide synthesizer. Fmos amino acids supplied by Bachem Inc. are also used as input amino acids. The other reagents used are almost the same as those used in the routine solid-phase peptide synthesis method described by Austem (1986, Peptide Synthesis: Methods in Moleculae Biology, pp. 311-331).

 Teflon-coated bioreactor used for peptide binding reaction; a standard bioreactor for solid-phase protein synthesis is used as a mixing chamber, where aliquots are mixed after binding is completed.

 Approximately 0.5 g of the Fmos-Trp-alkoxymethyl-polymer is allowed to swell in dimethylchloromethane. Then the specified biopolymer is washed twice with dichloromethane, once with a mixture of dichloromethylformamide, 1: 1 and three times only with dimethylformamide. After thoroughly washing the biopolymer with the removed protective groups with dimethylformamide (3 times), dichloromethane (3 times), 1: 1 dimethylchloromethane-dimethylformamide mixture, this biopolymer is divided into three separate aliquots of approximately 2.5 ml each and placed in three series of conjugation tubes .

 The amount of protected amino acid to be administered is calculated based on the number of moles of tryptophan already immobilized on said biopolymer. For each amino acid introduced, a five-fold excess of the indicated amino acid was introduced into each bioreactor into which aliquots of the washed biopolymer were already introduced. A five-fold excess of a specific amino acid is thus introduced into each bioreactor. After that, each bioreactor is subjected to shaker mixing for 2 minutes and diisopropylcarbodiimide is added in five-fold excess in 3 ml of dichloromethane (DCM), followed by stirring in a shaker for 1 hour.

To verify the degree of completion of binding, a sample from each tube was reacted with nhydhydrin, supplied by Pcerce Chemical according to the method described by Sarin et al. (1981, Anal. Biochem. 117: 147-157), which is specifically incorporated by reference. If the binding reaction is not complete, as shown by the data of the indicated sample, the reaction is brought to completion by the following methods known to those skilled in the art, including (a) conducting a second binding using a protected amino acid in 1-5-fold excess; (b) conducting additional binding using different or additional solvents (e.g., trifluoroethanol); or (c) adding chaotropic salts, e.g., NaClO ₄ or LiBr (Klis and Stewart, 1990, Peptides: Chemistry, Structure and biology, River and Marchal, 2nd edition, pp. 904-906).

 After binding is complete, the biopolymers from the three conjugation tubes are carefully transferred and mixed in a homo mixer. The resulting resin is washed twice with a mixture of DCM / DMF, 1: 1, three times with DCM, three times with DMF and protective groups are removed with a 20% solution of piperidine in DMF, v / v. After thorough washing with DCM and DMF according to the above procedure, the resulting mixture is divided into three aliquots and placed in three separate bioreactors. Then add a second series of amino acids. After complete binding, the obtained biopolymer is first unlocked with a 20% solution of DCM and DMF according to the above procedure. In a similar manner, a third series of amino acids is added.

 To remove peptides from solid phase carriers, 30 ml of 50%, v / v trifluoroacetic acid, 5% anisole and 0.9% (v / v) ethanedithiol in DMX are added to the indicated biopolymer. The resulting mixture was stirred in a shaker for 4 hours, and the peptide supernatant was collected. Thereafter, the indicated peptide supernatant was evaporated on a rotary evaporator and the peptides precipitated in ether. After thorough washing, the peptide precipitate is dried and prepared for subsequent analysis. Lyophilized peptide (in powder form) is stored frozen.

7. Example: Comparative analysis of the proposed method and the traditional method of synthesis of peptides
A library of randomized peptides is obtained according to the procedure described in example 6 above. Moreover, a tetrapeptide library is prepared using standard solid phase peptide synthesis (hereinafter referred to as "SPPS") described by Austen (see above). Na-Fmos-tryptophan-alkoxymethyl-biopolymer, supplied by Bachem, Inc., is used as a solid-phase carrier-amino acid combination. Equimolar amounts of a five-fold excess of Na-Fmos-valine, Na-Fmos-serine (O-Bu) and Na-Fmos-alanine are added to the bioreactor during each binding step. After three successive binding steps, the resulting tetrapeptides are cleaved in a mixture containing 50% (v / v) trifluoroacetic acid, 5% (v / v) anisole and 0.9% (v / v) ethanedithiol in DCM according to the Austen method described above.

7.2 Results
Both peptide libraries are analyzed by C-18 reverse phase HPLC (Vydac) to determine the number of peptide species (by the number of peaks), the relative concentration of peptides (peak zone and the relative hydrophilic nature of these peptides (by early or late elution from the column). The results are presented in Fig. 3). The chromatogram in the upper panel shows the sample obtained together with the peptide library synthesized according to the method according to the present invention, and the chromatogram in the lower panel represents the sample obtained by the standard method of solid-phase peptide synthesis.

 Both chromatograms show 21 distinct peaks, indicating the presence of at least 21 different peptide species within each library. The picture obtained by the SPPS method, however, shows significantly larger peaks at # 1, 2, 3, 4, 5, 6, and 7, indicating that the library obtained by the SPPS method contains a higher concentration of peptides 1-7 compared with peptides 8-21. The increased content of peptides 1-7 shows that these peptides have a preferred synthesis advantage over the remaining 21 peptides. Moreover, these protruding peaks are eluted first, that is, these peptides exhibit a shorter retention time in the column, indicating a more hydrophilic nature of these peptides.

 Such a result cannot be expected when using the SPPS method. Valine is known to be a hydrophobic and bulk substance and has a significantly slower binding rate (as suggested by steric hindrance) than is established for both valine and serine. Thus, in the traditional synthesis of disordered peptides carried out in this invention, in which valine “competes” with alanine and serine for binding sites, the synthesized peptides are valine-depleted, and the resulting peptide library does not exhibit an equimolar distribution of disordered peptides.

In contrast, the chromatogram of a library of disordered peptides created according to the method of the present invention confirms their equimolar distribution. Although peaks 3, 6, 12, 13, and 18 are approximately two times larger than the zone of other peaks, which indicates the presence of two peptides in that area, the remaining 16 peaks have an almost identical picture. Moreover, all 21 peaks lie in the retention time range, which also indicates the equimolar distribution of these peptides. Sequencing of samples with given peaks also confirms this result. Samples with small peaks 8, 9 and 21 and sample 6 with a large peak were sequenced on a 477A protein sequencer from Applied Biosysmems, as follows
# 8 = Val-Ala-Ser-Trp
# 9 = Val-Ser-Ala-Trp
# 21 = Val-Val-Val-Trp
# 6 = (Ser-Val) - (Ser-Ala) - (Ser-Ala) -Trp
These valine-containing sequences confirm the possibility of using the method of the invention for randomized synthesis of peptides, even when known slowly binding amino acids are used.

 The sequence of the largest sample 6 was not confirmed, apparently due to the presence of several peptides at this peak. Most likely, the most important peaks refer to the sequence Ser-Ala-Trp and Val-Ser-Ser-Trp.

7.3. conclusions
The results obtained indicate that the proposed method for the synthesis of disordered peptides allows one to obtain a library of randomized peptides in practically equimolar amounts, in contrast to the standard SPPS method, in which it acquires a set of peptides containing amino acids with a faster binding rate.

8. Examples: Isolation of a Peptide Ligand Binding to a Receptor Molecule
In order to confirm the use of the method of the present invention to isolate a specific peptide, a 12-amino acid peptide with a given sequence is synthesized based on the V-mos gene product. The V-mos gene product relates to an oncogen isolated from murine sarcoma, and refers to the Molony sarcoma virus of mice. It is known that V-mos-
the gene product exhibits serine / threonine kinase activity.

8.1.Materials and methods
The Leu-Gly-Ser-Gly-Gly-Phe-Ser-Val-Tyr-Lys-Ala sequence is synthesized on a polyacrylamide macrosphere (approximately 300 μm diameter) using Na-Fmos chemistry and standard reagents and methods for performing solid-phase peptide synthesis . The side chain protecting groups are removed using a 50% trifluoroacetic acid solution, and the peptide remaining covalently attached to the polyacrylamide resin via a linker at the outlet has the following structure: Leu-Gly-Ser-Gly-Gly-Phe-Ser-Val-Tyr-Lys-Ala α-aminocaproic acid is an ethylene diamine polymer (hereinafter referred to as the “long” V-mos microsphere). The indicated peptide sequence corresponds to the amino acid residue of 100-111 of the V-mos gene product.

 Using the same technique, a shortened peptide corresponding to amino acid residues 106-111 of the V-mos gene product (Gly-Ser-Val-Tyr-Lys-Ala) on a polyacrylamide microsphere with the same linker (hereinafter referred to as the “short” V- mos microsphere). This peptide is used as a negative control.

 A hybridoma cell line producing a murine monoclonal antibody against the "long" V-mos peptide, known as anti-V-mos (Hybridoma N-165-28E7, SCRF 354, Lot N 165-119) is commercially available from Biological Associates Inc . , Maryland. In the enzyme-linked immunosorbent assay (ELISA - the indicated antibody recognizes a homologous sequence from V-mos, MOS, Neu HER-1, HER-2-gene products. It is known that this antibody exhibits negligible affinity for the short V-mos peptide. Secondary goat anti-mouse JgG labeled with alkaline phosphatase obtained from Sigma.

 Using traditional methods for the production of monoclonal antibodies, as described, for example, in the journal Methods in Enzymology, no. 121, 1986, monoclonal antibodies are obtained in ascites form and then purified on a G-protein column obtained from Pharmacia.

8.2. results
Long V-mos microspheres are mixed with a 1000-fold excess of short -mos microspheres 2 ml of purified anti-V-mos monoclonal antibody (1 μg / ml) in phosphate buffered saline with 0.1% Tween 20 are added to the resulting mixture of microspheres and incubated at room temperature for one hour with gentle stirring. After that, the microspheres are washed on a small removable polypropylene column (obtained from Isolab), where they are held in frit. Then the microspheres are mixed with 2 ml of the second antibody at a dilution of 1: 2000 for 1 hour. After washing, the microspheres are poured into polystyrene Petri dishes and allowed to settle. The resulting supernatant was removed and a solution containing 5-bromo-4-chloro-3-indolyl phosphate and nitrosine tetrazolium was added as a substrate.

 After incubation at room temperature for 15 minutes, long V-mos microspheres become purple as opposed to short V-mos microspheres, which remain unpainted. This allows you to immediately detect only one dark microsphere in the lawn of thousands of unpainted microspheres. The differences between the microspheres are shown in FIG. 4-6, each of which is a photograph of microspheres in Petri dishes, at 40x magnification. In FIG. 4 shows a lawn of long V-mos microspheres labeled with a monoclonal antibody; in FIG. 5 shows a mixture of long and short V-mos microspheres labeled with anti-V-mos monoclonal antibody, and FIG. 6 shows a single blue microsphere after detecting unpainted microspheres in a lawn. Accordingly, microspheres. containing the studied peptide, the desired peptide sequence, are easily recognized and isolated from other microspheres in the specified library.

 After isolation, the 477A protein sequencer from Applied Biosystems was used to determine the N-terminal amino acid sequences of a single Vsp-long microsphere.

8.3. conclusions
This example shows a powerful tool of the present invention for selecting a microsphere containing a bio-oligomeric ligand, in this case a peptide that is detected from a thousand-fold excess of non-binding, irrelevant microspheres. Moreover, in the indicated example, the possibility of isolating the reactive microsphere and determining the sequence of the peptide was demonstrated.

9. Example: Isolation of a Shortened Peptide Ligand Binding to a Receptor Molecule
To further illustrate the use of the proposed method for isolating a specific peptide, a hexapeptide with a given sequence of Gly-Phe-Gly-Ser-Val-Tyr was synthesized on a standard 100 μm polyacrylmethacrylate resin using Na-Fmos chemistry and other reagents from traditional solid-phase peptide synthesis.

9.1. Materials and methods
The binding reaction is carried out according to the method described above in section 8.1. Alpha-amino-protecting groups are removed with 20% piperidine, the side chain protecting groups are removed with a 50% trifluoroacetic acid solution, while the peptide that remains covalently attached to the polyacrylamide resin via a linker has the following structure: Gly-Phe-Gly-Ser-Var-Tyr-aminocaproic Acid Beta Ala Oligomer. The indicated peptide sequence corresponds to amino acid residues 104-109 of the V-mos gene product described above in example 8.

 Analogously to example 8, anti-V-mos antibodies are harvested from a hybridoma cell line producing a murine monoclonal antibody specific for the V-mos peptide. A second alkaline phosphatase-labeled goat anti-mouse IgG antibody was obtained from Sigma.

9.2. results
About 0.1 mg of the PAM / V-mos peptide described previously is mixed with a 100-fold excess of N-beta-F-mos-PAM microspheres obtained from Bachem Inc. Two ml of purified monoclonal antibody (1 μg / ml in phosphate-buffered saline with 0.1% Tween 20 is added to the resulting peptide / carrier mixture and incubated at room temperature for 45 minutes with gentle stirring.

After that, the microspheres are washed on a small removable polypropylene column (obtained from Isolab), where they are held on a frit (glass filter). Then the microspheres are mixed with 2 ml of a second antibody labeled with alkaline phosphatase at a dilution of 1: 100 for 1 hour. After washing, the microspheres are laid out on a piece of filter paper and dried in 2,2'-azinobis (3-ethylbenzothiazoline sulfonic acid (ABTS) substrate with H ₂ O _2. After incubation at room temperature for 15 minutes, PAM / V-mos-peptide microspheres become dark green. A small light green band forms on the glass filter. Most of the solid-phase carriers where the V-mos peptide is absent does not interact with the monoclonal antibody and thus does not stain. V-mos microspheres are easy to detect .

9.3. conclusions
In this example, it is shown that the analyzed acceptor molecule is not specific for a solid phase carrier, but rather specific for a carrier / peptide combination. Similarly to the above example 8, a positively reacting microsphere can be isolated, and the peptide immobilized on it can be sequenced.

10. Example: Determination of ligands for streptavidin and MAB against β-endorphin
This example illustrates a completely different approach to the identification of the peptide ligand of the present invention. In the described methods, instead of using a biological system (for example, a hybrid filamentous phage) to generate a randomized library, the synthesis of huge peptide libraries is effectively used, where each peptide that is different from the others is on a separate microsphere. Then, the individual microspheres specifically associated with the peptide are physically isolated from one microsphere and the sequence of the peptide associated with it is determined.

 This method depends on the possibilities of chemical synthesis of a huge randomized peptide library and its binding for the corresponding detection, isolation and structure determination system.

 The means to solve this problem provided by the present invention is that the polymer microspheres are divided into a series of separate equal aliquots during each binding cycle, and each aliquot of the polymer is reacted until it is complete with a separate activated amino acid. After binding is complete, various aliquots of the polymer are thoroughly mixed, washed, unblocked, washed and aliquoted again for a new binding cycle. According to this technique, no polymer microsphere is exposed to more than one amino acid in any one binding cycle, and upon completion of several such steps, each microsphere will contain one single peptide sequence. The peptide library thus obtained will theoretically be completely randomized. In addition, as a result of this procedure, equimolar ratios of all peptide species will be obtained. The total number of permutations, and therefore the number of peptides will depend on the number of aliquots and amino acids selected in each stage of binding, and on the total number of stages of synthesis synthesis (peptide length).

 The new method for the simultaneous synthesis of a wide range of peptides not only makes it possible to obtain an absolutely randomized and equimolar library, but, more importantly, makes it possible to obtain a library of solid-phase peptide polymer microspheres, where each microsphere contains only one unique peptide sequence. This last of these factors is completely undeniable, since during each cycle of peptide synthesis, each microsphere is in contact with only one separate amino acid at a given time, and each binding reaction proceeds until its completion. The success of the described method is mainly based on the idea of binding one microsphere to one peptide.

 In addition, thanks to the described method, almost any peptide library with a well-defined composition can be synthesized. For example, up to 20 natural L-amino acids in separate aliquots can be used in each binding step, or one or more amino acids can be used in some binding steps.

10.1. Materials and methods
10.1.1. Peptide Library Synthesis
A large library was synthesized having the following structure XXXXX-β-aminocaproic acid - ethylenediamine polymer (X = 19 of the 20 known amino acids, except cysteine, in each stage of binding). In the peptide synthesis, polydimethyl acrylamide (PDA) was used as solid phase polymer microspheres (M: 11: Gen. Inc., USA).

 A polymer chemical peptide synthesis method was carried out according to Atherton (1988, "Solid Phase Peptide Synthesis. Practical Methods", Solid Phase Peptide Synthesis, A. Practical Approach, IDL-Press).

Three grams of polymer (approximately 2 million microspheres) was lightly stirred overnight with ethylenediamino. After thorough washing, aminocaproic acid and then β-alanine were coupled to the polymer using Fmos chemistry, but without the use of a cleavable linker. Randomization was carried out with successive five stages of binding, and all 19 Fmos amino acid-OPFp, with the exception of cysteine, were used separately during each stage of binding. After the completion of these five binding steps, the Fmos group was removed in 20% piperidine (v / v) in DMF. Side chain protecting groups were removed with a mixture of 90% TFA (v / v), 1% anisole (v / v) and 0.9% ethanedithiol (v / v). The polymer was neutralized with 10% diisopropylethylamine (in DMF) and stored in DMF at 4 ^° C.

Linker β-alanine-aminocaproic acid - ethylenediamine as a whole consists of 11 C atoms and 4 atoms with a maximum shoulder length of 17.6 A. Since 19 different amino acids were used in each of the five stages of randomized binding, the theoretical number of peptides in this library was 19 ⁵ or 2476099.

 As indicated above, the main idea of this method is that a huge library of randomized peptides located on separate solid-phase polymer microspheres is synthesized in such a way that each polymer microsphere contains one kind of peptide. Each individual polymer microsphere interacting with an acceptor molecule can then be identified and physically isolated, and the amino acid sequence of the peptide ligand can then be determined by Edman degradation. Therefore, for the successful implementation of this technique, accurate identification of the peptide sequence on one sphere is necessary. Using an automatic peptide sequencer (Model 477A-01 Pulsed Ligued Automatic Protein (Peptid Seguencer, Applied. Biosystems, Foster City, California), 50-500 pM peptides were usually isolated from each polymer microsphere. In addition, preliminary analysis (DiMarch et al. 1990, "Peptids Chemistry, Structure and Biolody", Proccedings of the Eleventh American Peptide Symposium, July 9-14, 1988, La Solla, Ca ESCOM, Leiden pp. 229-230) of sequencing data showed that the binding efficiency in the synthesis of solid phase peptides exceeded 98%.

10.1.2. Specific Identification and Selection of Peptide Ligands from the Library
The identification and selection of specific peptide ligands from a randomized library can be easily carried out using immunological techniques, such as, for example, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, or using immunomagnetic microspheres. For the experiments described in this example, an immunohistochemical technique was used in the detection system. In the present experiment, (i) a biotin-binding protein (streptavidin) and (ii) a monoclonal antibody (MAT) against β-endorphin were used as specific binding acceptor molecules. Using a library of hybrid filamentous phage epitopes (Cwirla et al., De Vlin et al., Supra, part 2) and both of these acceptor molecules, peptide ligands were successfully identified.

 An immunohistochemical technique was used to detect streptavidin-binding microspheres. A randomized library was carefully mixed with a gradually increasing amount of distilled water added to dilute DMF. The microspheres were then thoroughly washed with PBS, and gelatin (0.1% w / v) was used to block any nonspecific binding. Then, 1: 200,000 dilution of streptavidin-alkaline phosphatase (Pilrce, Rockford, Illinois) was added to these microspheres over 1 hour, slightly stirring. After that, the microspheres were thoroughly washed and a standard substrate 5-bromo-4-chloro-3-indolylphosphate / nitrosine tetrazolium (BCIP / NBT) was added. The microspheres along with the substrate solution were transferred to 15 polystyrene Petri dishes (100 x 20 mm) and the reaction was carried out for almost 2 hours. Microbuses with bound streptavidin-alkaline phosphatase were stained dark blue, while most microbuses in the library remained unpainted.

 10.1.3. Determination of the affinity of peptide ligands.

The binding affinity of the peptide ligand to a monoclonal antibody (MAT) against β-endorphin was determined in the solution phase. In the anti-β-endorphin mAb binding assay, the inhibition of peptide ligand binding of 5.0 nM [ ³ H] [Leu] enkephalin (specific activity = 39.0 Ci / mM, New England Nuclear, Boston, MA) with 125-200 ng was measured / ml anti-β-endorphin MAT in 1.0 ml of 40 mm Tris-HCl, 150 mm NaCl, pH 7.4 buffer containing 1.0 mg / ml bovine serum albumin, 0.1% (vol / vol). Tween-20 and 0.05% (w / v) sodium azide. Specific binding was defined as the difference between the binding measured in the presence or absence of 1.0 μM unlabeled [Leu] enkephalin. The bound radioligand was precipitated by adding a 10-fold excess of Protein-G-Sepharose (Pharmacia), followed by incubation overnight (23-24 ^o C). Protein-G-Sepharose was collected by centrifugation (13000 x g, for 5 minutes), and the pellet was suspended in 250 μl of 5% (v / v) acetic acid before being transferred to the vessels for scintillation fluid counting. K ₀ values (n = 3) were determined by saturation analysis using 5 concentration of radioligand (1.87 - 30 nM) for [ ³ H] [Leu] enkephalin with duplication of each of the samples for complete and non-specific binding. The average K ₀ value was 9.79 ± 4.63 nM. The inhibition curve of the peptide ligand was built for eight concentrations of the peptide in the 400-ct range. Binding data for saturation and inhibition was analyzed by nonlinear regression using the appropriate single site models (Khapp et al., 1990, J. Pharmacol. Exp. Ter .; 255: 1278-1282). Ki — affinity inhibitory binding constant values were calculated by the method of Cheng and Prusoff (1973, Biochem Pharmacol, 22: 3099-3102). Each K - value was calculated from 3-4 independent measurements.

10.2 Results
A large synthetic disordered peptide library was screened (XXXXX polymer, where X = 19 known amino acids (except cysteine) for a total of 19 ⁵ = 2476099 permutations). About 2 million microspheres were present in a portion of the screened library. In the first stage of selection, using only streptavidin-alkaline phosphatase (see part 10.1.2, see above), approximately 75 microspheres were stained with different color intensities; they were physically isolated and removed under a dissecting magnifier using a micromanipulator. Then, each microsphere was washed with 8 M guanidine hydrochloride to remove the streptavidin-enzyme conjugate. After that, each microsphere was separately placed on a glass filter for the Applied Biosystem Protein Sequencer (ABI) cartridge. In the table. 1 shows sequences of 28 and 75 microspheres. All of these microspheres had a generic typical sequence of either HPO or HPM. The photomicrogram of FIG. 7 illustrates how a positive (dark blue) microsphere can be easily identified against the background of many thousands of negative (colorless) microspheres in the process of screening a library of peptide ligands.

 To prove that a typical generalizing sequence actually binds to the biotin-binding site in the streptavidin molecule, LMPQF-β-AIa-aminocaproic acid - ethylene diamine polymer (LHPQF polymer) was synthesized. The LHPQF polymer was then mixed with streptavidin-alkaline phosphatase in the presence of various concentrations of biotin. The results are shown in FIG. 8. At 10 nM, biotin completely blocked the staining of the LHPQF polymer. At concentrations of 10 and 1.0 nM, biotin partially inhibited staining, and at a concentration of 0.1 nM, biotin had no effect on staining of the LHPQF polymer with streptavidin-alkaline phosphatase. Inhibition analysis showed that a generic generic sequence binds to the streptavidin biotin-binding site.

 Before using the same disordered peptide library for screening using anti-β-endorphin mAbs, all blue microspheres that were stained with only one streptavidin-alkaline phosphatase were removed. To remove any bound protein, the remaining microspheres were then treated with 8 M guanidine hydrochloride.

This recycled library was then mixed with biotinylated anti-β-endorphin MAT (anti-β-endorphin, clone 3-E7, purchased from Boehringer Mannhein, Indiana) for 16 hours. After extensive washing, a second stage was performed using streptavidin-alkaline phosphatase in order to stimulate the staining reaction for ELISA. In this screening, 6 peptides with a typical summary sequence were identified that are very similar to the native ligand, YGGFL-encaphaline (YQGM): YGALQ, YGGLS, VGGFA, YGGFT, YGGFQ. Peptide analogues of the above ligands with different carboxy-ends were synthesized, and their affinities (Ki) were determined using [ ³ H] (New - encafaline (New England Nuclear, Boston, Mass.) As unlabeled ligand and unlabeled peptides as a competitive ligand ( analysis using anti-β-endorphin, see part 10.1.2) The results of these studies are summarized in Table 2.

 YGGI is [Leu] encafalin, a native ligand for anti-β-endorphin MAT.

10.3. Discussion
Using the methodology of the present invention, the key to success is the ability to synthesize individual peptides on each microsphere in combination with a sensitive and specific detection and selection system. This new methodology is called the "selective way." A single peptide microsphere selected from the library and treated with 8 M guanidine hydrochloride (to remove bound protein) was efficiently purified, and the remaining peptide was covalently coupled to the polymer and prepared for sequencing. Standard automated peptide sequencers are suitable for peptide sequencing at concentrations up to 5-10 nM. In addition, the blue dye, which irreversibly binds to the microsphere, does not interfere with sequencing. During each randomization cycle, all efforts were aimed at optimizing the synthesis methodology, including the use of large excesses of N ^α -Fmos amino acids. A preliminary analysis of a series of sequencing data clearly showed that the binding efficiency in synthetic chemical reactions exceeded 98%.

 Since the colored microspheres are clearly visible against the background of colorless microspheres (for example, in Fig. 7), then visually viewing 2 million microspheres in a series of 10-15 Petri dishes under a dissecting magnifier and selecting reactive microspheres for sequencing is almost effortless. In addition, the relative affinity of various ligands can be estimated by assessing the relative staining intensity of each positive microsphere. This allows you to select microspheres with a specific color intensity for sequencing.

 Devlin et al. (1990, Science 259: 404-406, part 2, supra) pointed to the importance of typical HPQ, HPM, and HPN generic sequences in their 20 streptavidin-binding ligands isolated by hybrid filamentous phage. Of the 20 isolates, 15 had HPQ, 4 had HPM, and 1 had HPN. It is interesting to note that the peptide library produced 28 different peptides, of which 23 had the HPO sequence, and 5 had the HPM sequence (Table 1). Obviously, the position of the HPQ / HPM sequence in the peptapentide is not important for binding to streptavidin. Of all the identified HPQ or HPM pentapeptide sequences, only two were repetitive (TpHPQ and MHPMA). These results run counter to the data of Devlin et al. (See above), where among 20 isolates there are many repetitions, which suggests a standard error in the selection according to the biosynthetic method used by them.

 In the anti-β-endorphin system, 6 peptides were identified with sequences very similar to the native ligand YGGF1 (Table 2). These results are similar to those obtained using a hybrid filamentous phage where the same monoclonal antibody (clone 3-E7) was used (Cwirla et al., 1990, Proc. Nate. Acad. Sci. USA 87: 6378-6382, see above). Although this peptide library produced fewer ligand sequences than was obtained by Cwirla et al., However, 50% of the obtained ligands had much greater affinity for the antibody than the ligands selected by the phage method.

11. Example: Limited Peptide Library
In another series of experiments, the present invention was tested using a different antibody system, where the epitope was located in the middle of the peptide chain and not at its N-end (as in the case of β-endorphin). The antibody used was an anti-V-mos-peptide monoclonal antibody (anti-V-mos-MAT) (see below, part 11.1). This antibody was obtained by immunizing mice with a 12 amino acid peptide (LQSGGFGSYKA) corresponding to residues 100-111 of the oncogenic product V-mos. This peptide was conjugated to a carrier protein before immunization. In the ELISA test, anti-V-mos-MAT detects a homologous sequence of V-mos, mos, mos neu / HER1 and HER-2 gene products.

 11.1 Materials and methods.

 Using a commercially available epitope mapping kit (Cambridge Research Biochenricol, Boston) in a multi-pin system (Geysen et al., 1986, Mol. Ymmunol, 23: 709-715) to synthesize overlapping peptides (a series of tetrapeptides, pentapeptides and hexapeptides), the epitope was mapped within the 12 amino acid V-mos peptide recognized by the anti-V-mos-MAT for the pentapeptide sequence (FOSSVV) (i.e., residues 6-10 of the V-mos dodecapapeptide).

In the present experiment, a limited randomized library was used where the amino acids valine and serine, which are present in the V-mos epitope, were deliberately excluded. This limited randomized library had the following composition: GXXXXX-β-Ala-aminocaproic acid - ethylene diamine polymer, where X = Glu, Pro, Asn, Phe, Leu, Thr, Lys, Leu, Gly, Tyr, Ala, Met, Arg, Trp. These 14 amino acids were chosen so that valine and serine were excluded, but all functional groups of the side chains were included, that is, (i) Asn was selected, but not Glu; (ii) Glu, but not Asn; (iii) Thr, but not Ser; (iv) Leu was chosen, but not Phe or Val; or (V) chose Met, but not Cys. Since 14 amino acids were selected in each of the five stages of randomized binding, the theoretical number of peptides was 14 ⁵ or 537824 individual peptides.

 A hybridoma cell line that produces anti-V-mos-MAT was purchased from Microbiological Associatod. Jnc., Maryland (Hybridoma N 165-28E7, SCRF 354, Series N 165-119).

The affinity of the peptide ligand for anti-V-mos-MAT was measured by binding in the solution phase using the [ ³ H] acetyl-V-mos peptide ([ ³ H] -V-mos-) as the radioligand. This radioligand was obtained by - terminal acetylation of a V-mos peptide before unblocking the side chains using an equimolar amount of [ ³ H] sodium acetate (specific activity = 2.52 Ci / mM, Neu England Nugleur, Boston, MA). The [ ³ H] Ac-V-mos product, which was isolated from the unreacted V-mos peptide using reverse phase HPLC, had a specific activity of 2.50 Ci / mM. The binding affinity of ³ H Ac-V-moc- for anti-V-mos MAT (= 10 μg / ml) was measured in 1.0 ml of PBS-gelatin buffer (0.05% gelatin in PBS) at 23-24 ^o C, three hours after incubation. Specific binding was defined as the difference between the binding of [ ³ H] Ac-V-mos in the presence of (non-specific) and in the absence of (complete) 1000 μM unlabeled V-mos peptide for each concentration of [ ³ H] Ac-V-mos]. The bound radioligand was isolated by centrifugation using a 10-fold excess (binding capacity to the immunoglobulin used) of protein-G-Sepharose (Pharmacia) to precipitate the antibody. An analysis of saturation binding by five determinations in a concentration range of 125-5000 nM showed that [ ³ H] -Ac-V-mos bound to anti-V-mos MAT with a Kd value of 850 ± 160 nM. Peptide ligand binding affinities were determined by binding inhibition assays using seven concentrations of peptide ligand in competition for 10 μg / ml anti-V-mos MAT with 20 nM [ ³ H] Ac-V-mos under the conditions described above. Inhibition analysis showed that over 50% of total binding was specific. Inhibition and saturation binding constants were determined for binding to a single site by nonlinear regression analysis as previously described (Knapp and Yamamura, 1990, Life, 46: 1457-1463).

11.2. results
Approximately 230,000 microspheres were screened using an anti-V-mos-alkaline phosphatase conjugate. Therefore, less than 43% of permutations were investigated. After incubation with the substrate, about 50 microspheres showed an intense blue color, 4 of these microspheres were physically isolated and the amino acid sequence of 11 of them was determined. In addition, 17 unpainted microspheres were randomly selected for sequencing.

The results of sequencing the anti-V-mos ligand are presented in table. 3. Since valine and serine were not intentionally included in this peptide library, there was nothing unexpected in the fact that none of the sequences of 11 peptide ligands resembled the native epitope (FCSW). Although there were no repeats in these 11 peptide ligands, their sequences were non-randomized. Arginine and tyrosine are often found in these sequences. In addition, at least one and sometimes two arginines are present in the second and / or third position of each of these peptide ligands. On the other hand, randomly selected negative microspheres did not reveal any common types of amino acid sequences. Although the size of the samples was limited, the statistical chi-quadratic correspondence for sequences from negative microspheres was small (x ² = 18.27, df = 13, P = 0.15), which indicates the absence of any evidence of an uneven distribution of amino acids for unpainted arbitrary peptide microspheres.

 Some of the positive ligands were synthesized, and their affinity for anti-V-mos MAT was determined by analysis of binding in the solution phase (see part 11.1 above). The results of these analyzes are presented in table. 4.

 In the table. 4A shows the binding affinities of the V-mos epitope (determined by epitope mapping) (for anti-V-mos monoclonal antibodies (MAT). The effect of the carboxy-terminal change is also shown. Table 4B shows the binding affinities of mimeotopes identified from the peptide library, where several amino acids are missing from the V-mos epitope, a β-alanine amine was included in some of the tested ligands to model the structure of the identified ligand to which the antibody binds on the microsphere.

In the table. 4, the affinity of the best anti-V-mos mimeotope is approximately 2.5 times less than the affinity of the epitope of the native peptide. Although none of the studied peptide ligands had the same K _i value as the native V-mos ligand, the results clearly show that using a randomized library that lacks some of the amino acids present in the native epitope, the series can be identified structurally different mimeotopes with affinity for an acceptor molecule, i.e., anti-V-mos-MAT.

11.3. Discussion
The anti-V-mos MATs used in this study had a relatively low affinity for the V-mos peptide (immunogen). Serine and valine were present in the linear epitope (FGSVY), but were deliberately excluded from the library used for screening, however, the described method allows to determine the linear mimeotope of a completely different sequence and with a different composition of amino acids, but with a comparable affinity for the antibody. This shows that macromolecular-peptide interactions have a complex and multifaceted nature.

12. Example: Selectively Detachable Linker
A series of four peptides was prepared and tested, including a UV-cleavable GNb linker (see above, part 5.4).

12.1. Materials and methods
Using the solid-phase synthesis technique (for example, see parts 6 and 7 above), the following four peptides were obtained on two polymers:
(i) Fmos-Trp-Tyr (OBu ^t ) -Phe-ONb-β-Ala-ACA-EDA-Pep-SynK
(ii) TPP-Tyr-Phe-ONb-β-Ala-ACA-EDA-Pep-SynK
(iii) Fmos-Tyr (OBu ^t ) -Phe-ONb-β-Ala-ACA-4-MBHA
(iv) Trp-Tyr-Phe-ONb-β-Ala-ACA-4-MBHA
For 1 and 3 hours, each peptide was subjected to ultraviolet (UV) radiation and visible light (VIS) in 0.3 ml of water or 3/7 of a mixture of chloroethanol and dichloromethane. In general, 16 + 16 experiments were performed and analyzed.

 After irradiation, the supernatant was filtered off, lyophilized and dissolved again until equal volumes of MeOH (0.3 ml) were obtained. Products were analyzed by HPLC.

12.2. results
The analyzes carried out using HPLC clearly showed that when irradiated with visible light in water, the tripeptide did not stand out, and under UV irradiation an almost complete release of the tripeptide was observed for 1 hour.

 In particular, peptide i in water was cleaved to a simple product. In some cases, two products were observed prior to elution (HPLC). With a longer exposure time (UV), the mutual conversion of the two products was observed in at least a few cases.

12.3. Discussion
Obviously, the UV-sensitive Lenker promotes the release of the peptide in aqueous solution. Exposure for 1 hour would be sufficient for incomplete release of the peptide. The results also showed that the polyamide resin is stable and compatible with aqueous systems.

13. Example: Identification of a simulated enzyme
13.1. Materials and methods
A disordered library of pentapeptides containing 19 and 20 known amino acids (excluding cysteine) was obtained in accordance with the present invention (see above, part 10.1).

 This peptide library was exposed to a chromogenic substrate, nitro blue tetrazolium chloride, in the absence of an exogenous enzyme and under conditions acceptable for product formation; and identified positive microspheres. These microspheres were selected and sequenced.

13.2. results
The sequences of the five microspheres, which obviously catalyze the reduction of nitro-blue tetrazolium to its dark blue diformazan product, are presented in table. 5.

13.3. Discussion
The results obtained suggest that the peptide PNNNH microsphere has the ability to reduce nitro-blue tetrazolium (NBT) to dark blue diformazan pigment either chemically or enzymatically. Thus, the peptide or peptide on the microsphere has demonstrated its activity as an “artificial enzyme” or as an enzyme mimic.

14. Example: Screening for anti-cancer peptide sequence
A limited library containing pentapeptides including tyrosine, followed by an unordered sequence of five amino acids selected from the group of the following amino acids: glutamic acid, serine, valine, glycine, agrinin and asparagine, has peptide sequences with activity against a cancer cell line (i.e. with antitumor activity).

14.1. Materials and methods
The disordered peptide library of the present invention was synthesized using a hydrolyzable diketopiperazine linker. In screening for anticancer (antitumor) drugs, 96-well plates were used.

After the side chain protecting group and the N ^α -Fmos group were unlocked, the peptide microsphere library was neutralized with DIEA (diisopropylethylamine) and extensively washed with DMF to remove any remaining, possibly toxic, chemicals. The library was then gradually replaced in 0.01 M HCl (conditions under which the linker is stable) and finally washed with 0.001 M HCl. After that, approximately 10 microspheres of the library were transferred to each well of the tablets. Then, 50 μm RPMI medium (with 25 mM HEPES buffer) was added to neutralize the pH of the solution. At a neutral or slightly alkaline pH, the peptides will be released (for example, after 16-24 hours).

Then, either (I) part or all of the medium was transferred to a separate plate with a specific cancer cell line, or (2) the cancer cell line was added directly to the microsphere wells. In this case, approximately 2500 lung cancer cells per well were used. These plates were then incubated for 8 days at 37 ^° C and MTT analysis was performed to quantify the relative cytotoxicity of the isolated peptides in each well.

 Various standard cell lines of carcinoma, lymphoma, human myeloma and leukemia can be used for screening. An example of this is the following list of tumors (NCl): lymphocytic leukemia L 1210, melanoma B16, Lewis lung carcinoma, M5076 sarcoma, colon carcinoma 38, and human breast carcinoma MX-1. As other examples, the following tumors may be mentioned: breast cancer MCF-7, myeloma cell line 8226, leukemia (mouse) cell line P388, and non-small cell lung cancer cell line Hodkens.

14.2. results
A library containing about 8000 peptides with the YXXXXX microsphere sequence was screened, where Y is Tyr and X is Glu, Val, Gly, Arg or Aru. In two experiments, 3-4 supernatants containing the released peptide showed growth inhibition of the non-small cell Hodkens lung cancer line. Since each well contained approximately 10 microspheres, basically, all 8000 possible sequences were investigated, resulting in the identification of at least 3 active peptides. Similar results were obtained by direct incubation of the microspheres with cells and by transferring the supernatant with the released peptide.

14.3. Discussion
The results obtained indicate that a limited (shortened) peptide library may contain several peptide sequences exhibiting cytotoxic activity. In the above example, 0.05% of the possible sequences showed activity against cancer cells. By bioanalysis of supernatants containing the released peptide in a variety of bioassays, cytotoxicity can be established against cancer cells and against mature tissue cells.

 Accordingly, peptide sequences with broad cytotoxic activity to tumor cells or with cytotoxic activity to a specific tumor cell line can be identified; sequences of low toxicity to intact cells are preferred as medicaments. The indicated method can be used for screening antimicrobial, antiparasitic agents and growth factor antagonists.

 A similar method for screening a growth factor agonist uses the growth factor of cell line dependencies. In such a bioassay, peptide sequences with growth factor agonist activity stimulate the growth of cells cultured in the absence of a major growth factor.

 The present invention is not limited to these specific embodiments described herein. Various embodiments of the invention, other than those disclosed herein, should be within the scope of the invention as defined by the claims.

Claims (15)

 1. A library of peptides immobilized on solid-phase carriers, characterized in that it is an unordered library of unprotected peptides containing numerous solid-phase carriers, in which one type of unprotected peptide is associated with each solid-phase carrier, all statistically possible combinations of the subunits making up these peptides included in the specified library.  2. The peptide library according to claim 1, characterized in that subunits selected from the group consisting of glycine, alpha amino acids, D-amino acids, non-classical amino acids and peptidomimetics are used as subunits.  3. A method of obtaining a library of peptides immobilized on solid carriers, characterized in that the library is an unordered library of unprotected peptides containing numerous solid phase carriers, in which one type of unprotected peptide is associated with each solid phase carrier, wherein I) receive at least two aliquots of solid-phase carriers having a reactive site, II) introduce a set of subunits separately into the indicated aliquots of the solid-phase carrier so that each aliquot of it only one subunit having a protective group is introduced, wherein at least said subunit has a protecting group or a plurality of protective groups, III) completely sub-covalently binds the subunit to essentially all reactive sites of the solid-phase carrier, IV) the degree of binding is assessed, and if necessary, continue the binding reaction until it is complete; V) carefully mix aliquots of a new combination of subunits on a solid-phase support, remove blocking groups so that espechit active sites and, after repeating steps (II) - (V) the desired number of times, a final step is carried out deprotection whereby unprotected peptides remain covalently bound to solid carriers.  4. The method according to claim 3, characterized in that the solid phase carrier is selected from the group consisting of polystyrene resin, polyhappa resin, polyamide resin grafted with polystyrene polyethylene glycol and polydimethacrylic resin.  5. The method according to claim 4, characterized in that the solid phase carrier is polydimethylacrylamide.  6. The method according to claim 3, characterized in that the solid-phase carrier is silicon dioxide.  7. The method according to claim 3, characterized in that the solid-phase carrier contains a linker.  8. The method according to p. 7, characterized in that the linker comprises a linker selected from the group consisting of aminobutyric acid, aminocaproic acid, 7-aminoheptanoic acid, ethylene glycol and 8-aminocaproic acid.  9. The method of claim 8, wherein the linker comprises aminocaproic acid.  10. The method according to claim 3, characterized in that the disordered library of peptides is obtained by a single repeat of stages (II) to (V) before the final stage of removal of the protective groups.  11. The method according to claim 3, characterized in that the disordered library of peptides is obtained by more than one repetition of stages (II) - (V) before the final stage of removal of the protective groups.  12. A method for identifying a peptide synthesized by solid-phase synthesis, characterized in that an acceptor molecule is introduced into the disordered library of unprotected peptides so that the acceptor molecule recognizes and binds to one or more peptides of the library immobilized on a solid carrier, the peptide complex immobilized on a solid carrier is isolated a ligand exhibiting the ability to bind to an acceptor molecule and determine the sequence of the peptide isolated from the complex, immobilized on a solid carrier.  13. The method according to p. 12, characterized in that the acceptor molecule is selected from the group consisting of antibodies, receptors, species, bacteria, proteins, hydrocarbons, nucleic acids, lipids, drugs, metals and small molecules.  14. The method according to item 13, wherein the acceptor molecule is an antibody.  15. The method according to item 13, wherein the acceptor molecule is a receptor.

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