JPH10501818A - Template-directed cyclization - Google Patents

Abstract

  (57) [Summary] A novel and efficient method of cyclizing peptides on a solid support is disclosed. The method is useful for preparing a cyclic peptide and preparing a cyclic peptide library for screening.

Description

DETAILED DESCRIPTION OF THE INVENTION                              Template-directed cyclization                                Field of the invention   The present invention relates to cyclic peptides, cyclic peptides bound to solids, and cyclic peptides. A method for preparing a library.                                   background   The peptide forms a disulfide bond that requires two thiol-containing residues, 2 Amide or ester bond formation between two side chain functional groups, one side chain functional group and Amide or ester bond formation between the backbone α-amino or carboxy groups, or Many methods, such as amide bond formation between the backbone α-amino or carboxy groups To convert to a cyclic structure (cyclic homodetic peptide). Traditionally, these Various cyclization reactions were performed in highly diluted solutions. For example, Brady ( Brady, et al., J. Org. Chem., 1979, 44, Pp. 3101-3105, and McMurray et al. t. Lett.), 1993, 34, 8059-8062. New resin And with the advent of the orthogonal protecting group method, the peptide remains attached to the solid support. Some of these cyclizations can now be performed. Most solid phase papers Since the peptide synthesis method requires attachment of the carboxy terminus to a solid support, cyclization is This is most suitable when side-chain functional groups and / or α-amino groups are required. I have. For example, a side chain amide bond is formed between an amine containing and a carboxyl containing side chain. Formed, while the peptide remains attached to the resin. Thus, Syrah (Sc Hiller et al., J. Med. Chem., 1985, Vol. 28, 1766-1771, Bouvier et al., J. Med. . Chem.), 1992, 35, 1145-1155, Pla. ue, S.), Int. Peptide Res. Protein Res.), 1990, 35, 510-517, and Bloomba. Bloomberg et al., Tet. Lett., 1993, volume 34. Pp. 4709-4712 describe a method for cyclization of a side chain functional group of a peptide on a resin. ing. Similarly, for cyclized peptides via disulfide bonds, (Ploux) et al., Int.Peptide Protein Res. 1987, Vol. 29, pp. 162-169, and Alberici (Alberici). o) et al., Int. Peptide Protein Res. 1991, Vol. 37, pages 402-413, shows that peptides remain attached to the resin. A method for cyclization of a peptide is described.   Solid phase synthesis of cyclic homodetic peptides involves free amino and More trials are needed because both need a boxyl end. Against this One approach to this is to attach the C-terminal amino acid to the resin via a side chain functional group, And deprotection with orthogonal carboxyl protecting groups and amino and carboxy This was the cyclization of the terminal. This approach is based on McMurray, J. Tet Lett., 1991, Vol. 32, 7679-7682. Page, Trzeciak et al., Tet. Lett., 1992. 33, 4557-4560, and Tromelin et al., Tet. * According to Tet. Lett., 1992, Vol. 33, pp. 5197-5200. Are listed. This is because only amino acids whose side chains can be attached to the resin are Position limits the nature of the peptide that can be prepared.   Another approach to preparing cyclic homodetic peptides is the oxime resin Including the preparation above, it was necessary to cleave the peptide from the resin following cyclization. Enables intramolecular aminolysis of the peptide-resin bond by the α-amino group. This This is described in Osapay et al., Tet. Lett., 1990, No. 43, 6121-6124, and He et al., Jay Ameri Chem. Sosa (J. Amer. Chem. Soc.), 1993, Vol. 115, 8066-8072. Page. This method allows for greater flexibility in the prepared sequences. Allows facilitation but may be hindered by low yield. Otherwise, In this method, a cyclic intermediate having a protected side chain is removed from the resin, and a solution deprotection step is performed. And synthesis of a resin-bound cyclic homodetic peptide is not possible.   New methods of cyclizing peptides, specifically new cyclization of peptides on resins A new way is needed. The present invention relates to the synthesis and synthesis of homodetic peptides on resins. A simple and versatile method for cyclization is provided. Further, in this specification Amino acid residues that direct or promote cyclization in the peptide Incorporation may improve the yield of cyclic peptide.                                Summary of the Invention   An object of the present invention is to provide a novel method for synthesizing a cyclic homodetic peptide. It is. Another object of the present invention is to provide a new resin-bound cyclic homodetic peptide. It is to provide a method for normal synthesis. Yet another object of the present invention is to provide biological activity By producing a library of cyclic peptides used for molecular screening is there.   A feature of the invention provides for a selective, cleavable bond between the peptide and the resin. Provides a means for attaching the peptide to the resin via the first cross-linking group and the side-chain group This is a combination with the second crosslinking group. Yet another feature of the present invention is the The use of amino acid sequences and amino acid residues in tides that promote cyclization reactions. In one embodiment, a preferred feature of the present invention is to promote cyclization with a second bridging group. This is a combination with the template residue to be used.                                Detailed description   We have prepared cyclic homodetic peptides and peptide libraries. Two new approaches to manufacturing have been developed and combined. Thus, the present invention The authors applied template-based cyclization to solid-phase synthesis and applied cyclic homogenization on a solid support. Allows for the complete synthesis and deprotection of the native peptide. A variant of this method is Allows release of the chain deprotected cyclic peptide from the resin. These can be free or tree Synthesis of any of the lipid-linked cyclic homodetic peptides, K Enables peptide library creation.   In one embodiment, the present invention provides a structure comprising: [In the formula A peptide of 10 amino acid residues; L is a linker; and A method for synthesizing a resin-bound cyclic homodetic peptide represented by The above method 1) Formula (I):   L1 is a linker between the solid support and the carboxy terminus of the peptide;   L2 is a linker on the amino acid side chain;   Pg¹Is one or more protecting groups on the reactive side chain function of the peptide;   Pg^TwoMay optionally be a protecting group at the amino terminus of the peptide;   S1 may optionally be a protective functional group attached to the solid support, May be via the linker L1, but not attached to the peptide or L2,     And S1 can form a covalent bond and react with S2;   S2 is a protector on L2, which can optionally form a covalent bond and react with S1 May be active group]   Preparing a linear resin-binding peptide represented by: 2) If necessary, remove any protecting groups from S1 and S2 and II): Forms a covalent bond between S1 and S2 to form the compound represented by thing; And, if necessary, Pg to provide a free amino terminus.^TwoRemove And formula (III): Forming a compound represented by: 4) Cyclizing the amino and carboxy termini to form a compound of formula (IV): Forming a compound represented by: and 5) Protecting group Pg¹To remove the compound of formula (V): To form a compound represented by the formula:   This method involves performing the above steps and then cleaving the peptide from the resin. Cleavage of these peptides and the side chain protecting group Pg¹Removal of the desired free cyclic peptide Can be done to get The peptide comprises linker L2 and It is removed from the resin by cleaving the bond between the attached amino acids.   For convenience, some abbreviations are used herein. Thus, Aloc means allyloxycarbonyl, β-Ala is β-alanine (3- BHA means benzhydrylamine-polystyrene resin Fat, Boc means t-butyloxycarbonyl, Bn means benzyl BOP means benzotriazol-1-yloxy-hexafluorophosphate Tris (dimethylamino) phosphonium, DBU is 1.8diazobic [5.4.0] undec-7-ene, and DPPA is diphenylphosphoryla Zide, DCC is dicyclohexylcarbodiimide, and DMAP is N-ethyl-N '(dimethylaminopropyl) L) -carbodiimide, DMF means dimethylformamide, Fmo c represents 9-fluorenylmethyloxycarbonyl, and HBTU represents (hexaf 1-hydroxybenztriazolyltetramethyl-uronium phosphorophosphate) Said HMBA is 4- (4-hydroxymethyl-3-methoxy-phenyloxy) ) Means butyric acid, HOBT means 1-hydroxy-benzotriazole, S uc means succinyl, Hyp means hydroxyproline, Z is benzyl Means luoxycarbonyl. In general, amino acid abbreviations are Iochem (Eur. J. Biochem.), Vol. 158, p. 9, (1984). As noted, IUPAC-IUB Joint Commission on Bio Chemical Nomenclatu (Joint Commission On Biochemical Nomenclatu) re).   As used herein, amino acids are amino-terminal and carboxy-terminal. The ends are preferably 1,2-, 1,3- or 1,4-substituted patterns on the carbon skeleton And the compounds having the formula: Alpha amino acids are most preferred and are found in proteins. 20 natural amino acids (L-amino acids excluding glycine), corresponding to D-amino acids, biosynthetically available amino acids not found in proteins Acids (eg, 4-hydroxy-proline, 5-hydroxy-lysine, citrulline) , Ornithine, canavanine, dencolic acid, β-cyanoalanine), and Mino-isobutyric acid, norleucine, norvaline, Cα alkylation and Nα alkyl Syntheses such as fluorinated amino acids, Dtc, Tpr, homocysteine and homoserine Α-amino acids. β-alanine and γ-aminobutyric acid are 1,3- and Examples of 1,4-amino acids, many others are well known in the art . Statin-like equivalents (two of which have a CONH bond replaced by CHOH) Dipeptide consisting of amino acids), hydroxyethylene equivalent (CONH bond is C HOHCH_TwoA dipeptide consisting of two amino acids, which is replaced by Reduced amide equivalent (CONH bond is CH_TwoSubstituted by an NH bond, 2 Horn Dipeptides consisting of amino acids) and thioamide equivalents (CONH bond is CS Dipeptides consisting of two amino acids, which are replaced by NH) Is a useful residue.   As used herein, peptides are linked by amide bonds. 1 shows the amino acid sequence. The peptide of the present invention has 3 to 10 amino acid residues, preferably It preferably consists of an amino acid sequence of 5 to 7 residues, each of which is amino and amino. And carboxy termini.   As used herein, square brackets surrounding an amino acid sequence refer to a ring. A peptide having an amino terminus linked to the carboxy terminus, for example, In the case of α-amino acids, the α-amino group of the first residue is linked to the carboxy group of the last residue. Is tied. Pins, or other such forms suitable for solid phase peptide synthesis. Take Examples of resins include benzhydrylamine polystyrene, aminomethyl polystyrene, Polyethylene glycol-polystyrene and the like. Such solid supports are generally commercially available. Sold. Typical of such resins are tentagel^R(TentaGel^R), PEG-PS , Merrifield and Shepard resins. Po Polystyrene-based resins are particularly useful.   As used herein, the term linker refers to a single atom or molecular break. Attach a piece (a collection of atoms) to another atom or molecular fragment by a covalent bond Any molecular fragments are meant. Typically, the linker will be At an intervening functional site such as a sulfide, disulfide, disulfide, ester or amide It will consist of an optionally substituted alkylene or allylalkyl group. Or Such functional sites usually create a linker (or join two molecular fragments), Or used to break existing linkers (and separate them into molecular fragments) Will be.   Coupling methods for forming peptide bonds are generally well known in the art. Are known. In general, the practice by Bodansky et al. The Practice of Peptide Synthesis, Split Springer-Verlag, Berlin, 1984 The methods of peptide synthesis depicted illustrate the technique and are incorporated herein by reference. It is captured. Typical coupling methods include carbodiimide, activated anhydride and Ester and acyl halide are used. EDC, DCC, DPPA, PP A, BOP reagent, HOBt, N-hydroxysuccinimide, and oxa chloride Reagents such as Lil are typical. Ali et al., J. Med Chem d. Chem.), 29, 984 (1986), and Shay Med Chem ( J. Med. Chem.), 30: 2291 (1987). Amino acid side chain anti Responsive functional groups, such as those that undesirably react or interfere during synthesis Hydroxyl, mercaptan, carboxylic acid, amino, guanidino, imidazole Or a functional group such as an indole group is generally protected during the synthesis. The protecting group is Is a group that masks the reactivity of such functional groups, but is removed at some point later in the synthesis. It restores the original functional group after being removed. Appropriate protection for reactive functional groups Groups and reagents for deprotecting these functional groups are described by Greene et al. PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (PROTECTIVE  GROUPS IN ORGANIC SYNTEHSIS), 2nd edition, John Wiley and Sons (John Wiley and Sons), New York, 1991 ing. Deprotection refers to removal of the protecting group and replacement with a hydrogen atom.   Pg¹Is used to protect side chain functional groups on amino acid residues during solid phase synthesis Is a selectively removable protecting group.   Pg^TwoProtects the α-amino group of the amino acid residues of the peptide during solid phase synthesis. Is a selectively removable protecting group used for   Linker L2 is attached to an amino acid in the peptide by a site that can be selectively cleaved. Containing a second attachment site S2 that attaches and forms a covalent bond with the attachment site S1 It is a carl group. In a preferred embodiment, L2 causes cyclization of the peptide Because it is attached to a residue that acts as a template.   S1 is an attachment site that forms a covalent bond with the second attachment site S2 on the linker L2. Rank. If the functional group of S1 can react with the functional group of S2, then S1 is a solid support Will be on the linker or attached to a solid support. L1 and When the bond between the peptides is cleaved, the function of the S2 site is that the peptide binds to the resin S1 resides on the peptide itself because it provides a second pathway to Must not be present. In a preferred embodiment, S1 is on linker L1. You. As used herein, the linker L is a bond between S1 and S2 It will be clear that there is a link between the peptide and the solid support resulting from the formation . If S1 is on linker L1, L will consist of L1 and L2 If S1 is on the solid support itself, L will consist only of L2. Reactive functionality where the attachment site S1 reacts differently under the conditions of solid phase synthesis of the peptide If protected, is protected by a protecting group that is removed before forming a bond with S2 Will. Such protecting groups should be orthogonal to other protecting groups, for example, Is Pg¹Or Pg^TwoCan be removed under conditions that do not affect other protecting groups in the molecule, such as And it is due to the α-amino group of the constituent amino acid residues in the growing peptide chain Stable to the conditions used to remove other protecting groups such as Should be. For example, if the bond between S1 and S2 is a peptide bond, The attachment site S1 is suitably a suitable protecting group Pg such as an Aloc group^ThreeProtected by The attachment site S2 is suitably an allyl alcohol ester Suitable protecting group Pg^FourIs a carboxyl group protected by The Fmoc group Suitable protecting group Pg for the α-amino group of the constituent amino acids^TwoWill. Other suitable straight Intersecting protection methods are known in the art.   However, in an unusual embodiment, the attachment site S1 is on a solid support, The attachment site is on a linker attached to the solid support. In a preferred embodiment, The attachment site is attached to linker L1 itself.   S2 is an attachment site that forms a covalent bond with S1, and is present on the linker L2 . The reactivity at which the attachment site S2 reacts in another way under the conditions of solid phase synthesis of the peptide If a functional group, it can be removed before forming a bond with the reactive group at S1. Protection group Pg^FourWill be protected by Such protecting groups may be Step Should be orthogonal to the cleavage site in the tide-resin complex, but these groups Since activated at the same point in the procedure, such a protecting group is Pg^ThreeMust be orthogonal to There is no. The appropriate groups for the attachment sites S1 and S2 are illustrated: These can be cleaved by Pd (O) and Pg¹Suitable orthogonal protecting groups for T-butyl groups (pentamethylenechroman-6-sulfonyl) (Excluding Arg protected by a (Pmc) group), The Fmoc group for is cleaved by a base. Other reactive groups and protecting groups Combinations will be apparent to those skilled in the art. Couples the first N-protected amino acid via the carboxyl group with linker L1 Solid phase by coupling the remaining residues one after the other. It is synthesized by the usual method of peptide synthesis. The peptide is typically an N-protected amino acid. Acid, performed by sequential deprotection of the amino group of the last residue added to the resin It is synthesized from peptide synthesis and coupling of the following N-protected amino acids. is there In an example, one or more amino acids are attached to linker L1 and then It would be more effective to attach L1 to the carrier. Amino acids are reactive functional groups Having a side chain containing a suitable protecting group Pg¹By Protected. The amino group of the amino acid is a suitable protecting group Pg^TwoProtected by   For a template that directs cyclization, peptide Q has a residue or combination of residues. Conformations in which the amino and carboxy termini of the peptide are close It is synthesized in such a way that the peptide adopts a secondary structure that prefers Thus, the mold Residues are amino acid residues or combinations of residues that conform Tethered to facilitate cyclization of linear peptides. For example, the linear peptide HDT rp-D-Asp-Pro-D-Val-Leu-OH has a Pro residue with an amino acid. Low-energy γ-ta Conformation, making it very easy and with almost quantitative yield Cyclize. Suitable templates include Pro, Hyp, Aib, reduced dipeptide equivalents. Or a γ-turn derivative containing amino acids such as thioamide equivalents, D-Pr o-Pro (or more generally, DX is a D-form amino acid and N- ( Alk) DXN, wherein Y is an amino acid Y that is alkylated on an amino group Β-turn derivatives including dipeptides such as-(Alk) Y) Peptide turn mimics; and backbone connectors extended in cyclic hexapeptides Would include D-Pro-Gly-Pro to induce formation. An example For example, such elements are described in Peishoff et al., J. Med Chem. . Chem.), 35, 3962 (1992). This These elements represent the large clumps they possess (eg, Aib, Ca-methylamino Steric hindrance by acid, Nα-methylamino acid) or ring structure (eg Pr o) Incorporation of carbon skeleton elements or formation of hydrogen bonds at adjacent residues (Eg, thioamide equivalents, dipeptide equivalents) The effect is obtained by restricting the rotation of. Other conformational systems The limiting element will be designed or selected from technologies that use these principles.   The template must be introduced into the linear peptide sequence to promote cyclization. No. First, attachment of the template to the resin, followed by bidirectional peptide chain extension from the template Chief is a tricky approach. Successive extension from the C-terminus Danger of racemization in between, making coupling completion not easily measured . Coupling the complete C-terminal peptide moiety to the template linked to the resin, or Alternatively, coupling the template-C-terminal peptide moiety to the resin can be either Means fragment coupling, which is essentially an inefficient method, and Not suitable for creating tide libraries. Attachment of peptide to resin and linker To manipulate the point of attachment of L2, the template or secondary structure derived element in the sequence It is possible to take advantage of.   In a preferred embodiment, coupling of the peptide to the resin via the backbone linker L2 Like the reactive site S1 used for the linker, the linker L1 has two unstable linkages to the resin. The method is performed in such a way as to consist of knots. Furthermore, the linker L2 is a template residue Adheres to In the following examples, the template amino acid residue is depicted by a C, As referenced in formula (I), the linker L1 is represented by BA, The functional group S2 is present on B-A^FourIs protected by the linker L2 And the reactive functional group S1 is present on D^ThreeProtected by; In the peptide Q, the functional group on the side chain of the amino acid represents a protecting group for the terminal amino group of the peptide. Pg¹And Pg^TwoProtected by [AA¹-AA^Four] -C- [AA^Five-A A⁸].   According to the method, a suitably protected linker BA is attached to a peptide synthetic resin. To wear. The first amino acid then attaches to the linker, and then the peptide synthesis It is performed by a usual solid phase peptide synthesis method. At a suitable point in time, C is an amino acid and D is a tether (eg, L2) , Template-tether Pg^Two-C (D-Pg^Four) Is coupled to the growing peptide chain It is. After completion of the linear peptide synthesis, the protecting group Pg on the reactive functional group of tezah^FourAnd Pg on the reactive functional group of linker and linker (L1)^ThreeIs removed. Then tether And the linker are coupled to each other and cyclized on the resin via the linker. To make the peptide. Then the bond between the C-terminal amino acid and the linker BA is The bond is cleaved to yield a linear peptide attached to the resin via the template-teza. Ami No terminal protecting group Pg^TwoResin-bonded linear peptide with both ends released by cleavage And a cyclization is possible. Then, after cyclization, the side chain protecting group Pg¹ Cleavage yields a completely deprotected resin-bound cyclic homodetic peptide. You. At this point, the bond between the template amino acids C and tether D is optionally Razinolysis, hydrogenolysis, selection by conventional methods such as trifluoroacetic acid or HF Alternatively, it can be cleaved to give the free peptide.   The salient features of this scheme are as follows. Resin has two types of peptide attachment Incorporates linkers with sites, one of which is temporarily obscured ing. Peptides can be synthesized by the usual methods of peptide synthesis and provide high yields of The coupling method is performed, and the completion of coupling can be evaluated by the ninhydrin test. You. The protected linear peptide is attached to the linker and the template portion of the peptide. Attached to the resin via a newly synthesized bond between the tethers. This allows Amide and carboxyl termini of tides are released, resulting in highly effective template directivity Cyclization can be performed on the peptide attached to the resin.   The method requires many orthogonal cleavable protecting groups and linkers. Ami Protecting group Pg^TwoIs a side chain protecting group Pg¹, Teza protecting group Pg^Four, Linker protecting group Pg^Three, Link between template C and tether D, and carboxyl-terminal amino acid and linker It must be able to cleave in the presence of the bond between -BA. Furthermore, it is Pg^Three And Pg^FourOf carboxyl-terminal amino acid and linker B It must be stable to the bond cleavage conditions during -A. Similarly, Pg^ThreeYou And Pg^FourIs Pg^TwoStable to removal conditions, Pg^Two, Pg¹, And mold C and te The bond between the D and between the carboxyl terminal amino acid and the linker BA It must be able to selectively cleave in the presence of a bond. Carboxyl terminal amino acid The bond between and linker BA is Pg^TwoAnd Pg¹And mold C and tether It must be able to selectively cleave in the presence of a bond between D.   In a specific embodiment, the approach is illustrated in Schemes I-III below. Have been. Scheme I illustrates the partial synthesis of an acid labile linker (eg, L1), And the first residue of the peptide for attachment to the resin is illustrated. Scheme II is A linker incorporated into the peptide to direct cyclization of the peptide (eg, , L2), illustrates the synthesis of conformationally restricted template residues I do. Scheme III illustrates the generality of template-directed cyclization on resin for model peptides. Fig. 2 illustrates a schematic method.   In the specific embodiment shown in Scheme III, the resin is benzhydrylamine Resin. Linker BA (eg, L1) consists of two parts: A is ε- Aloc-Lys, whose side chain provides a second attachment site to the resin And the carboxyl of the Lys residue is attached to the BHA resin via an amide bond; and And B is 4- (4'-hydroxymethyl-3-methoxy) phenoxybutyric acid (HM PB), and the carboxyl of HMPB is attached to the α-amine of Lys residue. The growing peptide attaches to the hydroxymethyl group. t-butyl based protection An Fmoc amino acid having a side chain protected by a protecting group can be prepared by this method, ie, Pg^TwoIs Fmoc and Pg¹Is t-butyl based or PMC Used in the method. The template residue is 4-hydroxyproline (Hyp) Tezah L2 is succinyl-β-alanine, which is a β-Ala carboxylate. Attaches to the hydroxyl of Hyp via a sil group. The succinylcarboxyl group is Allyl ester (Pg^Four) As protected. The Fmoc group is acid stable, Stable to d (O) reagent, but cleavable with secondary amines. Allyl ester And Aloc groups are stable to secondary amines and acids, but have a Pd (O) Can be cleaved with drugs. The HMPB linkage to the peptide was made with dilute acid (2% TFA / CH_TwoC l_Two) And are stable to secondary amines and Pd (O). t -Butyl and Pmc side chain protecting groups for secondary amines and Pd (O) Stable, but strong acid (95% TFA / CH_TwoCl_Two). The ester linkage between the Hyp template and the tether β-Ala Stable, but cleavable by hydrazinolysis. Those skilled in the art will recognize that orthogonality Other useful protecting groups that can be used as well, provided that all requirements are met You will recognize that there are potential schemes.   Prepare and test combinatorial libraries in liquid or on solid supports Methods are known, for example, Lam et al., Nature 354, 82 (1991) and PCT / US91 / 04666 (W. O92 / 00091); Houghton et al., Nature, 3rd. 54, p. 84 (1991) and PCT / US91 / 08694 (WO92) / 09300). Such a method involves the use of a cyclic homodetic peptide. May be combined with the method of the present invention for obtaining a library of the present invention. For example When the peptide chain is completed, the peptide coupling generated each solid support The amino acids into several equal molar groups, and react different amino acids thoroughly with each group. And the groups are recombined and mixed, then the groups are ready for the next coupling. It may be divided again. Thus, the obtained linear peptide resin complex is Each bead has a different amino acid sequence, but each bead has a unique peptide. Will have a peptide sequence.   The library is a library of solutions of peptide-resin complexes or free cyclic peptides. It can be a burrary. As is known in the art, these licenses The library binds to the target molecule or prevents the binding of a second molecule to the target molecule. Eye to screen target molecule or surface to identify harmful peptides It may be used regularly. PCT / US92 / 09345 (WO94 / 08051) )), The encryption method is based on such combinatorial libraries. It may be combined with the method of the present invention which has improved detection of active peptides.   The library is prepared according to the method of Geysen et al., US Pat. No. 94,392 and U.S. Pat. No. 4,562,157. By using the minotope method, peptides of partially known composition May be constructed and screened by preparing groups of For assaying resin beads by partially cleaving active peptides Alternatively, other methods known in the art may be applied.   The following examples illustrate methods of making and using compounds, libraries, and methods of the present invention. It shows the law and does not limit it in any way. For convenience of reference For example, compound 5 in Scheme III is converted to III-5 Some compounds are labeled by the numbers shown in Schemes I-III.                                 Example I Preparation of Fmoc-leucine preloaded with an acid labile linker 4 [4-N^α-(9-Fluorenylmethoxycarbonyl) -leucinyl-oxy Methyl] -3-phenyloxy] butyric acid (1) Scheme 1   Dry CH_TwoCl_Two(100 ml) and pyridine (2.3 ml, 28 mmol) To a stirred solution of Fmoc-leucine (10 g, 28 mmol) Null (3.3 ml, 28 mmol) was added dropwise. Reaction at room temperature in the presence of argon Stir for 4 hours, filter off the inert precipitate and add cold H_TwoWash with O (2x250ml) And dried (MgSO 4)_Four) And then evaporated to dryness.   0 ° C dry CH_TwoCl_Two(100 ml) and 4 (4-hydroxyme) in pyridine. Stirring of tyl-3-methoxy-phenyloxy) butyric acid (6.80 g, 28 mmol) CH solution_TwoCl_Two(50 ml) of a solution of Fmoc-leucinyl fluoride Was added. The reaction was warmed to room temperature and then stirred for 16 hours. Then, anti The reaction was acidified with cold aqueous 0.5N HCl, washed with brine, dried (MgSO 4). O_Four) And then evaporated under reduced pressure. Silica gel (30: 70: 1) Et Purification by flash chromatography on OAc / n-hexane / HOAc And the evaporation from toluene, the title compound was obtained. Was obtained as a white solid (10.25 g, 63%).¹H-NMR (CDCl_Three, 400 MHz) δ 0.92 (2xd, 6H), 1.55 (m, 1H), 1.68 ( m, 2H), 2.10 (m, 2H), 2.58 (t, 2H), 3.77 (s, 3 H), 3.99 (t, 2H), 4.19 (t, 1H), 4.40 (m, 3H), 5. 10 (d, J = 11.9 Hz, 1H), 5.16 (d, J = 11.9 Hz, 2H) , 5.28 (d, J = 8.8 Hz, 1H), 6.38 (m, 1H), 6.41 (s, 1H), 7.19 (m, 1H), 7.30 (t, 2H), 7.39 (t, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H)                                 Example 2 N^α-(9-Fluorenylmethoxycarbonyl) -O- (2-allyloxycal Bonyl-propionyl-β-alaninyl) hydroxyproline (Compound II-5 ) —Template residue with linker a) N^α-(T-butoxycarbonyl) -hydroxyproline benzyl ester (II-1)   N in methanol (100 ml) and water (50 ml)^α-T-butoxycal To a stirred solution of bonyl-hydroxyproline (5 g, 21.6 mmol) was added C sHCO_Three(4.2 g, 21.6 mmol) were added. After stirring for 15 minutes, the solution was Evaporate to dryness, twice from methanol (2 × 100 ml), then And finally re-evaporated from DMF (100 ml). The residue was treated with DMF (10 Benzyl bromide (3.1 ml, 26 mm) while stirring at room temperature. Mol) was added all at once. The reaction was then stirred at 50 ° C. for 16 hours in the presence of argon. Stirred. The resulting suspension was evaporated under reduced pressure and ethyl acetate (150 ml) H inside_TwoO (150 ml), dried (MgSO_Four), Evaporate did. Flash chromatography on silica gel (50 in n-hexane) % Ethyl acetate) to give the title compound (1) Scheme 1 as a clear oil. Obtained as quality (6.75 g, 21 mmol).¹ H-NMR (CDCl_Three, 250 MHz) Mixture of amilotomers δ 1.35 And 1.46 (2 × s, 9H), 1.80 (brs, 1H), 2.08 (m, 1 H), 2.30 (m, 1H), 2.92-3.69 (m, 2H), 4.48 (m, 2H) H), 5.16 and 5.19 (s and dd, 2H), 7.35 (s, 5H) b) N^α-(T-butoxycarbonyl) -O- [N^β-(Benzyloxycarbo Nyl) -β-alaninyl) hydroxyproline benzyl ester (II-2)   Cbz-β-alanine (5 g, 22.4 mmol) in toluene (50 ml) Oxalyl chloride (20 ml) was added all at once to the stirred solution of One drop of DMF was added to start the reaction. The reaction was stirred for 16 hours, then And evaporated under reduced pressure. Re-evaporation from toluene (2x50ml) This gave the corresponding acid chloride to be used in the next step.   CH_TwoCl_TwoCompound of Example 1 (6.75 g, 21 mmol) in (75 ml) To a stirred 0 ° C. solution of pyridine (1.82 ml, 22.5 mmol) was subsequently added. And CH_TwoCl_TwoA solution of the above acid chloride in (25 ml) was added. Then the reaction The thing was warmed to room temperature and stirred for 16 hours. After evaporation under reduced pressure, the residue was treated with acetic acid. Put in ethyl (150 ml) and add H_TwoO (150 ml), 1N HCl aqueous solution (1 50 ml), washed with brine (150 ml), dried (MgSO 4)_Four), Evapo Rated. Flash chromatography on silica gel (in n-hexane (40% ethyl acetate) to give the title compound as a clear oil. (4.30 g, 38%). ¹H-NMR (CDCl_Three, 250MHz) Amidro A mixture of Tomers δ 1.36 and 1.45 (2 × s, 9H), 2.19 (m, 1 H), 2.37 (m, 1H), 2.54 (t, 2H), 3.46 (m, 2H), 3. 69 (m, 2H), 4.42 (dt, 1H), 5.09 (s, 2H), 5.16 ( s, 2H), 5.16-5.30 (m, 2H), 7.35 (s, 10H) c) N^α-(T-butoxycarbonyl) -O- (2-allyloxycarbonyl- Propionyl-β-alaninyl) hydroxyproline (II-4)   The compound of Example 2 (b) (4.20 g, 8 mmol) in methanol (75 ml) Solution) at 55 psi H using a Parr apparatus._Two6:00 on Pd on 10% carbon For a while. The catalyst was removed by filtration through a celite pad, and glacial acetic acid (4 × 50 ml) was used. After washing, the filtrate was evaporated under reduced pressure and methanol (2 × 50 ml) was added. Re-evaporate to give Nα- (t-butoxycarbonyl) -O- (β-alaninyl ) Hydroxyproline benzyl ester (II-3) (2.36 g, 98%) Obtained as a white solid.   Succinic anhydride (24 g, 240 mmol) was added to allyl alcohol (25 ml, 3 60 mmol) was added. Reflux the reaction in the presence of Ar for 16 hours and cool to room temperature And saturated NaHCO_ThreeDissolve in aqueous solution (400ml) and add Et_TwoO (200ml) Wash, acidify with cold 3N aqueous HCl and extract with ethyl acetate (300 ml) And washed with brine, dried (MgSO 4)_Four), Then evaporate under reduced pressure This provided monoallyl succinate (36.02 g, 95%).   Monoallyl succinate (13.75 g, 87 in toluene (50 ml)) Oxalyl chloride (20 ml, 230 mmol) to the stirred solution of Was added. The reaction was stirred at room temperature for 16 hours, then evaporated under reduced pressure . Re-evaporation from toluene (2 × 50 ml) gave the following couplings: Succinyl chloride monoallyl ester (14.68 g, 9 6%) as a clear liquid.   (4: 1) dioxane / H_TwoNα- (t-butoxycal) in O (100 ml) Bonyl) -O- (β-alaninyl) hydroxyproline benzyl ester (2. 36 g, 7.8 mmol) in a stirred solution at 0 ° C._Three(1.6 g, 1 9 mmol), followed by succinyl chloride monoallyl ester (2.0 g, 11 mmol) was added dropwise over 15 minutes. Bring the reaction to room temperature, Stirred for 16 hours. After acidification with 1N aqueous HCl, the solution was washed with CHCl_Three( 150 ml), washed with brine, dried (MgSO 4)_Four), Until dry Was evaporated. Flash chromatography on silica gel (97: 3: 1) CHCl_ThreePurification with / MeOH / HOAc gave the title compound (4) Scheme 1 was obtained as a white solid (1.81 g, 53%).¹ H-NMR (CDCl_Three, 400 MHz) Amidlotomers mixture δ 1.44 And 1.48 (2 × s, 9H), 2.11-2.50 (m, 2H), 2.75 (t , 4H), 3.01 (t, 2H), 3.70 (m, 2H), 4.01 (m, 2H) , 4.35 (m, 2H), 4.60 (d, J = 5.6 Hz, 2H), 5.24 (d, J = 7.5 Hz, 1H), 5.32 (dd, 2H), 5.90 (m, 1H); MS ( ES) m / z 443.0 [M + H]⁺, M / z 441.2 [MH]^- d) N^α-(9-Fluorenylmethoxycarbonyl) -O- (2-allyloxy Carbonyl-propionyl-β-alaninyl) hydroxyproline (II-5)   CH_TwoCl_TwoCompound (1.81 g, 4.1 mmol) of Example 2 (c) in (5 ml). Trifluoroacetic acid (45 ml) was added to the stirred solution of Stir for 45 minutes Thereafter, the reaction was evaporated to dryness under reduced pressure. CHCl_Three/ With toluene Re-evaporate several times to foam the deprotected TFA salt (1.87 g) Obtained as a solid.   CHCl_Three(75 ml) to a stirred solution of the above TFA salt in Et._ThreeN (1.1 5 ml, 8.2 mmol) followed by N- (9-fluorenylmethoxycarbo) (Nyl) succinimide (1.52 g, 4.5 mmol) was added. After stirring for 6 hours The solution was acidified with 1N aqueous HCl (10 ml) and washed with brine. , Dried (MgSO 4)_Four), Evaporated to dryness. On silica gel Rush chromatography (100: 4: 1) CHCl_Three/ MeOH / HOA Purification by c provided title compound (5) Scheme 1 as a white solid (2 .11 g, 91%).¹H-NMR (CDCl_Three, 400 MHz) δ 2.30 (m, 1H), 2.40 (m, 1H), 2.46 (t, 2H), 2.53 (t, 2H), 2.68 (t, 2H), 3.50 (m, 2H), 3.72 (m, 2H), 4.22 ( dt, 1H), 4.32-4.53 (m, 3H), 4.55 (d, J = 5.6 Hz, 2H), 5.22-5.35 (m, 3H), 5.88 (m, 1H), 6.28 (m, 1H), 7.23-7.45 (m, 4H), 7.55 (m, 2H), 7.71 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H); MS (ES) m / z 565.2 [M + H]⁺, M / z 563.0 [M-H]^-                                 Example 3 Preparation of Model Cyclic Peptide (4) Scheme III Cyclo- (D-tryptophanyl-D-aspartyl-hydroxyprolyl-D -Valinyl-leucine) (III-4)   Protected linear peptide acid labile with lysine reattachment site (III-1) The resin is Fmoc-Nε- (Aloc) -lysine according to the following general synthetic method: , Fmoc-leucyl-HMBA, Fmoc-D-valine, Fmoc- (O-β Ala-Suc-O allyl) -hydroxyproline (II-5), Fmoc- ( Ot-Bu) -D-aspartic acid, and Fmoc-Nⁱⁿ-Boc-D- Commercial Benz hydride by continuous coupling and deprotection of liptophan Built on Luamine resin (1.18 mmol / g). General coupling method   Shake HBTU (3 molar equivalents) in a small Erlenmeyer flask Was dissolved in DMF. Fmoc-amino acid (3 molar equivalents) is added to this solution followed by N-methylmorpholine (8 molar equivalents) was added. Next, add this solution to rocker -Add to the resin (1 molar equivalent) and HOBt (3 molar equivalent) in the vessel Shake for hours. The reaction was checked by Kaiser test, and after completion of the reaction, DMF (4 × ) Then CH_TwoCl_TwoWashed with (2x). General Fmoc deprotection method   Treating the Fmoc protecting group with 20% piperidine in DMF of the peptide resin ( (1 × 5 minutes and 1 × 15 minutes). Next, the resin was added to DMF (4 Washed in x).   (1: 1) DMSO / CH_TwoCl_Two(10 ml) prepared resin peptide, Fmoc- (Boc) D-Trp- (Ot-Bu) D-Asp- (O-β-A la-Suc-O allyl) Hyp-D-Val-Leu-HMBA- (Aloc ) Lys-BHA (III-1) (2 g, 0.8 mmol) was added to acetic acid (460 μl). 1, 8 mmol), (Ph_ThreeP)_TwoPdCl_Two(24 mg, 34 μmol), continued And Bu_ThreeSnH (862 μl, 3.2 mmol) was stirred at room temperature in the presence of Ar. Was added. After stirring for 30 minutes, the resin was filtered and (1: 1) DMSO / CH_TwoCl_Two (2 × 50 ml). The reaction was repeated twice, (1: 1) DMSO / CH_Two Cl_Two(2x50ml), CH_TwoCl_Two(2x50ml), CH_TwoCl_Two(1x50 ml) N-methylmorpholine, CH_TwoCl_Two(2 × 50 ml), then n -Washed with hexane (2x50ml). Partially deprotected peptide resin 1 .97 g were obtained after drying under reduced pressure. The free amine was positive in the Kaiser test.   To the above resin peptide (1.84 g, 0.75 mmol) in DMF (10 ml) HOBt (0.22 g, 1.6 mmol), N-methyl Lumorpholine (0.45 ml, 1.6 mmol) followed by HBTU (0.6). (0 g, 1.6 mmol). After stirring for 16 hours, the resin peptide was filtered, DMF (2 × 50 ml), CH_TwoCl_Two(2 × 50 ml), and n-hexane ( 2 × 50 ml). Free amine negative in the Kaiser test.   Treat the resin peptide with 20% piperidine in DMF (50 ml) for 5 minutes Then again treated with 20% piperidine in DMF (50 ml) for 15 minutes and filtered. And washed with DMF, CH_TwoCl_Two5% trifluoroacetic acid in (4 x 10 ml) And 5% anisole solution for 2 minutes each. Next, the obtained hydroxy The prolyl side chain attached linear peptide resin was converted to CH_TwoCl_Two(2x50ml), CH_Two Cl_Two10% N-methylmorpholine in CH_TwoCl_Two(2x50ml), and And n-hexane (2 × 50 ml), and dried under reduced pressure. I got a peptide. The free amine was positive in the Kaiser test.   To the above resin peptide (1.20 g, 0.74 mmol) in DMF (10 ml) While stirring at room temperature in the presence of Ar, HOBt (0.17 g, 1.2 mmol), N -Methylmorpholine (0.33 ml, 3.0 mmol) followed by HBTU ( (0.45 g, 1.2 mmol). After stirring for 24 hours, the resin was filtered, and DMF was added. (2 × 50 ml), (1: 1) CHCl_Three/ MeOH (2 × 50 ml), CH_TwoC l_Two(2 × 50 ml), and n-hexane (2 × 50 ml). 1.2 2 g of peptide resin (2) scheme 3 was obtained after drying under reduced pressure. In the Kaiser test Free amine negative.   Peptide resin (2) in EtOH (5 ml) 1 g, 0.31 mmol), NH_TwoNH_TwoMonohydrate (200 μl, 4.1 mmol) Was added. After stirring for 48 hours, the resin was filtered and washed with MeOH (2 × 20 ml) did. Crude protected cyclic peptide (66 mg, 29%) was added to the filtrate by evaporation and And dried under reduced pressure. Flash chromatography on silica gel (CHCl_ThreePurification with 3% MeOH in) affords analytically pure material (3% MeOH). ) Scheme 3 (44 mg) was obtained as a white solid. TLC Rf = 0.28 ( 5% MeOH / CHCl_Three);¹H-NMR (CDCl_Three, 400 MHz) δ0. 71 (d, J = 6.5 Hz, 6H), 0.86 (d, J = 6.6 Hz, 3H), 0.8. 93 (d, J = 6.7 Hz, 3H), 1.20 (m, 1H), 1.40 (m, 1H ), 1.43 (s, 9H), 1.53 (m, 2H), 1.67 (s, 9H), 1.8 5 (m, 2H), 2.40 (dd, 1H), 2.75 (m, 1H), 2.82 (d d, 1H), 3.16 (m, 1H), 3.28 (d, J = 6.1 Hz, 2H), 3. 50 (D, J = 4.5 Hz, 2H), 3.78 (dd, 1H), 4.00 (t, 1 H), 4.62 (m, 1H), 4.78 (dd, 1H), 4.95 (dd, 1H) 5.12 (m, 1H), 6.12 (d, J = 8.0 Hz, 1H), 7.10 (m, 1H), 7.24 (m, 2H), 7.33 (t, 1H), 7.44 (s) 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 9.7 Hz, 1H), 8.15 (d, 1H); MS (ES) m / z 783.4 [M + H].⁺, M / z 781 .2 [MH]^-   To the remainder of the resin peptide (III-2) (0.71 g, 0.44 mmol), A solution of 10% anisole in fluoroacetic acid (10 ml) was added. Stir for 15 minutes After stirring, the resin was filtered and washed with 10% anisole in trifluoroacetic acid (10 ml). For 15 minutes. The completely deprotected peptide resin is converted to CH_TwoCl_Two(2x 25 ml), CH_TwoCl_TwoN-methylmorpholine (2 × 25 ml) in CH_Two Cl_Two(2 × 25 ml), and n-hexane (2 × 25 ml).   To the above peptide resin in EtOH (5 ml) was added NH_TwoNH_TwoMonohydrate (300 μl) was added. After stirring for 48 hours, the resin was filtered and MeOH (2 × 20 ml) And washed. The filtrate was evaporated under reduced pressure to obtain a crude product as a hydrazine salt (77.0 mg, 29%). Flash on YMC-ODS (S-50) column Purification by press chromatography (30-50% CH_ThreeCN, 0.1% TFA, H 2O) to give the title compound (III-4) as a lyophilized white powder. (37 mg). An additional 103 mg of crude cyclic peptide (68% total) was added to the remaining tree. The fat was applied to anhydrous hydrazine (2 ml) in methanol (2 ml) and further added to Ar Stir at 60 ° C. for 48 h in the presence, filter, wash with methanol (2 × 20 ml) And the filtrate was evaporated to dryness. HPLCK '= 5.21 Hamilton PRP-1 column, 30% CH_ThreeCN, 0.1% TFA / 0.1% TFA, H_TwoElution with O; K '= 5.31 Gradient 20-80% CH for 20 minutes_Three CN, 0.1% TFA / 0.1% TFA, H_TwoO; TLCRf = 0.81 (4: 1: 1) n-butanol / HOAc / H_TwoO, Rf = 0.81 (15: 3: 12: 10 ) N-Butanol / HOAc / H_TwoO / pyridine;¹H-NMR (d6-DMSO , 400 MHz) δ 0.61 (d, J = 6.6 Hz, 3H), 0.73 (d, J = 6.3 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H), 1.00 (m, 1H), 1.15 (m, 1H), 1.21 (m , 1H), 1.60 (m, 1H), 1.68 (m, 1H), 2.32 (dd, 1H) ), 2.51 (m, 1H), 2.79 (dd, 1H), 2.88 (dd, 1H), 3.09 (dd, 1H), 3.19 (dd, 1H), 3.47 (dd, 1H), 3. 98 (m, 1H), 4.11 (dd, 1H), 4.26 (m, 2H), 4.34 ( dd, 1H), 5.00 (m, 1H), 6.98 (t, 1H), 7.05 (t, 1H) H9, 7.14 (d, J = 1.9 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H) H), 7.38 (d, J = 10.3 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 9.2 Hz, 1H), 8.77 (d, J = 5.0 Hz, 1H), 8.80 (d, J = 8.1 Hz, 1H); MS (ES) m / z 627.2 [ M + H]⁺, M / z 649.2 [M + Na]⁺, M / z 625.2 [MH]^-                                 Example 4 Preparation of model library   The following libraries were prepared according to the experimental procedure outlined in Example 3, except that The standard split-and-combine method described by Furka (D A. Furka, M. Sebestyen, M. As Gedom (M. Asgedom), G. Dibbs (G. Dibσ), Int Jay Pept. Proto Res (Int. J. Pept. Prot. Res.) 1991, Vol. 37, 487-49. 3). All libraries were of the general formula cyclo [AA1-AA2-Hyp-AA4-AA5 ]. Library 1 (8 compounds)   Cyclo [AA1-AA2-Hyp-AA4-AA5]   [Where A = D-Trp or D-Gln         B = D-Asp or D-Ala         C = D-Val or D-Thr         D = Leu]   2.0 g of Fmoc-Leu-HMPB-Lys (ε-Aloc) -BHA Starting from the above, the resin was split in half and the amino terminus was deprotected as in Example 3. Was. One was coupled with Fmoc-D-Val and the other was coupled with Fmoc-D-Val. -Thr (t-Bu) was coupled. The two resins are then re-combined. In the beginning, deprotected, and with Fmoc-Hyp (β-Ala-Suc-O-allyl) Coupled. The resin was split in half again, deprotected and Fmoc-D-As Coupling was performed with either p (t-Bu) or Fmoc-D-Ala. The resin was recombined, mixed, split in half and Fmoc-D-Trp (Boc ) Or Fmoc-D-Gln. Resin again In the meantime, the synthesis of the mixture of peptidyl resin was continued as in Example 3. .   294 mg of isolated product after hydrazine decomposition from 1 g of the peptidyl resin Was obtained (98% of theory). The products are hplc and hplc / mass Spectrometry analysis shows that it contains only the 8 expected peptides in approximately equal proportions. I was Library 2 (10,000 compounds)   Cyclo [AA1-AA2-Hyp-AA4-AA5]   [Wherein AA1, AA2, AA4 = D-Asp, D-Asn, Gly,     D-Nva, D-Ser, D-Ser (Me), D-Phe, D-Tyr,     D-Trp or D-Lys     AA5 = Asp, Asn, Gly, Nva, Ser, Ser (Me),     Phe, Tyr, Trp, or Lys]   After coupling of the N-terminal amino acids of the sequence, the resin sample is not Separate them separately and make the 10 sublibraries listed below. Sub-library 2.1a   AA-1 = Gly   37.6 mg of product were obtained from 300 mg of peptidyl resin (theoretical 46 %). Sub-library 2.1b   AA-1 = D-Nva   75.1 mg of product was obtained from 300 mg of peptidyl resin (86 theoretical). %). Sub-library 2.1c   AA-1 = D-Ser   77.2 mg of product were obtained from 300 mg of peptidyl resin (theoretical 92%). %). Sub library 2.1d   AA-1 = D-Ser (Me)   69.6 mg of product were obtained from 300 mg of peptidyl resin (75 theoretical). %). Sub-library 2.1e   AA-1 = D-Asp   58.5 mg of product were obtained from 300 mg of peptidyl resin (theoretical 56 %). Sub-library 2.1f   AA-1 = D-Asn   53.6 mg of product were obtained from 300 mg of peptidyl resin (50 theoretical). %). Sub library 2.1g   AA-1 = D-Phe   61.5 mg of product were obtained from 300 mg of peptidyl resin (theoretical 57%). %). Sub library 2.1h   AA-1 = D-Trp   67.0 mg of product were obtained from 300 mg of peptidyl resin (theoretical 62 %). Sub-library 2.1i   AA-1 = D-Tyr   73.7 mg of product were obtained from 300 mg of peptidyl resin (theoretical 69). %). Sub-library 2.1j   AA-1 = D-Lys   69.4 mg of product was obtained from 300 mg of peptidyl resin (theoretical 64). %).   According to the above method, the resin is not cut at the positions of AA2, AA4 and AA5. Perform each sub-library separately using the same procedure Rally 2.2.a-j, 2.4.a-j and 2.5.a-j (2.x.a-j Various sub-libraries in which known amino acids are isolated and identified at position x of the tide Used to represent the color.                                 Example 5   Binding the peptide library of Example 4 to neurokinin 3 (NK3) Inhibits the phosphorylation of test substances by compounds or protein kinase C (PKC) Screen in an assay to identify the offending compound. Enzyme inhibition The assay was performed for PKC by Egan et al., Anal Biochem (Anal. Biochem.), 1988, 175, 552, and Alexander. ander) et al., Biochem. J., 1990, 268, 3; Clones for human PKCα and PKCβ receptors described on page 03 Perform the procedure using the Binding assays for the NK3 receptor are described in Mac McKnight et al., Bull J. Pharmacol., 1991, Vol. 104, pages 335-360. .   The results of such an assay using the sublibrary 2.1.aj of Example 4 were It is shown in Table 1 below.   Analysis of the library was performed by Zuckermann et al., J. Med Chem. (J. Med. Chem.), 1994, 37, 2678-2685, and Erb et al., The Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), 1994, No. 91. Vol., Pp. 11422-11426. This is done by repeating the resynthesis of the active sublibrary until defined.   The present invention is not limited to the embodiments described above, but is intended to Various modifications will be apparent to those skilled in the art. Such modified methods are encompassed within the present invention. And is limited only by the intent of the following claims. Various listed in this specification Publications describe the state of the art and should not be construed as an entirety. Incorporated herein.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Newlander, Kenneth Allen             United States 19382 Pennsylvania             West Chester, Sage Road             West 911

Claims (1)

[Claims] 1. formula: [In the formula L is a linker of 10 amino acid residues; L is a linker; and A method for producing a cyclic homodetic peptide bound to a solid support represented by the following formula: 1) Formula (I): [Where, L1 is a linker between the carboxy terminus of the solid support and peptide; L2 is a linker on an amino acid side chain; Pg ¹ is 1 or more protective groups on reactive side chain functional groups of the peptide Pg ² is an optional protecting group at the amino terminus of the peptide; S1 is an optionally protected functional group attached to a solid support; optionally, via a linker L1, the peptide Or an optionally protected functional group on L2 that does not attach to L2 and is capable of reacting with S2 to form a covalent bond; S2 is capable of reacting with S1 to form a covalent bond. 2) If necessary, remove any protecting groups from S1 and S2 and form a covalent bond between S1 and S2 to form a compound of formula ( II) : Forming a compound of the formula: If Pg ² is removed, a free amino terminus is obtained, having the formula (III): 4) Cyclizing the amino and carboxy termini of the peptide to form a compound of formula (IV): And 5) removing any protecting group Pg ¹ to form a compound of formula (V): Forming a compound represented by the formula: 2. The method according to claim 1, further comprising cleaving the cyclic peptide from the linker L2 to prepare a cyclic homodetic peptide. The described method. 4. The method of claim 1 wherein Pg ¹ and Pg ² is a functional group protected orthogonally to pg ³ and Pg ^4. The method of claim 1. 6. The residue that directs cyclization is Pro, Hyp, Aib, Pro-D-Pro or DXN (Alk) Y, DX is a D-configuration amino acid, and N- (Alk) Y Is the amino acid Y alkylated on the α-amino group or a derivative thereof. Method. 8. The method according to claim 1, wherein the attachment site S1 is on the linker L1. 9. L2 The method of claim 1, wherein Ten. L1 The method of claim 1, wherein 11． L is _{-CO (CH 2) 2 NHCO (} CH 2) 2 CONH (CH 2) 4 N (HMBA) The method of claim 1 wherein the CO-. 2. The method according to claim 1, for preparing a library of peptides. 13. The method of claim 1, wherein the first reactive site S1 is an optionally protected amino group and the second reactive site S2 is an optionally protected carboxyl group. 14. The method according to claim 1, wherein the first reactive site S1 is an optionally protected carboxyl group and the second reactive site S2 is an optionally protected amino group. 15． The method of claim 1 wherein the cleaved without protecting groups in S1 and S2 to remove the Pg ¹ or Pg ^2. The method of claim 1. 17． 7. The method of claim 6, wherein the residue that directs cyclization is Hyp. 18． A cyclic peptide produced by the method of claim 2. 19. A library of cyclic peptides produced by the method of claim 2. 20. Formula: Cyclo [AA1-AA2-Hyp-AA5-AA6] [where AA1, AA2, AA3 = D-Ala, D-Asp, D-Asn, Gly, D-Nva, D-Ser, D-Ser ( And AA4 means Ala, Asp, Asn, Gly, Nva, Ser, Ser (Me), Phe, Tyr, Trp or Lys; Me), D-Phe, D-Tyr, D-T rp or D-Lys; ] The library of the cyclic peptide shown by these. twenty one. A library of cyclic peptides attached to a solid support produced according to claim 1. twenty two. formula: [Where, At least one residue Chi is Hyp, a peptide of from 10 amino acid residues no 3; L is the _{formula: -CO (CH 2) 2 NHCO} (CH 2) 2 CONH (CH 2) 4 N (HMBA) CO A linker of- And a library of cyclic peptides bound to a solid support prepared according to claim 1. 24. The library of claim 23, wherein AA1, AA2, AA4 and AA5 are amino acid residues selected from the group of D- and L-form natural amino acids. twenty four. formula: [Wherein Pg means Boc or Fmoc].