TEMPLATE DIRECTED CYCLIZATION

Abstract: A new and efficient method for cyclizing a peptide on a solid support is disclosed. The method is useful for preparing cyclic peptides, and for preparing libraries of cyclic peptides for screening.

Description: TITLE TEMPLATE DIRECTED CYCLIZATION Field of the Invention This invention relates to methods for preparing cyclic peptides, cyclic peptides bound to a solid substrate and cyclic peptide libraries. Background Peptides can be converted to cyclic structures by a number of strategies: disulfide bond formation involving two thiol-bearing residues; amide or ester bond formation between two side chain functional groups; amide or ester bond formation between one side chain functional group and the backbone α-amino or carboxyl function; or amide bond formation between the backbone α-amino or carboxyl functions (cyclic homodetic peptide). Traditionally, these various cyclization reactions have been carried out in solution at high dilution. See, for instance, Brady et al., J. Org. Chem. 1979, 44, 3101- 3105, and McMurray et al, Tet. Lett. 1993, 34, 8059-8062. With the advent of new resin and orthogonal protecting group strategies, it has become possible to perform some of these cyclizations while the peptide is still attached to the solid support. This is most feasible when the cyclization involves side chain functional groups and/or the α-amino group, since most solid phase peptide synthesis strategies involve attachment of the carboxyl terminus to the solid support. For example, side chain amide bonds have been formed between amine- and carboxyl-bearing side chains while the peptide has remained attached to the resin. Thus, Schiller et al, J. Med. Chem. 1985, 28, 1766-1771, Bouvier et al, J. Med. Chem. 1992, 35, 1145-1155, Plauέ, S. Int. J. Peptide Protein Res. 1990, 35, 510-517, and Bloomberg et al, Tet. Lett. 1993, 34, 4709-4712, describe methods for on- resin cyclization of side chain functional groups of peptides. Similarly, for a peptide cyclized through a disulfide bond, Ploux et al, Int. J. Peptide Protein Res. 1987, 29, 162- 169, and Albericio et al, Int. J. Peptide Protein Res. 1991, 37, 402-413 describe methods for cyclizing peptides while the peptide has remained attached to the resin. Solid phase synthesis of cyclic homodetic peptides presents more of a challenge since it requires both a free amino and carboxyl terminus for the cyclization reaction. One approach to this has been to attach the C-terminal amino acid to the resin through a side chain functional group and, using an orthogonal carboxyl protecting group, deblock and cyclize the amino and carboxyl termini. This approach is described by McMurray, J., Tet. Lett. 1991, 32, 7679-7682, Trzeciak et al, Tet. Lett. 1992, 33, 4557-4560, and Tromelin et al, Tet. Lett. 1992, 33, 5197-5200. This naturally imposes a limitation on the nature of the peptide which can be prepared, since only amino acids whose side chains can be attached to the resin can be placed at the C-terminus. Another approach to the preparation of cyclic homodetic peptides involves their preparation on an oxime resin, which allows for intramolecular aminolysis of the peptide- resin bond by the α-amino group to cleave the peptide off the resin with concomittant cyclization . This is described by Osapay et al, Tet. Lett. 1990, 43, 6121-6124, and He et al, J. Amer. Chem. Soc. 1993, 115, 8066-8072. This method allows more flexibility in the sequences prepared, but can be hampered by low yields. Nevertheless, this method removes the side chain-protected cyclic intermediate from the resin, necessitating a solution deprotection step, and does not allow for the synthesis of a resin-bound cyclic homodetic peptide. There exists a need for new methods for cyclizing peptides, in particular cyclizing peptides on a resin. The instant invention provides a convenient and versatile method for the synthesis and cyclization of homodetic peptides on a resin. In addition, as described herein, by incorporating amino acid residues in the peptide which direct or facilitate cyclization, improved yields of the cyclic peptide may be obtained. Summary of the Invention An object of this invention is to provide a new method for preparing cyclic homodetic peptides. Another object of this invention is to provide a new method for preparing resin-bound cyclic homodetic peptides. Still another object of this invention is to produce cyclic peptide libraries which may be used for screening for bioactive molecules. A feature of this invention is the combination of a first linker group which provides a selectively cleavable bond between the peptide and the resin, and a second linker group which provides a means for attaching the peptide to the resin through a side chain group. Yet another feature of this invention is the use of amino acid sequences and amino acid residues in the peptide which facilitate the cyclization reaction. In one embodiment, a preferred feature of this invention is the combination of the second linker group with a template residue which facilitates cyclization. Detailed Description We have developed and combined two new approaches to the preparation of cyclic homodetic peptides and peptide libraries. Thus, we have applied template-based cyclization to solid phase synthesis to allow for the complete synthesis and deprotection of a cyclic homodetic peptide on a solid support. A variation of this procedure permits the release of the side chain-deprotected cyclic peptide from the resin. These allow for the preparation of either free or resin-bound cyclic homodetic peptides, and the preparation of cyclic homodetic peptide libraries. In one aspect, this invention is a method for preparing a resin bound cyclic homodetic peptide of the structure: wherein is a peptide of from 3-10 amino acid residues cyclized from its amino to carboxy terminus; L is a linker; and ® is a solid support; comprising: 1) preparing a linear resin bound peptide of the formula (I): Pg 1 (I) wherein I Q I is a linear peptide of from 3-10 amino acid residues; LI is a linker between the solid support and the carboxy terminus of the peptide; L2 is a linker on an amino acid side chain; Pg 1 is one or more protective groups on any reactive side chain functional groups of the peptide; Pg 2 is an optional protective group on the amino terminus of the peptide; SI is an optionally protected functional group which is attached to the solid support, optionally through the linker LI, but is not attached to the peptide or L2, and which can react with S2 to form a covalent bond; S2 is an optionally protected functional group on L2 which can react with SI to form a covalent bond; 2) removing any protecting groups from SI and S2, if necessary, and forming a covalent bond between SI and S2 to form a compound of the formula (II); 3) cleaving the carboxy terminus of I Q I from LI to provide a free carboxyl group, and, if necessary, removing Pg 2 to provide a free amino terminus to form a compound of the formula (IE); L1 L2 ® Q Pg 1 (III) 4) cyclizing the amino and carboxy termini to form a compound of the formula (IV); 5) and removing any protective groups Pg 1 to form a compound of the formula (V): Jo ~ K This method may also be used to prepare the free cyclized peptide V > ) by performing the above steps and subsequently cleaving the peptide from the resin. The cleavage of the peptide from the resin and removal of the side chain protecting groups Pg 1 can be performed in any order to yield the desired free cyclic peptides. The peptide is removed from the resin by cleaving the bond between the linker L2 and the amino acid to which it is attached. Certain abbreviations are used herein for convenience. Thus, Aloe means allyloxycarbonyl, β-Ala means β-alanine (3-amino propanoic acid), BHA means a benzhydrylamine-polystyrene resin, Boc means t-butyloxycarbonyl, Bn means benzyl, BOP is benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, DBU is 1.8 diazobicyclo[5.4.0]undec-7-ene; DPPA is diphenylphosphoryl azide, DCC is dicyclohexylcarbodiimide, DMAP is dimethylaminopyridine, EDC is N-ethyl- N'(dimethylaminopropyl)-carbodiimide;DMF means dimethyl formamide, Fmoc means 9-fluorenylmethyloxycarbonyl, HBTU refers to (1-hydroxybenztriazolyltetramethyl- uronium hexafluorophosphate), HMBA means 4-(4-hydroxymethyl-3-methoxy- phenyloxy)butyric acid, HOBT means 1-hydroxy-benzotriazole, Sue means succinyl, Hyp means hydroxyproline, Z means benzyloxycarbonyl. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984). Amino acids, as used herein, include compounds with have an amino terminus and carboxy terminus, preferably in a 1,2-, 1,3-, or 1,4- substitution pattern on a carbon backbone, α- Amino acids are most preferred, and include the 20 natural amino acids (which are L- amino acids except for glycine), which are found in proteins, the corresponding D-amino acids, the biosynthetically available amino acids which are not found in proteins {e.g., 4-hydroxy-proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, β-cyanoalanine), and synthetically derived α-amino acids, such as amino-isobutyric acid, norleucine, norvaline, C α alkylated and N α alkylated amino acids, Dtc, Tpr, homocysteine and homoserine. β-Alanine and γ-amino butyric acid are examples of 1 ,3- and 1 ,4- amino acids, and many others are well known to the art. Statine-like isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOH), hydroxyethylene isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOHCH2), reduced amide isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CH2NH linkage) and thioamide isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CSNH linkage) are also useful residues for this invention. Peptide, as used herein, indicates a sequence of amino acids linked by amide bonds. The peptides of this invention comprise a sequence of amino acids of 3 to 10 amino acid residues, preferably 5-7 residues, each residue being characterized by having an amino and a carboxy terminus. As used herein the designation of square brackets surrounding an amino acid sequence indicates a cyclic peptide wherein an amino terminus is joined to a carboxy terminus, e.g., in the case of α-amino acids the α-amino group of the first residue is joined to the carboxy group of the last residue. A cyclic peptide formed in such a manner is termed a homodetic peptide. ® is a solid support which may be used for solid phase peptide synthesis. The solid support may be in the form of a bead, a pin or other such form which is adaptable for solid phase phase peptide synthesis. Exemplary of such resins are benzhydrylamine- polystyrene, aminomethylpolystyrene, polyethyleneglycol-polystyrene, and the like. Such solid supports are generally commercially available. Typical of such resins are the TentaGel®, PEG-PS®, Merrifield and Sheppard resins. Polystyrene-based resins are especially useful. As used herein, the term linker means any molecular fragment which connects one atom or molecular fragment (collection of atoms) to another via covalent bonds. Typically, the linker will be composed of an optionally substituted alkylene or arylalkyl group with intervening functionalities, such as ether, sulfide, disulfide, ester or amide functionalities, within the fragment. Such functionalities will normally be used to create the linker (or make the connection between two molecular fragment) or to cleave an established linker (and separate the molecular fragments). Coupling methods to form peptide bonds are generally well known to the art. The methods of peptide synthesis generally set forth by Bodansky et al, THE PRACTICE OF PEPTIDE SYNTHESIS, Springer- Verlag, Berlin, 1984, are illustrative of the technique and are incorporated herein by reference. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical. Ali et al. in J. Med. Chem., 29, 984 (1986) and J. Med. Chem., 30, 2291 (1987). Reactive functional groups of the side chains of amino acids, e.g., those groups which undergo reaction inappropriately or interfere during a synthesis, such as the hydroxyl, mercaptan, carboxylic acid, amino, guanidino, imidazole or indole groups, are generally protected during synthesis. Protecting groups are those groups which mask the reactivity of such functional groups, yet may be removed at a later point in the synthesis to restore the original functional group. Suitable protecting groups for the reactive functional groups, and reagents for deprotecting these functional groups are disclosed in Greene et al, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS , Second Edition, John Wiley and Sons, New York, 1991. Deprotection indicates the removal of the protecting group and replacement with an hydrogen atom. Pg 1 is a selectively removable protecting group which may be used to protect the reactive side chain functional groups on the amino acid residues during solid phase synthesis. Pg 2 is a selectively removeable protecting group which may be used to protect the α-amino group of the amino acid residues of the peptide during solid phase synthesis. The first linker, LI, is a radical group which is attached by a selectively cleavable bond to an amino acid carboxyl group of the peptide to support peptide synthesis. The linker L2 is radical group which is attached by a selectively cleavable site to an amino acid in the peptide and which contains a second attachment site, S2, which may form a covalent bond with an attachment site SI. In a preferred embodiment, L2 is attached to a residue which acts as a template to promote cyclization of the peptide. SI is an attachment site, which may form a covalent bond with a second attachment site, S2, on the linker, L2. SI may be located on the solid support or on a linker attached to the solid support, provided that it is possible for the functional group at SI to react with the functional group at S2. It is necessary that SI not be on the peptide itself because the function of the S2 site is to provide a second path of attachment to the resin for the peptide when the bond between LI and the peptide is cleaved. In a preferred embodiment, SI is located on the linker LI . It will be apparent that the linker L, as used herein will be the resultant connection between the peptide and the solid support following the formation of a bond between SI and S2. If SI is located on the linker LI then L will be comprised of LI and L2; if S 1 is located on the solid support itself, then L will be comprised merely of L2. If the attachment site S 1 is a reactive functional group which might otherwise react under the conditions of the solid phase synthesis of the peptide, it may be protected by a protecting group which may be removed prior to forming the bond with S2. Such a protecting group should be orthogonal to other protecting groups, e.g., it should removable under conditions which would not affect other protecting groups in the molecule, such as Pg 1 and Pg 2 , and it should be stable to conditions which may be used to remove other protecting groups, such as the protecting group for the α-amino group of the component amino acid residues in the growing peptide chain. For instance, if the bond between SI and S2 is to be a peptide bond, then the attachment site SI is suitably an amino group protected by a suitable protecting group, Pg 3 , such as an Aloe group; and the attachment site at S2 is suitably a carboxyl group, protected by a suitable protecting group, Pg 4 , such as an allyl alcohol ester. The Fmoc group would be a suitable protecting group Pg 2 for the α-amino group of the component amino acids. Other suitable orthogonal protection strategies are known in the art. The attachment site SI may be on the solid support, however, in a unique embodiment, the attachment site is on a linker attached to the solid support. In a preferred embodiment the attachment site is attached to the linker LI itself. S2 is an attachment site which may form a covalent bond with SI , and is located on the linker L2. If the attachment site S2 is a reactive functional group which might otherwise react under the conditions of the solid phase synthesis of the peptide, it may be protected by a protecting group, Pg 4 which may be removed prior to forming the bond with the reactive group at SI. Such a protecting group should be orthogonal to other protecting groups and cleavable sites in the peptide-resin complex, except that such protecting group need not be orthogonal to Pg 3 since these groups will be activated at the same point in the synthetic sequence. Illustrative of suitable groups at the attachment sites 51 and S2 are: site reactive group protective group SI NH 2 -Aloc (Pg 3 ) 52 CO 2 H -O-allyl (Pg 4 ) which are cleavable by Pd(0), and suitable orthogonal protecting groups for Pg 1 are t- butyl based groups (except for Arg which is protected by a pentamethylenechroman-6- sulfonyl (Pmc) group) which are cleavable with acid, and the Fmoc group for the α- amino group of the amino acids, which is cleavable with base. Other combinations of reactive groups and protective groups will be apparent to one skilled in the art. I Q I , the peptide chain, is composed of 3-10 component amino acid residues. The peptide is built up in the usual manner for solid phase peptide synthesis by coupling the first N-protected amino acid through its carboxyl group to the linker LI, and coupling the remaining residues sequentially. Since the peptide is typically built up from N- protected amino acids, peptide synthesis proceeds by sequential deblocking of the amino group of the last residue added to the resin and coupling the next N-protected amino acid. In certain instances, it may be more efficient to attach one or more of the amino acids to the linker, LI, and then attach LI to the support. Where the amino acids possess side chains which contain reactive functional groups, these functional groups are protected by a suitable protecting group, Pg 1 . The amino group of the amino acids are protected by a suitable protecting group, Pg 2 . For template directed cyclization, the peptide Q is synthesized in such a manner that a residue or combination of residues imposes a secondary structure upon the peptide which favors conformations in which the amino and carboxyl termini of the peptide are in proximity. Thus, a template residue is an amino acid residue or combination of residues that imposes a conformational constraint, and facilitates cyclization of a linear peptide. For example, the linear peptide H-D-Trp-D-Asp-Pro-D-Val-Leu-OH cyclizes quite readily and in nearly quantitative yield because the Pro residue adopts a low energy γ-turn conformation which brings the amino and carboxyl termini into proximity, facilitating the cyclization reaction. Suitable templates would include derivatives of γ-turn inducing amino acids such as Pro, Hyp, Aib, a reduced dipeptide isostere, or a thioamide isostere, β-turn inducing dipeptides such as D-Pro-Pro (or more generally D-X-N-(Alk)Y, ' wherein D-X is an amino acid of the D configuration and N-(Alk)Y is an amino acid Y which is alkylated on it amino group) or non-peptide turn mimetics; and D-Pro-Gly-Pro, which induces an extended backbone conformation in a cyclic hexapeptide. Such elements are disclosed, for instance, by Peishoff et al, J. Med. Chem., 35, 3962 (1992). These elements have their effect by restricting rotation about certain bonds by steric hindrance due to their possessing a bulky group (e.g., Aib, C α -methyl amino acids, N α - methyl amino acids), or by incorporating elements of the carbon backbone in a ring structure (e.g., Pro) or by forming a hydrogen bond with adjoining residues (e.g. , thioamide isostere, dipeptide isostere). Other conformationally constrictive elements may be designed or selected from the art using these principles. The template must be introduced internally in the linear peptide sequence in order for the cyclization to be facilitated. Attachment of the template to the resin first, followed by growing the peptide chain in both directions from the template is a cumbersome approach. Sequential elongation from the C-terminus incurs the risk of racemization during coupling and also does not allow for a facile determination of the completeness of coupling. Coupling the complete C-terminal peptide moiety to the resin linked template or coupling the template-C-terminal peptide moiety to the resin both involve a fragment coupling, which are inherently less efficient procedures and in addition are not well suited to peptide library generation. It is possible to take advantage of the template or secondary structure-inducing element in the sequence to engineer the point of attachment of the peptide to the resin, and the point of attachment of the linker, L2. In a preferred embodiment, the method is carried out in such a manner that the linker LI comprises both the labile linkage to the resin as well as the reactive site SI, which is used to bind the peptide to the resin through a backbone linker L2. In addition the linker L2 is attached to the template residue. In the example which follows, the template amino acid residue is illustrated by C, and with reference to formula (I), the linker LI is represented by B-A and the reactive functional group S2 is located on B-A and is protected by Pg 4 ; the linker L2 is represented by D and the reactive functional group SI is located on D and is protected by Pg 3 ; and the peptide Q is represented by [AAι-AA4]-C-[AA5-AA8] wherein the functional group of the side chains of the amino acids are protected by Pg 1 and Pg 2 represents a protecting group for the terminal amino group of the peptide: Pg 4 D Pg 3 I I Pg 2 — [AA1-AA4] — C [AA 5 -AA 8 ] — [B-A]- ® Pg 1 Pg 1 1 ) removal of Pg 3 and Pg 4 2) coupling of D and A D- I Pg 2 — [AA 1 -AA 4 ] — c- -[AA 5 -AA 8 }— {B-A]- ® Pg 1 Pg 1 1) cleavage of link between B and peptide chain 2) removal of Pg 2 [B-A]- ® D I cyclization of peptide [B-A]-® D I [AA 1 -AA 4 ]— C — [AA 5 -AA 8 ]— ! Pg 1 Pg 1 removal of side chain protecting groups Pg 1 [B-A]— ® D Resin-linked cyclic homodetic peptide optional cleavage of link between tether D and template C I [AA 1 -AA 4 ] C [AA 5 -AA 8 ] - Free cyclic homodetic peptide In accordance with the method, a suitably protected linker B-A is attached to the peptide synthesis resin. The first amino acid is then attached to the linker and peptide synthesis is carried out according to the usual methods of solid phase peptide synthesis. At the appropriate time the preformed protected template-tether Pg -C(D-Pg 4 ), wherein C is an amino acid and D is a tether (e.g., L2), is coupled to the growing peptide chain. Upon completion of the linear peptide synthesis, the protecting group Pg 4 on the reactive functional group of the tether and the protecting group Pg 3 on the reactive functional group of the linker (LI) are removed. The tether and linker are then coupled to each other, creating a peptide which has been cyclized onto the resin via the linker. The bond between the C-terminal amino acid and the linker B-A is then cleaved, to yield a linear peptide now attached to the resin through the template-tether. Cleavage of the amino terminal protecting group Pg 2 gives a resin-bound linear peptide with both termini free, allowing for cyclization to be carried out. After carrying out the cyclization, cleavage of the side-chain protecting groups Pg 1 then gives the fully deprotected, resin-bound cyclic homodetic peptide. At this point, the bond between the template amino acid C and the tether D can be selectively cleaved by conventional methods if desired, such as by hydrazinolysis, hydrogenolysis, trifluoroacetic acid or HF, yielding the free peptide. The salient features of this scheme are as follows. The resin incorporates a linker with two sites of peptide attachment, one of which is temporarily masked. The peptide can be synthesized by the usual methods of peptide synthesis, employing high yielding coupling strategies and allowing for assessment of completeness of coupling by ninhydrin test. The protected linear peptide is attached to the resin via a newly synthesized bond ■ between the linker and a tether attached to the template component of the peptide. This allows for the amino and carboxyl termini of the peptide to be freed and high efficiency, template directed cyclization can take place on a resin-bound peptide. The method requires a number of orthogonally cleavable protecting groups and linkers. The amine protecting group Pg 2 must be cleavable in the presence of the side chain protecting groups Pg 1 , the tether protecting group Pg 4 , the linker protecting group Pg 3 , the bond between template C and tether D and the bond between carboxyl terminal amino acid and linker B-A. It must also be stable to cleavage conditions for Pg 3 and Pg 4 and to the bond between the carboxyl terminal amino acid and linker B-A. Similarly, Pg 3 and Pg 4 must be stable to the conditions for removal of Pg 2 and selectively cleavable in the presence of Pg 2 , Pg 1 and the bonds between template C and tether D, and the carboxyl terminal amino acid and linker B-A. The bond between carboxyl terminal amino acid and linker B-A must be selectively cleavable in the presence of Pg 2 and Pg 1 and the bond between template C and tether D. In a specific embodiment the approach is illustrated in Schemes I-III below. Scheme I illustrates the partial synthesis of an acid labile linker (e.g., LI) and the first residue of a peptide for attachment to the resin. Scheme II illustrates the synthesis of a conformationally-constraining template residue attached to a linker (e.g., L2) for incorporation into a peptide for directing cyclization of the peptide. Scheme m illustrates the general method of on-resin template directed cyclization for a model peptide. Scheme I pyridine, CH 2 CI 2 Fmoc-Leu-HMBA-OH Scheme II Synthesis of a Protected Hydroxyproline Template with a Linker Boc-Hyp-OH 1 " CsHC ° 3 ' H *°- MeOH^ Boc . Hyp . OBn Z-NHCH 2 CH 2 COCI, 2. BnBr, DMF 50 ° C (1) pyridine, CH2CI2 (2) (3) moc (5) Scheme III Template Directed Cyclization (1.18 mMol/ g) BHA resin 1. Fmoc-(Aloc)-Lys-OH, HBTU, HOBt, NMM, DMF 2. 20% piperidine/ DMF 3. Fmoc-Leu-HMBA, HBTU, NMM, HOBt, DMF Fmoc-l_eu-HMBA-(Aloc)-Lys-BHA resin Repeated Fmoc couplings and deprotections. β-Ala-Suc-OAIIyl Fmoc-(Boc)-D-Trp-(o-f-Bu)-D-Asp-Hyp-D-Val-Leu-HMBA-(Aioc)-Lys-BHA resin (1) (PPh 3 ) 2 PdCI 2 , Bu 3 SnH, HOAc (1 :1 ) DMSO, CH 2 CI 2 β-Ala-Suc-OH NH ; Fmoc-(Boc)-D-Trp-(o-f-Bu)-D-Asp-Hyp-D-Val-Leu-HMBA-Lys-BHA resin HBTU, HOBt, NMM, DMF β-Ala-Suc r Π* ~ ™ I Fmoc-(Boc)-D-Tφ-(o-f-Bu)-D-Asp-Hyp-D-Val-Leu-HMBA-Lys-BHA resin 1. 20 % piperidine in DMF 2. 5 % TFA, 5 % anisole in CH 2 CI 2 β-Ala-Suc NH 2 -(Boc)-D-Trp-(o-f-Bu)-D-Asp-Hyp-D-Val-Leu-OH HMBA-Lys-BHA resin HBTU, HOBt, NMM, DMF • β-Ala-Suc Cyclo [(Boc)-D-Tφ-(o-f-Bu)-D-Asp-Hyp-D-Val-Leu-] HMBA-Lys-BHA resin (2) Leu-] (3) Cyclo [ D-Trp-D-Asp-Hyp-D-Val-Leu-] (4) In the specific example cited in Scheme in, the resin is benzhydrylamine resin. The linker B-A (e.g., LI) is composed of two moieties: A is ε-Aloc-Lys, in which the side chain of the Lys provides for the secondary site of attachment to the resin and the carboxyl of the Lys residue is attached to the BHA resin via an amide bond; and B is 4- (4'-hydroxymethyl-3-methoxy)phenoxybutyric acid (HMPB), in which the carboxyl of the HMPB is attached to the α-amine of the Lys residue and the growing peptide is attached to the hydroxymethyl group. Fmoc amino acids with side chains protected by t- butyl based protecting groups are used in this strategy, i.e. Pg 2 is Fmoc and Pg 1 is t-butyl- based or PMC. The template residue is 4-hydroxyproline (Hyp) and the tether L2 is succinyl-β-alanine, which is attached to the hydroxyl of Hyp via the β-Ala carboxyl group. The succinyl carboxyl group is protected as an allyl ester (Pg 4 ). The Fmoc group is acid stable and stable to Pd(0) reagents while cleavable with secondary amines. The allyl ester and Aloe groups are stable to secondary amines and acid but cleavable with Pd(0) reagents. The HMPB linkage to the peptide is cleavable with dilute acid (2% TFA/CH2CI 2 ) but stable to secondary amines and Pd(0). The t- butyl and Pmc side chain protecting groups are stable to secondary amines and Pd(0) but are cleavable by strong acid (95% TFA/CH2CI2). The ester linkage between the Hyp template and the β-Ala of the tether is stable to all the above conditions but can be cleaved by hydrazinolysis. One skilled in the art will recognize that there are other potentially useful protecting group schemes which could be used as well, provided that all the requirements for orthogonality are met. Strategies for preparing and testing combinatorial libraries in solution or on solid phase supports are known and are disclosed, for example, by Lam et al, Nature, 354 82, (1991) and PCT/US91/04666 (WO 92/00091); Houghton et al, Nature, 354, 84 (1991) and PCT/US91/08694 (WO 92/09300). Such strategies may be combined with the method of the instant invention to yield libraries of cyclic homodetic peptides. For instance, as one build the peptide chain one may divide the solid support into several equimolar groups at each occurrence of peptide coupling, exhaustively react a different amino acid with each group, recombine and mix the groups, and redivide the groups for the next coupling. In this way, the resultant linear peptide resin complex will be composed of beads which each have different amino acid sequences, although each bead will have a unique peptide sequence. The libraries may be of the peptide-resin complex or of solutions of the free cyclic peptides. These libraries may be used for the purpose of screening a target molecule or surface to identify those peptides which bind to the target molecule, or inhibit binding of a second molecule to the target molecule, as is known in the art. Encoding strategies, such as those disclosed in PCT/US92/09345 (WO 94/08051) may also combined with the method of this invention for improved detection of the active peptides of such combinatorial libraries. Libraries may also be constituted and screened in accordance with the instant method by preparing groups of peptides of partially known composition by using the mimotope strategy disclosed by Geysen et al, U.S. Patent 5,194,392 and U.S. Patent 4,562,157. Other methods known to the art for assaying resin beads by partial cleavage of the active peptide may also be appropriate. The Examples which follow are intended to illustrate how to make and use the compounds, libraries and methods of this invention and are in no way considered to be a limitation. For convenience of reference, certain of the compounds are labeled by the numbers indicated in Schemes 1-iπ, e.g., compound 5 in scheme El is iπ-5. Example 1 Preparation of the Fmoc-leucine preloaded acid labile linker 4[4-[N α -(9-fluorenylmethoxycarbonyl)-leucinyl-oxymethyl]-3-methoxy- phenyloxy] butyric acid (1) Scheme 1 To a stirred solution of Fmoc-leucine (10 g, 28 mMol) in dry CH2CI2 (100 mL) and pyridine (2.3 mL, 28 mMol) was added dropwise cyanuric fluoride (3.3 mL, 28 mMol). The reaction was stirred under Ar at room temperature for 4 h, filtered to remove insoluble precipitates, washed with cold H2O (2 x 250 mL), dried (MgSO_ι) and evaporated to dryness. To a stirred solution of 4(4-hydroxymethyl-3-methoxy-phenyloxy)butyric acid (6.80 g, 28 mMol) in dry CH2CI2 (100 mL) and pyridine at 0°C was added a solution of the Fmoc-leucinyl fluoride in CH2CI2 (50 mL). The reaction was allowed to warm to room temperature and stirred for 16 h. The reaction was then acidified with cold aq. 0.5N HCI, washed with brine, dried (MgSO 4 ) and evaporated under reduced pressure. Purification by flash chromatography on silica gel (30:70:1) EtOAc/n-hexane/HOAc and repeated evaporation from toluene gave the title compound. (10.25 g, 63%) as a white solid. ϊH-NMR (CDC13, 400 MHz) δ 0.92 (2 x d, 6H), 1.55 (m, IH), 1.68 (m, 2H), 2.10 (m, 2H), 2.58 (t, 2H), 3.77 (s, 3H), 3.99 (t, 2H), 4.19 (t, IH), 4.40 (m, 3H), 5.10 (d, J=l 1.9 Hz, IH), 5.16 (d, J=l 1.9 Hz, 2H), 5.28 (d, J=8.8 Hz, IH), 6.38 (m, IH), 6.41 (s, IH), 7.19 (m, IH), 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 Preparation of N α -(9-fluorenylmethoxycarbonyl)-O-(2-allyloxycarbonyl-propionyl-β- alaninyl)hydroxyproline (Compound II-5) - Template residue with linker a) N α -(t-butoxycarbonyl)-hydroxyproline benzyl ester (II- 1) To a stirred solution of N α -t-butoxycarbonyl-hydroxyproline (5g, 21.6 mMol) in methanol (100 mL) and water (50 mL) was added CSHCO3 (4.2 g, 21.6 mMol). After stirring for 15 min the solution was evaporated to dryness and re-evaporated twice from methanol (2 x 100 mL) then finally DMF (100 mL). The remaining residue was taken up in DMF (100 mL) and with stirring at room temperature benzylbromide (3.1 mL, 26 mMol) was added in one portion. The reaction was then stirred at 50°C under Ar for 16 h. The resultant suspension was evaporated under reduced pressure, taken up in ethyl acetate (150 mL), washed with H 2 O (150 mL), dried (MgSO 4 ) and evaporated. Purification by flash chromatography on silica gel (50% ethyl acetate in n-hexane) gave the title compound (1) Scheme 1 (6.75 g, 21 mMol) as a clear oil. *H-NMR (CDC13, 250 MHz) Mixture of amide rotomers δ 1.35 and 1.46 (2 x s, 9H), 1.80 (br s, IH), 2.08 (m, IH), 2.30 (m, IH), 2.92-3.69 (m, 2H), 4.48 (m, 2H), 5.16 and 5.19 (s and dd, 2H), 7.35 (s, 5H). b) N α -(t-butoxycarbonyl)-0-[Nβ-(benzyloxycarbonyl)-β-alaninyl)hydroxyproline benzyl ester (II-2) To a stirred solution of Cbz-β-alanine (5 g, 22.4 mMol) in toluene (50 mL) was added oxalyl chloride (20 mL) in one portion followed by 1 drop of DMF to initiate the reaction. The reaction was stirred for 16 h then evaporated under reduced pressure. Re- evaporation from toluene (2 x 50 mL) gave the corresponding acid chloride which was used as is in the next step. To a stirred solution of the compound of Example 1 (6.75 g, 21 mMol) in CH 2 CI2 (75 mL) at 0°C was added pyridine (1.82 mL, 22.5 mMol) followed by a solution of the above acid chloride in CH 2 CI 2 (25 mL). The reaction was then allowed to warm to room temperature and stirred for 16 h. After evaporation at reduced pressure the remaining residue was taken up in ethyl acetate (150 mL), washed with H 2 O (150 mL), aq. IN HCI (150 mL), brine (150 mL), dried (MgSO 4 ) and evaporated. Purification by flash chromatography on silica gel (40% ethyl acetate in n-hexane) gave the tide compound (4.30 g, 38%) as a clear oil. -NMR (CDCI 3 , 250 MHz) Mixture of amide rotomers δ 1.36 and 1.45 (2 x s, 9H), 2.19 (m, IH), 2.37 (m, IH), 2.54 (t, 2H), 3.46 (m, 2H), 3.69 (m, 2H), 4.42 (dt, IH), 5.09 (s, 2H), 5.16 (s, 2H), 5.16-5.30 (m, 2H), 7.35 (s, 10H). c) N α -(t-butoxycarbonyl)-0-(2-allyloxycarbonyl-propionyl-β-alaninyl)hydroxyproline (II-4) A solution of the compound of Example 2(b) (4.20 g, 8 mMol) in methanol (75 mL) was hydrogenated in a Parr apparatus over 10% Pd on carbon (2.5 g) at 55 psi H 2 for 6 h. After filtering off the catalyst through a pad of Celite and washing with glacial acetic acid (4 x 50 mL) the filtrate was evaporated under reduced pressure and re- evaporated from methanol (2 x 50 mL) to give N α -(t-butoxycarbonyl)-O-(β- alaninyl)hydroxyproline benzyl ester (II-3) (2.36 g, 98%) as a white solid. To succinic anhydride (24 g, 240 mMol) was added allyl alcohol (25 mL, 360 mMol). The reaction was reflected for 16 h under Ar, cooled to room temperature, dissolved in aq. sat. NaHCO 3 (400 mL), washed with Et2θ (200 mL), acidified with cold aq. 3N HCI, extracted with ethyl acetate (300 mL), washed with brine, dried (MgSO 4 ) and evaporated under reduced pressure to give succinic acid mono allyl ester (36.02 g, 95%). To a stirred solution of succinic acid mono allyl ester (13.75 g, 87 mMol) in toluene (50 mL) was added oxalyl chloride (20 mL, 230 mMol). The reaction was stirred for 16 h at room temperature and evaporated under reduced pressure. Re- evaporation from toluene (2 x 50 mL) gave succinyl chloride mono allyl ester (14.68 g, 96%) as a clear liquid which was used in the following coupling reaction. To a stirred solution of N α -(t-butoxycarbonyl)-0-(β-alaninyl)hydroxyproline benzyl ester (2.36 g, 7.8 mMol) in (4:1) dioxane/H2θ (100 mL) at 0°C was added NaHCO 3 (1-6 g, 19 mMol) followed by the above succinyl chloride mono allyl ester (2.0 g, 11 mMol) dropwise over 15 min. The reaction was allowed to go to room temperature and stirred for 16 h. After acidification with aq. IN HCI the solution was extracted with CHCI3 (150 mL) washed with brine, dried (MgSO 4 ) and evaporated to dryness. Purification by flash chromatography on silica gel (97:3:1) CHCl3/MeOH HOAc gave the title compound (4) Scheme 1 (1.81 g, 53%) as a white solid. -NMR (CDCi3,400 MHz) Mixture of amide rotomers δ 1.44 and 1.48 (2 x 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, IH), 5.32 (dd, 2H), 5.90 (m, IH); MS(ES) m/z 443.0 [M+H] + , m/z 441.2 [M-H]-. d) N α -(9-fluorenylmethoxycarbonyl)-O-(2-allyloxycarbonyl-propionyl-β- alaninyl)hydroxyproline (II-5) To a stirred solution of the compound of Example 2(c) ( 1.81 g, 4.1 mMol) in CH2CI2 (5 mL) was added trifluoroacetic acid (45 mL). After stirring for 45 min the reaction was evaporated to dryness under reduced pressure. Re-evaporation several times with CHCi toluene gave the deprotected TFA salt (1.87 g) as a foam. To a stirred solution of the above TFA salt in CHCI3 (75 mL) was added Et3N (1.15 mL, 8.2 mMol) followed by N-(9-fluorenylmethoxycarbonyloxy)succinimide (1.52 g, 4.5 mMol). After stirring for 6 h, the solution was acidified with aq. IN HCI (10 mL), washed with brine, dried (MgSO 4 ) and evaporated to dryness. Purification by flash chromatography on silica gel (100:4:1) CHCl3/MeOH/HOAc gave the title compound (5) Scheme 1 (2.11 g, 91%) as a white solid. Η-NMR (CDC-3,400 MHz) δ 2.30 (m, IH), 2.40 (m, IH), 2.46 (t, 2H), 2.53 (t, 2H), 2.68 (t, 2H), 3.50 (m, 2H), 3.72 (m, 2H), 4.22 (dt, IH), 4.32-4.53 (m, 3H), 4.55 (d, J=5.6 Hz, 2H), 5.22-5.35 (m, 3H), 5.88 (m, IH), 6.28 (m, IH), 7.23-7.45 (m, 4H), 7.55 (m, 2H), 7.71 (d, J=7.5 Hz, IH), 7.76 (d, J=7.5 Hz, IH); MS(ES) m z 565.2 [M+H]+, m/z 563.0 [M-H]". Example 3 Preparation of the model cyclic-peptide (4) Scheme IE: cyclo-(D-tryptophanyl-D-aspartyl-hydroxyprolyl-D-valinyl-leucine) (TJI-4) The protected linear peptide acid-labile resin with lysine reattachment site (III- 1 ) was constructed on a commercial benzhydrylamine resin (1.18 mMol/g) by sequential couplings and deprotections of Fmoc-N ε -(Aloc)-Lysine, Fmoc-leucinyl-HMB A, Fmoc-D- valine, Fmoc-(O-βAla-Suc-OAllyl)-hydroxyproline (II-5), Fmoc-(0-t-Bu)-D-aspartic acid and Fmoc-N^-Boc-D-tryptophan according to the following general synthetic methods. General Coupling Method HBTU (3 Mol equiv.) was dissolved in DMF with swirling in a small Erlenmeyer flask. To this solution was added the Fmoc-amino acid (3 Mol equiv.) followed by N- methylmorpholine (8 Mol equiv.). This solution was next added to the resin (1 Mol equiv.) and HOBt (3 Mol equiv.) in a rocker vessel and shaken for 2h. The reaction was checked by the Kaiser test and when complete washed with DMF (4x) then CH 2 CI 2 (2x). General Fmoc deprotection Method The Fmoc protecting group was removed by treatment of the peptide resin with 20% piperidine in DMF (1 x 5 min and 1 x 15 min). The resin was then washed with DMF (4x). To the prepared resin peptide Fmoc-(Boc)D-Trp-(0-t-Bu)D-Asp-(O-β-Ala-Suc- OAllyl)Hyp-D-Val-Leu-HMBA-(Aloc)Lys-BHA (IH-l) (2 g, 0.8 mMol) in (1:1) DMSO/CH 2 CI2 (10 mL) was added with stirring under Ar, at room temperature, acetic acid (460 μL, 8 mMol), (Ph P) 2 PdCl2 (24 mg, 34μMol) followed by Bu SnH (862 μL, 3.2 mMol). After stirring for 30 min the resin was filtered off and washed with (1:1) DMSO/CH2CI 2 (2 x 50 mL). The reaction was repeated a second time and washed with (1:1) DMSO/CH 2 Cl 2 (2 x 50 mL), CH 2 C1 2 (2 x 50 mL), 10% N-methylmorpholine in CH 2 C1 2 (1 x 50 mL), CH 2 C1 2 (2 x 50 mL) then n-hexane (2 x 50 mL). 1.97 g of the partially deprotected peptide resin was obtained after drying under vacuum. The Kaiser test was positive for free amine. To the above resin peptide (1.84 g, 0.75 mMol) in DMF (10 mL) was added with stirring under Ar, HOBt (0.22 g, 1.6 mMol), N-methylmorpholine (0.45 mL, 1.6 mMol) followed by HBTU (0.60 g, 1.6 mMol). After stirring for 16 h the resin peptide was filtered and washed with DMF (2 x 50 mL), CH 2 CI 2 (2 x 50 mL) and n-hexane (2 x 50 mL). The Kaiser test was negative for free amine. The above resin peptide was treated with 20% piperidine in DMF (50 mL) for 5 min then again with 20% piperidine in DMF (50 mL) for 15 min, filtered, washed with DMF and treated with a solution of 5% trifluoroacetic acid and 5% anisole in CH2CI 2 (4 x 10 mL) for 2 min each. The resulting hydroxyprolyl side-chain attached linear peptide resin was next washed with CH 2 CI2 (2 x 50 mL), 10% N-methylmorpholine in CH 2 CI 2 , CH 2 CI 2 (2 x 50 mL) and n-hexane (2 x 50 mL) to give 1.22 g resin peptide after drying under vacuum. The Kaiser test was positive for free amine. To the above resin peptide (1.20 g, 0.74 mMol) in DMF (10 mL) was added with stirring under Ar at room temperature 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 h the resin was filtered and washed with DMF (2 x 50 mL), (1:1) CHCl3/MeOH (2 x 50 mL), CH2CI 2 (2 x 50 mL) and n-hexane (2 x 50 mL). 1.22 g of peptide resin (2) Scheme 3 was obtained after drying under vacuum. The Kaiser test was negative for free amine. To part of the above peptide resin (2) Scheme 3 (0.51 g, 0.31 mMol) in EtOH (5 mL) was added NH2NH2 monohydrate (200 μL, 4.1 mMol). After stirring for 48 h the resin was filtered off and washed with MeOH (2 x 20 mL). The crude protected cyclic peptide (66 mg, 29%) was obtained from evaporation of the filtrate and drying under vacuo. Purification by flash chromatography on silica gel (3% MeOH in CHCI3) gave analytically pure material (3) Scheme 3 (44 mg) as a white solid. TLC Rf=0.28 (5% MeOH/CHCl 3 ); iH-NMR (CDC13, 400 MHz) δ 0.71 (d, J=6.5 Hz, 6H), 0.86 (d, J=6.6 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H), 1.20 (m, IH), 1.40 (m, IH), 1.43 (s, 9H), 1.53 (m, 2H), 1.67 (s, 9H), 1.85 (m, 2H), 2.40 (dd, IH), 2.75 (m, IH), 2.82 (dd, IH), 3.16 (m, IH), 3.28 (d, J=6.1 Hz, 2H), 3.50 (d, J=4.5 Hz, 2H), 3.78 (dd, IH), 4.00 (t, IH), 4.62 (m, IH), 4.78 (dd, IH), 4.95 (dd, IH), 5.12 (m, IH), 6.12 (d, J=8.0 Hz, IH), 7.10 (m, IH), 7.24 (m, 2H), 7.33 (t, IH), 7.44 (s, IH), 7.54 (d, J=7.8 Hz, IH), 7.74 (d, J=9.7 Hz, IH), 8.15 (d, IH); MS(ES) m/z 783.4 [M+H] + , m/z 781.2 [M-H]-. To the remainder of the resin peptide (III-2) (0.71 g, 0.44 mMol) was added a solution of 10% anisole in trifluoroacetic acid (10 mL). After stirring for 15 min the resin was filtered and retreated once more with 10% anisole in trifluoroacetic acid (10 mL) for another 15 min. The fully deprotected peptide resin was washed with CH2CI2 (2 x 25 mL), 10% N-methylmorpholine in CH2CI2 (2 x 25 mL), CH2CI2 (2 x 25 mL) and n- hexane (2 x 25 mL). To the above peptide resin in EtOH (5 mL) was added NH2NH2 monohydrate (300 μL). After stirring for 48 h the resin was filtered off and washed with MeOH (2 x 20 mL). The filtrate was evaporated under reduced pressure to give the crude product (77.0 mg, 29%) as the hydrazine salt. Purification by flash chromatography on a YMC- ODS (S-50) column (30-50% CH3CN, 0.1% TFA, H 2 O) gave the title compound (D3-4) (37 mg) as a lyophilized white powder. A further 103 mg crude cyclic peptide (68% total) was obtained from subjecting the remaining resin with a solution of anhydrous hydrazine (2 mL) in methanol (2 mL) and stirring for an additional 48 h at 60°C under Ar, filtering, washing with methanol (2 x 20 ml) and evaporating the filtrate to dryness. HPLC K'=5.21 Hamilton PRP-1 column eluted with 30% CH3CN, 0.1% TFA/ 0.1% TFA, H 2 0; K'=5.31 20 min gradient 20-80% CH3CN, 0.1 % TFA/ 0.1 % TFA, H 2 0; TLC R f =0.81 (4:1:1) n-butanol/HOAc/H 2 O, Rf=0.81 (15:3:12:10) n-butanol/HOAc/H 2 O/ pyridine; 1H-NMR (c -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, IH), 1.15 (m, IH), 1.21 (m, IH), 1.60 (m, IH), 1.68 (m, IH), 2.32 (dd, IH), 2.51 (m, IH), 2.79 (dd, IH), 2.88 (dd, 1H), 3.09 (dd, IH), 3.19 (dd, IH), 3.47 (dd, IH), 3.98 (m, IH), 4.11 (dd, IH), 4.26 (m, 2H), 4.34 (dd, IH), 5.00 (m, IH), 6.98 (t, IH), 7.05 (t, IH), 7.14 (d, J=1.9 Hz, IH), 7.32 (d, J=8.1 Hz, IH), 7.38 (d, J=10.3 Hz, IH), 7.52 (d, J=7.9 Hz, IH), 7.80 (d, J=9.2 Hz, IH), 8.77 (d, J=5.0 Hz, IH), 8.80 (d, J=8.1 Hz, IH); MS(ES) m/z 627.2 [M+H]+, m/z 649.2 [M+Na]+, m/z 625.2 [M-H]-. Example 4 Preparation of model libraries. The following libraries have been prepared according to the experimental procedures outlined in Example 3 but using the standard split-and-combine methodology described by Furka (A Furka, M. Sebestyέn, M. Asgedom, G. Dibό, Int. J. Pept. Prot. Res. 1991, 37, 487-493). The libraries all have the general formula cyclo [AA1-AA2-Hyp-AA4-AA5]. Library 1 (8 compounds) cyclo[AAl-AA2-Hyp-AA4-AA5] wherein A = D-Trp or D-Gln B = D-Asp or D-Ala C = D-Val or D-Thr D = Leu Starting with 2.0 g Fmoc-Leu-HMPB-Lys(ε-Aloc)-BHA, the resin was split into two equal portions and the amino terminus is deprotected as in Example 3. One portion was coupled to Fmoc-D-Val; and the other was coupled to Fmoc-D-Thr(t-Bu). The two resin aliquots were then recombined, deprotected and coupled to Fmoc-Hyp(β-Ala-Suc- O-allyl). The resin was again split into two equal portions which were deprotected and coupled either to Fmoc-D-Asp(t-Bu) or Fmoc-D-Ala. The resins were again combined, mixed, and divided into two equal portions, to which were coupled either to Fmoc-D- Trp(Boc) or Fmoc-D-Gln. The resins were again combined and the synthesis continued as in Example 3 on the mixture of peptidyl resins. A 1 g aliquot of the peptidyl resin yielded 294 mg of isolated product after • hydrazinolysis (98% of theoretical). The product contained only the eight expected peptides by hplc and hplc/mass spectrometry analysis, and in approximately equimolar ratios. Library 2 (10,000 compounds) cyclo[AAl-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- Tip or D-Lys AA5 = Asp, Asn, Gly, Nva, Ser, Ser(Me), Phe, Tyr, Trp or Lys After coupling of the N-terminal amino acid in the sequence, the resin samples were not recombined but rather carried along individually, giving rise to the 10 sublibraries listed below. Sublibrary 2. a in which AA1 = Gly 300 mg of peptidyl resin yielded 37.6 mg product (46% of theoretical). Sublibrary 2. l.b in which AAl = D-Nva 300 mg of peptidyl resin yielded 75.1 mg product (86% of theoretical). Sublibrary 2. l.c in which AAl = D-Ser 300 mg of peptidyl resin yielded 77.2 mg product (92% of theoretical). Sublibrary 2. l.d in which AAl = D-Ser(Me) 300 mg of peptidyl resin yielded 69.6 mg product (75% of theoretical). Sublibrary 2.1. e in which AAl = D-Asp 300 mg of peptidyl resin yielded 58.5 mg product (56% of theoretical). Sublibrary 2. l.f in which AAl = D-Asn 300 mg of peptidyl resin yielded 53.6 mg product (50% of theoretical). Sublibrary 2. l.g in which AAl = D-Phe 300 mg of peptidyl resin yielded 61.5 mg product (57% of theoretical). Sublibrary 2. l.h in which AAl = D-Trp 300 mg of peptidyl resin yielded. 67.0 mg product (62% of theoretical). Sublibrary 2. l.i in which AAl = D-Tyr 300 mg of peptidyl resin yielded 73.7 mg product (69% of theoretical). Sublibrary 2. l.j in which AAl = D-Lys 300 mg of peptidyl resin yielded 69.4 mg product (64% of theoretical). Following the above procedure, except splitting the resin at positions AA2, AA4 and AA5 and carrying each sublibrary through individually thereafter using the same procedure, Sublibraries 2.2.a-j, 2.4a-j and 2.5.a-j (wherein 2.x.a-j is used to express the various sublibraries which are isolated and identified with a known amino acid in position x of the peptide) may be prepared. Example 5 The peptide libraries of Example 4 are screened in assays to identify compounds which bind to neurokinin 3 (NK3) or inhibit phosphorylation of a test substrate by protein kinase C (PKC). The enzyme inhibition assays are run according to the procedures described in Egan et al, Anal. Biochem. 1988, 175, 552 and Alexander et al, Biochem. J. 1990, 268, 303 for PKC using clones for the human PKCα and PKCβ receptors. Binding assays for NK3 receptors can be performed as in McKnight et ah, Br. J. Pharmacol, 1991, 104, 335-360. The results of such assays using sublibraries 2.1.a-j of Example 4, are given in Table 1 below: Table 1 NA = not active at 110 ug/mL Library deconvolution is performed by iterative resynthesis of active sublibraries until single compounds are identified, according to the methods disclosed in. Zuckermann et al. J. Med. Chem., 1994, 37, 2678-2685 and Erb et al, Proc. Natl Acad. Sci. USA 1994, 91, 11422-11426. This invention is not limited in scope to the foregoing embodiments described, and various modifications of the procedures described will be apparent to those skilled in the art. Such modifications are included within this invention which is limited only by the spirit of the claims which follow. The disclosures of the various publications which are cited herein are intended to describe the state of the art and are incorporated herein in their entirety as if fully set forth.

Claims: What is claimed is: 1. A method for preparing cyclic homodetic peptide bound to a solid support comprising: wherein . is a peptide of from 3-10 amino acid residues cyclized from its amino to carboxy terminus; L is a linker; and ® is a solid support; comprising: 1) preparing a linear resin bound peptide of the formula (I): Pg 1 (I) wherein ED i,s a linear peptide of from 3-10 amino acid residues; LI is a linker between the solid support and the carboxy terminus of the peptide; L2 is a linker on an amino acid side chain; Pg 1 is one or more protective groups on any reactive side chain functional groups of the peptide; Pg 2 is an optional protective group on the amino terminus of the peptide; 51 is an optionally protected functional group which is attached to the solid support, optionally through the linker LI, but is not attached to the peptide or L2, and which can react with S2 to form a covalent bond; 52 is an optionally protected functional group on L2 which can react with SI to form a covalent bond; 2) removing any protecting groups from SI and S2, if necessary, and forming a covalent bond between SI and S2 to form a compound of the formula (II); 3) cleaving the carboxy terminus of ΓQ~] from LI to provide a free carboxyl group, and, if necessary, removing Pg 2 to provide a free amino terminus to form a compound of the formula (IH); L1 L2 Q Pg 1 (III) 4) cyclizing the amino and carboxy termini of the peptide to form a compound of the formula (IV); 5) and removing any protective groups Pg 1 to form a compound of the formula (V): 2. A method according to claim 1 for preparing a cyclic homodetic peptide further comprising cleaving said cyclic peptide from the linker L2. 3. A method according to claim 1 wherein the peptide I Q I contains a residue which directs cyclization of the linear peptide. 4. A method according to claim 1 wherein Pg 1 and Pg 2 are functional groups orthogonally protected to Pg 3 and Pg 4 . 5. A method according to claim 1 wherein the support ® is a polystyrene-based or a polyamide-based resin. 6. A method according to claim 3 wherein the residue which directs the cyclization is Pro, Hyp, Aib, Pro-D-Pro or D-X-N(Alk)Y, wherein D-X is an amino acid of the D- configuration and N-(Alk)Y is an amino acid Y which is alkylated on its α-amino group, or a derivative thereof. 7. A method according to claim 1 wherein the peptide I Q I is a penta- or hexa-peptide. ' 8. A method according to claim 1 wherein the attachment site SI is located on the linker LI. 9. A method according to claim 1 wherein L2 is 10. A method according to claim 1 wherein LI is 11. A method according to claim 1 wherein L is - CO(CH 2 )2NHCO(CH 2 )2CONH(CH 2 )4N(HMBA)CO-. 12. A method according to claim 1 for preparing a library of peptides, wherein the peptide I Q I comprises a plurality of peptides each on a different solid support. 13. A method according to claim 1 wherein the first reactive site SI is an optionally protected amino group, and the second reactive site S2 is an optionally protected carboxyl group. 14. A method according to claim 1 wherein the first reactive site SI is an optionally protected carboxyl group, and the second reactive site S2 is an optionally protected amino group. 15. A method according to claim 1 wherein the protecting groups on SI and S2 are cleavable without removing Pg 1 or Pg 2 . 16. A method according to claim 1 wherein the covalent bond between the peptide I Q I and LI is cleavable in the presence of Pg 1 . • 17. A method according to claim 6 wherein the residue which directs the cyclization is Hyp. 18. A cyclic peptide prepared according to the process of claim 2. 19. A library of cyclic peptides prepared according to the process of claim 2. 20. A library of cyclic peptides according to the formula: cyclo[AAl-AA2-Hyp-AA5-AA6] wherein AA1, AA2, AA3 = D-Ala, D-Asp, D-Asn, Gly, D-Nva, D-Ser, D-Ser(Me), D-Phe, D-Tyr, D-Trp or D-Lys; and AA4 is Ala, Asp, Asn, Gly, Nva, Ser, Ser(Me), Phe, Tyr, Trp or Lys. 21. A library of cyclic peptides bound to a solid support prepared according to claim 1. 22. A library of cyclic peptides bound to a solid support prepared according to claim 1 of the formula: wherein is a peptide of from 3-10 amino acid residues cyclized from its amino to carboxy terminus wherein at least one residue is Hyp; L is a linker of he formula -CO(CH 2 )2NHCO(CH 2 ) 2 CONH(CH 2 )4N(HMBA)CO-; and ® is a benzhydrylamine, polyethyleneglycol-polystyrene or aminomethylpolystyrene resin. 23. A library according to claim 22 wherein I Q I is a peptide of the formula AA1-AA2-Hyp-AA4-AA5, wherein AAl, AA2, AA4 and AA5 are amino acid residues selected from the group of the D- and L- forms of the natural amino acids. 24. A compound of the formula: wherein Pg is Boc or Fmoc.

Inventors: MOORE MICHAEL LEE, NEWLANDER KENNETH ALLEN, MOORE, MICHAEL, LEE, NEWLANDER, KENNETH, ALLEN
Applicants: MOORE MICHAEL LEE, NEWLANDER KENNETH ALLEN, SMITHKLINE BEECHAM CORP, MOORE, MICHAEL, LEE, NEWLANDER, KENNETH, ALLEN, SMITHKLINE BEECHAM CORPORATION
Application year: 1995
Main classification: C07K7
IPC codes: C07D207, C07K1, C07K7, C07K17