Peptidomimetic Macrocycles

Abstract

The present invention provides novel peptidomimetic macrocycles and methods of using such macrocycles for the treatment of disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 13/816,880, filed Feb. 13, 2013 (or Apr. 25, 2013, which is the 371 date), which is a national stage of PCT/US2011/047692, filed Aug. 13, 2011, which claims the priority benefit of U.S. Provisional Application Nos. 61/373,701, filed Aug. 13, 2010, 61/373,638, filed Aug. 13, 2010, and 61/374,163, filed Aug. 16, 2010, each of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 6, 2013, is named 35224-757.401_SL.txt and is 586,346 bytes in size.

BACKGROUND OF THE INVENTION

[0003] The human transcription factor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 negatively regulates p53 function through a direct binding interaction that neutralizes the p53 transactivation activity, leads to export from the nucleus of p53 protein, and targets p53 for degradation via the ubiquitylation-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or HDM2 overexpression, is the most common defect in human cancers. Tumors that express wild type p53 are vulnerable to pharmacologic agents that stabilize or increase the concentration of active p53. In this context, inhibition of the activities of HDM2 has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. HDMX (HDM4) has more recently been identified as a similar negative regulator of p53, and studies have revealed significant structural homology between the p53 binding interfaces of HDM2 and HDMX.

[0004] The p53-HDM2 and p53-HDMX protein-protein interactions are mediated by the same 15-residue alpha-helical transactivation domain of p53, which inserts into hydrophobic clefts on the surface of HDM2 and HDMX. Three residues within this domain of p53 (F19, W23, and L26) are essential for binding to HDM2 and HDMX. The present invention provides p53-based peptidomimetic macrocycles that modulate the activities of p53 by inhibiting the interactions between p53 and HDM2, p53 and HDMX, or p53 and both HDM2 and HDMX proteins, and that may be used for treating diseases including but not limited to cancer and other hyperproliferative diseases.

SUMMARY OF THE INVENTION

[0005] Described below are stably cross-linked peptides related to a portion of human p53 ("p53 peptidomimetic macrocycles"). These cross-linked peptides contain at least two modified amino acids that together form an intramolecular cross-link that can help to stabilize the alpha-helical secondary structure of a portion of p53 that is thought to be important for binding of p53 to HDM2 and for binding of p53 to HDMX. Accordingly, a cross-linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked. The p53 peptidomimetic macrocycles are thought to interfere with binding of p53 to HDM2 and/or of p53 to HDMX, thereby liberating functional p53 and inhibiting its destruction. The p53 peptidomimetic macrocycles described herein can be used therapeutically, for example to treat cancers and other disorders characterized by an undesirably low level or a low activity of p53, and/or to treat cancers and other disorders characterized by an undesirably high level of activity of HDM2 or HDMX. The p53 peptidomimetic macrocycles may also be useful for treatment of any disorder associated with disrupted regulation of the p53 transcriptional pathway, leading to conditions of excess cell survival and proliferation such as cancer and autoimmunity, in addition to conditions of inappropriate cell cycle arrest and apoptosis such as neurodegeneration and immunedeficiencies. In some instances, the p53 peptidomimetic macrocycles bind to HDM2 (e.g., GenBank.RTM. Accession No.: 228952; GI:228952) and/or HDMX (also referred to as HDM4; GenBank.RTM. Accession No.: 88702791; GI:88702791).

[0006] In one aspect, the present invention provides a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60%, 80%, 90%, or 95% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1, 2, 3, or 4. Alternatively, an amino acid sequence of said peptidomimetic macrocycle is chosen from the group consisting of the amino acid sequences in Table 1. Alternatively, an amino acid sequence of said peptidomimetic macrocycle is chosen as above, and further wherein the macrocycle does not include a thioether or a triazole. In some embodiments, the peptidomimetic macrocycle comprises a helix, such as an .alpha.-helix. In other embodiments, the peptidomimetic macrocycle comprises an .alpha.,.alpha.-disubstituted amino acid. A peptidomimetic macrocycle of the invention may comprise a crosslinker linking the .alpha.-positions of at least two amino acids. At least one of said two amino acids may be an .alpha.,.alpha.-disubstituted amino acid.

[0007] In some embodiments, the peptidomimetic macrocycle has the formula:

##STR00001##

[0008] wherein:

[0009] each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;

[0010] B is a natural or non-natural amino acid, amino acid analog,

##STR00002##

[--NH-L.sub.3-CO-], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];

[0011] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

[0012] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;

[0013] L is a macrocycle-forming linker of the formula -L.sub.1-L.sub.2-;

[0014] L.sub.1 and L.sub.2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted with R.sub.5;

[0015] each R.sub.4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

[0016] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;

[0017] each R.sub.5 is independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0018] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0019] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5, or part of a cyclic structure with a D residue;

[0020] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5, or part of a cyclic structure with an E residue;

[0021] v and w are independently integers from 1-1000;

[0022] u is an integer from 1-10;

[0023] x, y and z are independently integers from 0-10; and

[0024] n is an integer from 1-5.

[0025] In various embodiments, the peptidomimetic macrocycle includes L.sub.1 and L.sub.2 wherein L.sub.1 and L.sub.2 either alone or in combination do not include a thioether or a triazole.

[0026] In other embodiments, the peptidomimetic macrocycle may comprise a crosslinker linking a backbone amino group of a first amino acid to a second amino acid within the peptidomimetic macrocycle. For example, the invention provides peptidomimetic macrocycles of the formula (IV) or (IVa):

##STR00003##

[0027] wherein:

[0028] each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;

[0029] B is a natural or non-natural amino acid, amino acid analog,

##STR00004##

[--NH-L.sub.3-CO-], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];

[0030] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;

[0031] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;

[0032] L.sub.1 and L.sub.2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted with R.sub.5;

[0033] each R.sub.4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

[0034] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;

[0035] each R.sub.5 is independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0036] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0037] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;

[0038] V and w are independently integers from 1-1000;

[0039] u is an integer from 1-10;

[0040] x, y and z are independently integers from 0-10; and

[0041] n is an integer from 1-5.

[0042] Additionally, the invention provides a method of treating cancer in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention. Also provided is a method of modulating the activity of p53 or HDM2 or HDMX in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention, or a method of antagonizing the interaction between p53 and HDM2 and/or HDMX proteins in a subject comprising administering to the subject such a peptidomimetic macrocycle.

INCORPORATION BY REFERENCE

[0043] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0045] FIG. 1 describes the synthesis of Fmoc-Me-6-Chloro-Tryptophan & Fmoc-6-Chloro-Tryptophan.

[0046] FIG. 2 shows an LC-MS trace of Me-6-Chloro-(Boc)Tryptophan-Ni--S-BPB.

[0047] FIG. 3 shows a 1H-NMR spectrum of Me-6-Chloro-(Boc)Tryptophan-Ni--S-BPB.

[0048] FIG. 4 shows an LC-MS trace of Fmoc-Me-6-Chloro-(Boc)Tryptophan.

[0049] FIG. 5 shows a 1H-NMR spectrum of Fmoc-Me-6-Chloro-(Boc)Tryptophan.

[0050] FIGS. 6a-f describe the results of a cell viability assay, a competition ELISA assay, GRIP assay, Kd data, p21 activation assay, fluorescence polarization competition binding and circular helicity data for exemplary peptidomimetic macrocycles of the invention (SEQ ID NOS 38-178, respectively, in order of appearance).

[0051] FIGS. 7A-D provide data from a variety of macrocycles (FIGS. 7A-7B disclose SEQ ID NOS 42, 163, 177, 214, 217, 344, 289-290, 383, 533, 529, 543, 601, 544, 594, 279, 374 and 660, respectively in order of appearance, and FIGS. 7C-7D disclose SEQ ID NOS 702, 699, 704, 706, 689, 507, 624, 703, 716, 606, 605, 642, 691, 731, 375, 727, 662, 587 and 714, respectively in order of appearance).

[0052] FIGS. 8A-B provide data from a variety of macrocycles.

DETAILED DESCRIPTION OF THE INVENTION

[0053] As used herein, the term "macrocycle" refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.

[0054] As used herein, the term "peptidomimetic macrocycle" or "crosslinked polypeptide" refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule. Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the .alpha. carbon of the first amino acid residue (or analog) to the .alpha. carbon of the second amino acid residue (or analog). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle. A "corresponding uncrosslinked polypeptide" when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.

[0055] As used herein, the term "stability" refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo. Non-limiting examples of secondary structures contemplated in this invention are .alpha.-helices, .beta.-turns, and .beta.-pleated sheets.

[0056] As used herein, the term "helical stability" refers to the maintenance of a helical structure by a peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR. For example, in some embodiments, the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in .alpha.-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.

[0057] The term ".alpha.-amino acid" or simply "amino acid" refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the .alpha.-carbon. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.

[0058] The term "naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

[0059] The term "amino acid analog" or "non-natural amino acid" refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., .beta.-amino .beta.-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester). Non-natural amino acids include structures according to the following:

##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##

[0060] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation). An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.

[0061] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).

[0062] The term "capping group" refers to the chemical moiety occurring at either the carboxy or amino terminus of the polypeptide chain of the subject peptidomimetic macrocycle. The capping group of a carboxy terminus includes an unmodified carboxylic acid (ie --COOH) or a carboxylic acid with a substituent. For example, the carboxy terminus may be substituted with an amino group to yield a carboxamide at the C-terminus. Various substituents include but are not limited to primary and secondary amines, including pegylated secondary amines. Representative secondary amine capping groups for the C-terminus include:

##STR00011## ##STR00012##

[0063] The capping group of an amino terminus includes an unmodified amine (ie --NH.sub.2) or an amine with a substituent. For example, the amino terminus may be substituted with an acyl group to yield a carboxamide at the N-terminus. Various substituents include but are not limited to substituted acyl groups, including C.sub.1-C.sub.6 carbonyls, C.sub.7-C.sub.30 carbonyls, and pegylated carbamates. Representative capping groups for the N-terminus include:

##STR00013##

[0064] The term "member" as used herein in conjunction with macrocycles or macrocycle-forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.

[0065] The symbol "" when used as part of a molecular structure refers to a single bond or a trans or cis double bond.

[0066] The term "amino acid side chain" refers to a moiety attached to the .alpha.-carbon in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an .alpha.,.alpha. di-substituted amino acid).

[0067] The term ".alpha.,.sup.-.alpha. di-substituted amino" acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the .alpha.-carbon) that is attached to two natural or non-natural amino acid side chains.

[0068] The term "polypeptide" encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).

[0069] The term "macrocyclization reagent" or "macrocycle-forming reagent" as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the invention by mediating the reaction between two reactive groups. Reactive groups may be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II) salts such as Cu(CO.sub.2CH.sub.3).sub.2, CuSO.sub.4, and CuCl.sub.2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh.sub.3).sub.2, [Cp*RuCl].sub.4 or other Ru reagents which may provide a reactive Ru(II) species. In other cases, the reactive groups are terminal olefins. In such embodiments, the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated. Additional catalysts are disclosed in Grubbs et al., "Ring Closing Metathesis and Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452, and U.S. Pat. No. 5,811,515. In yet other cases, the reactive groups are thiol groups. In such embodiments, the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups such as halogen groups.

[0070] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine or a radical thereof.

[0071] The term "alkyl" refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C.sub.1-C.sub.10 indicates that the group has from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, "alkyl" is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it.

[0072] The term "alkylene" refers to a divalent alkyl (i.e., --R--).

[0073] The term "alkenyl" refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. For example, C.sub.2-C.sub.10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term "lower alkenyl" refers to a C.sub.2-C.sub.6 alkenyl chain. In the absence of any numerical designation, "alkenyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

[0074] The term "alkynyl" refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl moiety contains the indicated number of carbon atoms. For example, C.sub.2-C.sub.10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term "lower alkynyl" refers to a C.sub.2-C.sub.6 alkynyl chain. In the absence of any numerical designation, "alkynyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

[0075] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with aryl.

[0076] "Arylalkyl" refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C.sub.1-C.sub.5 alkyl group, as defined above. Representative examples of an arylalkyl group include, but are not limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.

[0077] "Arylamido" refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more --C(O)NH.sub.2 groups. Representative examples of an arylamido group include 2-C(O)NH2-phenyl, 3-C(O)NH.sub.2-phenyl, 4-C(O)NH.sub.2-phenyl, 2-C(O)NH.sub.2-pyridyl, 3-C(O)NH.sub.2-pyridyl, and 4-C(O)NH.sub.2-pyridyl,

[0078] "Alkylheterocycle" refers to a C.sub.1-C.sub.5 alkyl group, as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's hydrogen atoms has been replaced with a heterocycle. Representative examples of an alkylheterocycle group include, but are not limited to, --CH.sub.2CH.sub.2-morpholine, --CH.sub.2CH.sub.2-piperidine, --CH.sub.2CH.sub.2CH.sub.2-morpholine, and --CH.sub.2CH.sub.2CH.sub.2-imidazole.

[0079] "Alkylamido" refers to a C.sub.1-C.sub.5 alkyl group, as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's hydrogen atoms has been replaced with a --C(O)NH.sub.2 group. Representative examples of an alkylamido group include, but are not limited to, --CH.sub.2--C(O)NH.sub.2, --CH.sub.2CH.sub.2--C(O)NH.sub.2, --CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2, --CH.sub.2CH(C(O)NH.sub.2)CH.sub.3, --CH.sub.2CH(C(O)NH.sub.2)CH.sub.2CH.sub.3, --CH(C(O)NH.sub.2)CH.sub.2CH.sub.3, --C(CH.sub.3).sub.2CH.sub.2C(O)NH.sub.2, --CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3, --CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3--CH3, and --CH.sub.2--CH.sub.2--NH--C(O)--CH.dbd.CH.sub.2.

[0080] "Alkanol" refers to a C.sub.1-C.sub.5 alkyl group, as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's hydrogen atoms has been replaced with a hydroxyl group. Representative examples of an alkanol group include, but are not limited to, --CH.sub.2OH, --CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH(OH)CH.sub.3, --CH.sub.2CH(OH)CH.sub.2CH.sub.3, --CH(OH)CH.sub.3 and --C(CH.sub.3).sub.2CH.sub.2OH.

[0081] "Alkylcarboxy" refers to a C.sub.1-C.sub.5 alkyl group, as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's hydrogen atoms has been replaced with a--COOH group. Representative examples of an alkylcarboxy group include, but are not limited to, --CH.sub.2COOH, --CH.sub.2CH.sub.2COOH, --CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH(COOH)CH.sub.3, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH(COOH)CH.sub.2CH.sub.3, --CH(COOH)CH.sub.2CH.sub.3 and --C(CH.sub.3).sub.2CH.sub.2COOH.

[0082] The term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted. Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0083] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.

[0084] The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.

[0085] The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.

[0086] The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring are substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

[0087] The term "substituent" refers to a group replacing a second atom or group such as a hydrogen atom on any molecule, compound or moiety. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.

[0088] In some embodiments, the compounds of this invention contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included in the present invention unless expressly provided otherwise. In some embodiments, the compounds of this invention are also represented in multiple tautomeric forms, in such instances, the invention includes all tautomeric forms of the compounds described herein (e.g., if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included in the present invention unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present invention unless expressly provided otherwise.

[0089] As used herein, the terms "increase" and "decrease" mean, respectively, to cause a statistically significantly (i.e., p<0.1) increase or decrease of at least 5%.

[0090] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable is equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable is equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 takes the values 0, 1 or 2 if the variable is inherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or any other real values .gtoreq.0 and .ltoreq.2 if the variable is inherently continuous.

[0091] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or."

[0092] The term "on average" represents the mean value derived from performing at least three independent replicates for each data point.

[0093] The term "biological activity" encompasses structural and functional properties of a macrocycle of the invention. Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.

[0094] The details of one or more particular embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0095] In some embodiments, the peptide sequences are derived from the p53 protein.

[0096] A non-limiting exemplary list of suitable p53 peptides for use in the present invention is given below.

TABLE-US-00001 TABLE 1 (SEQ ID NOS 1-18, respectively, in order of appearance) Sequence (bold = critical residue; X = cross-linked amino acid) Design Notes Ac- Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp Arg Leu Leu Pro Gln Asn --NH2 linear Ac- X Gln Ser Gln X Thr Phe Ser Asn Leu Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 4 x-link #1 Ac- X Ser Gln Gln X Phe Ser Asn Leu Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 4 x-link #2 Ac- Gln Ser X Gln Thr Phe X Asn Leu Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 4 x-link #3 Ac- Gln Ser Gln X Thr Phe Ser X Leu Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 4 x-link #4 Ac- Gln Ser Gln Gln X Phe Ser Asn X Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 4 x-link #5 Ac- Gln Ser Gln Gln Thr Phe X Asn Leu Trp X Leu Leu Pro Gln Asn --NH2 i--> i + 4 x-link #6 Ac- Gln Ser Gln Gln Thr Phe Ser X Leu Trp Arg X Leu Pro Gln Asn --NH2 i--> i + 4 x-link #7 Ac- Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp X Leu Leu Pro X Asn --NH2 i--> i + 4 x-link #8 Ac- Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp Arg X Leu Pro Gln X --NH2 i--> i + 4 x-link #9 Ac- X Gln Ser Gln Gln Thr Phe X Asn Leu Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 7 x-link #1 Ac- X Ser Gln Gln Thr Phe Ser X Leu Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 7 x-link #2 Ac- Gln X Gln Gln Thr Phe Ser Asn X Trp Arg Leu Leu Pro Gln Asn --NH2 i--> i + 7 x-link #3 Ac- Gln Ser Gln X Thr Phe Ser Asn Leu Trp X Leu Leu Pro Gln Asn --NH2 i--> i + 7 x-link #4 Ac- Gln Ser Gln Gln X Phe Ser Asn Leu Trp Arg X Leu Pro Gln Asn --NH2 i--> i + 7 x-link #5 Ac- Gln Ser Gln Gln Thr Phe X Asn Leu Trp Arg Leu Leu X Gln Asn --NH2 i--> i + 7 x-link #6 Ac- Gln Ser Gln Gln Thr Phe Ser X Leu Trp Arg Leu Leu Pro X Asn --NH2 i--> i + 7 x-link #7 Ac- Gln Ser Gln Gln Thr Phe Ser Asn X Trp Arg Leu Leu Pro Gln X --NH2 i--> i + 7 x-link #8

TABLE-US-00002 TABLE 2 (SEQ ID NOS 19-31, respectively, in order of appearance) Design Sequence (bold = critical residue; X = cross-linked amino acid) Notes Ac- Leu Thr Phe Glu His Tyr Trp Ala Gln Leu Thr Ser --NH2 linear Ac- X Leu Thr Phe X His Tyr Trp Ala Gln Leu Thr Ser --NH2 i--> i + 4 x- link #1 Ac- X Thr Phe Glu X Tyr Trp Ala Gln Leu Thr Ser --NH2 i--> i + 4 x- link #2 Ac- Leu X Phe Glu His X Trp Ala Gln Leu Thr Ser --NH2 i--> i + 4 x- link #3 Ac- Leu Thr Phe X His Tyr Trp X Gln Leu Thr Ser --NH2 i--> i + 4 x- link #4 Ac- Leu Thr Phe Glu X Tyr Trp Ala X Leu Thr Ser --NH2 i--> i + 4 x- link #5 Ac- Leu Thr Phe Glu His Tyr Trp X Gln Leu Thr X --NH2 i--> i + 4 x- link #6 Ac- Leu Thr Phe Glu His Tyr Trp Ala X Leu Thr Ser X --NH2 i--> i + 4 x- link #7 Ac- X Thr Phe Glu His Tyr Trp X Gln Leu Thr Ser --NH2 i--> i + 7 X- link #1 Ac- Gln X Phe Glu His Tyr Trp Ala X Leu Thr Ser --NH2 i--> i + 7 X- link #2 Ac- Gln Thr Phe X His Tyr Trp Ala Gln Leu X Ser --NH2 i--> i + 7 X- link #3 Ac- Gln Thr Phe Glu X Tyr Trp Ala Gln Leu Thr X --NH2 i--> i + 7 X- link #4 Ac- Gln Thr Phe Glu His X Trp Ala Gln Leu Thr Ser X --NH2 i--> i + 7 X- link #5

TABLE-US-00003 TABLE 3 (SEQ ID NOS 32-37, respectively, in order of appearance) Design Sequence (bold = critical residue; X = cross-linked amino acid) Notes Ac- Phe Met Aib/His/ Tyr 6-ClTrp Glu Ac3c/Gln/Leu Leu --NH2 linear Asn Ac- X Phe Met Aib/His/ X 6-ClTrp Glu Ac3c/Gln/Leu Leu --NH2 i--> i + 4 x- Asn link #1 Ac- Phe X Aib/His/ Tyr 6-ClTrp X Ac3c/Gln/Leu Leu --NH2 i--> i + 4 x- Asn link #2 Ac- Phe Met X Tyr 6-ClTrp Glu X Leu --NH2 i-> i + 4 x- link #3 Ac- X Phe Met Aib/His/ Tyr 6-ClTrp Glu X Leu --NH2 i--> i + 7 x- Asn link #1 Ac- Phe X Aib/His/ Tyr 6-ClTrp Glu Ac3c/Gln/Leu Leu X --NH2 i--> i + 7 x- Asn link #2

[0097] In Table 3 and elsewhere, "Aib" represents a 2-aminoisobutyric acid residue, while "Ac3c" represents a aminocyclopropane carboxylic acid residue.

[0098] Peptidomimetic Macrocycles

[0099] In some embodiments, a peptidomimetic macrocycle of the invention has the Formula (I):

##STR00014##

[0100] wherein:

[0101] each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;

[0102] B is a natural or non-natural amino acid, amino acid analog,

##STR00015##

[--NH-L.sub.3-CO-], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];

[0103] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

[0104] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;

[0105] L is a macrocycle-forming linker of the formula -L.sub.1-L.sub.2-;

[0106] L.sub.1 and L.sub.2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted with R.sub.5;

[0107] each R.sub.4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

[0108] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;

[0109] each R.sub.5 is independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0110] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0111] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5, or part of a cyclic structure with a D residue;

[0112] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5, or part of a cyclic structure with an E residue;

[0113] v and w are independently integers from 1-1000;

[0114] u is an integer from 1-10;

[0115] x, y and z are independently integers from 0-10; and

[0116] n is an integer from 1-5.

[0117] In one embodiment, L.sub.1 and L.sub.2, either alone or in combination, do not form a triazole or a thioether.

[0118] In one example, at least one of R.sub.1 and R.sub.2 is alkyl, unsubstituted or substituted with halo-. In another example, both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and R.sub.2 are methyl.

[0119] In some embodiments of the invention, x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected. For example, a sequence represented by the formula [A].sub.x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound of the invention may encompass peptidomimetic macrocycles which are the same or different. For example, a compound of the invention may comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.

[0120] In some embodiments, the peptidomimetic macrocycle of the invention comprises a secondary structure which is an .alpha.-helix and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an .alpha.,.alpha.-disubstituted amino acid. In one example, B is an .alpha.,.alpha.-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

##STR00016##

[0121] In other embodiments, the length of the macrocycle-forming linker L as measured from a first C.alpha. to a second C.alpha. is selected to stabilize a desired secondary peptide structure, such as an .alpha.-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first C.alpha. to a second C.alpha..

[0122] In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

##STR00017##

[0123] wherein each R.sub.1 and R.sub.2 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

[0124] In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

##STR00018##

[0125] In other embodiments, the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:

##STR00019## ##STR00020## ##STR00021## ##STR00022##

wherein "AA" represents any natural or non-natural amino acid side chain and "" is [D].sub.v, [E].sub.w as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.

[0126] Exemplary embodiments of the macrocycle-forming linker L are shown below.

##STR00023##

[0127] In other embodiments, D and/or E in the compound of Formula I are further modified in order to facilitate cellular uptake. In some embodiments, lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.

[0128] In other embodiments, at least one of [D] and [E] in the compound of Formula I represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers. In a specific embodiment, a peptidomimetic macrocycle comprises two macrocycle-forming linkers. In an embodiment, u is 2.

[0129] In the peptidomimetic macrocycles of the invention, any of the macrocycle-forming linkers described herein may be used in any combination with any of the sequences shown in Tables 1-4 and also with any of the R-substituents indicated herein.

[0130] In some embodiments, the peptidomimetic macrocycle comprises at least one .alpha.-helix motif. For example, A, B and/or C in the compound of Formula I include one or more .alpha.-helices. As a general matter, .alpha.-helices include between 3 and 4 amino acid residues per turn. In some embodiments, the .alpha.-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues. In specific embodiments, the .alpha.-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In some embodiments, the macrocycle-forming linker stabilizes an .alpha.-helix motif included within the peptidomimetic macrocycle. Thus, in some embodiments, the length of the macrocycle-forming linker L from a first C.alpha. to a second C.alpha. is selected to increase the stability of an .alpha.-helix. In some embodiments, the macrocycle-forming linker spans from 1 turn to 5 turns of the .alpha.-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the .alpha.-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 .ANG. to 9 .ANG. per turn of the .alpha.-helix, or approximately 6 .ANG. to 8 .ANG. per turn of the .alpha.-helix. Where the macrocycle-forming linker spans approximately 1 turn of an .alpha.-helix, the length is equal to approximately 5 carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 2 turns of an .alpha.-helix, the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 3 turns of an .alpha.-helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 4 turns of an .alpha.-helix, the length is equal to approximately 20 carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 5 turns of an .alpha.-helix, the length is equal to approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 1 turn of an .alpha.-helix, the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming linker spans approximately 2 turns of the .alpha.-helix, the linkage contains approximately 7 atoms to 15 atoms, approximately 9 atoms to 13 atoms, or approximately 11 atoms. Where the macrocycle-forming linker spans approximately 3 turns of the .alpha.-helix, the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms. Where the macrocycle-forming linker spans approximately 4 turns of the .alpha.-helix, the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms. Where the macrocycle-forming linker spans approximately 5 turns of the .alpha.-helix, the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms. Where the macrocycle-forming linker spans approximately 1 turn of the .alpha.-helix, the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members. Where the macrocycle-forming linker spans approximately 2 turns of the .alpha.-helix, the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members. Where the macrocycle-forming linker spans approximately 3 turns of the .alpha.-helix, the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members. Where the macrocycle-forming linker spans approximately 4 turns of the .alpha.-helix, the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members. Where the macrocycle-forming linker spans approximately 5 turns of the .alpha.-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.

[0131] In other embodiments, the invention provides peptidomimetic macrocycles of Formula (IV) or (IVa):

##STR00024##

[0132] wherein:

[0133] each A, C, D, and E is independently a natural or non-natural amino acid, and the terminal D and E independently optionally include a capping group;

[0134] B is a natural or non-natural amino acid, amino acid analog,

##STR00025##

[--NH-L.sub.3-CO-], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];

[0135] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;

[0136] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;

[0137] L is a macrocycle-forming linker of the formula -L.sub.1-L.sub.2-;

[0138] L.sub.1 and L.sub.2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted with R.sub.5;

[0139] each R.sub.4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

[0140] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;

[0141] each R.sub.5 is independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0142] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

[0143] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;

[0144] v and w are independently integers from 1-1000;

[0145] u is an integer from 1-10;

[0146] x, y and z are independently integers from 0-10; and

[0147] n is an integer from 1-5.

[0148] In one example, L.sub.1 and L.sub.2, either alone or in combination, do not form a triazole or a thioether.

[0149] In one example, at least one of R.sub.1 and R.sub.2 is alkyl, unsubstituted or substituted with halo-. In another example, both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and R.sub.2 are methyl.

[0150] In some embodiments of the invention, x+y+z is at least 1. In other embodiments of the invention, x+y+z is at least 2. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected. For example, a sequence represented by the formula [A].sub.x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.

[0151] In some embodiments, the peptidomimetic macrocycle of the invention comprises a secondary structure which is an .alpha.-helix and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an .alpha.,.alpha.-disubstituted amino acid. In one example, B is an .alpha.,.alpha.-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

##STR00026##

[0152] In other embodiments, the length of the macrocycle-forming linker L as measured from a first C.alpha. to a second C.alpha. is selected to stabilize a desired secondary peptide structure, such as an .alpha.-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first C.alpha. to a second C.alpha..

[0153] Exemplary embodiments of the macrocycle-forming linker -L.sub.1-L.sub.2- are shown below.

##STR00027##

Preparation of Peptidomimetic Macrocycles

[0154] Peptidomimetic macrocycles of the invention may be prepared by any of a variety of methods known in the art. For example, any of the residues indicated by "X" in Tables 1, 2, 3, or 4 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.

[0155] Various methods to effect formation of peptidomimetic macrocycles are known in the art. For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. The .alpha.,.alpha.-disubstituted amino acids and amino acid precursors disclosed in the cited references may be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides. For example, the "S5-olefin amino acid" is (S)-.alpha.-(2'-pentenyl) alanine and the "R8 olefin amino acid" is (R)-.alpha.-(2'-octenyl) alanine. Following incorporation of such amino acids into precursor polypeptides, the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle. In various embodiments, the following amino acids may be employed in the synthesis of the peptidomimetic macrocycle:

##STR00028##

[0156] In other embodiments, the peptidomimetic macrocycles of the invention are of Formula IV or IVa. Methods for the preparation of such macrocycles are described, for example, in U.S. Pat. No. 7,202,332.

[0157] Additional methods of forming peptidomimetic macrocycles which are envisioned as suitable to perform the present invention include those disclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat. No. 5,446,128; U.S. Pat. No. 5,824,483; U.S. Pat. No. 6,713,280; and U.S. Pat. No. 7,202,332. In such embodiments, aminoacid precursors are used containing an additional substituent R- at the alpha position. Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then effected according to the indicated method.

Assays

[0158] The properties of the peptidomimetic macrocycles of the invention are assayed, for example, by using the methods described below. In some embodiments, a peptidomimetic macrocycle of the invention has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.

Assay to Determine .alpha.-Helicity.

[0159] In solution, the secondary structure of polypeptides with .alpha.-helical domains will reach a dynamic equilibrium between random coil structures and .alpha.-helical structures, often expressed as a "percent helicity". Thus, for example, alpha-helical domains are predominantly random coils in solution, with .alpha.-helical content usually under 25%. Peptidomimetic macrocycles with optimized linkers, on the other hand, possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide. In some embodiments, macrocycles of the invention will possess an alpha-helicity of greater than 50%. To assay the helicity of peptidomimetic macrocycles of the invention, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H.sub.2O, to concentrations of 25-50 .mu.M). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710) using standard measurement parameters (e.g. temperature, 20.degree. C.; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The .alpha.-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [.PHI.]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

[0160] A peptidomimetic macrocycle of the invention comprising a secondary structure such as an .alpha.-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Typically peptidomimetic macrocycles of the invention exhibit Tm of >60.degree. C. representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on melting temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H.sub.2O (e.g. at a final concentration of 50 .mu.M) and the Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95.degree. C.) on a spectropolarimeter (e.g., Jasco J-710) using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1.degree. C./min; path length, 0.1 cm).

Protease Resistance Assay.

[0161] The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries the amide backbone and therefore may shield it from proteolytic cleavage. The peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E .about.125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln[S] versus time (k=-1.times.slope).

Ex Vivo Stability Assay.

[0162] Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more. For ex vivo serum stability studies, a variety of assays may be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 mcg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37.degree. C. for 0, 1, 2, 4, 8, and 24 hours. To determine the level of intact compound, the following procedure may be used: The samples are extracted by transferring 100 .mu.l of sera to 2 ml centrifuge tubes followed by the addition of 10 .mu.L of 50% formic acid and 500 .mu.L acetonitrile and centrifugation at 14,000 RPM for 10 min at 4.+-.2.degree. C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N.sub.2<10 psi, 37.degree. C. The samples are reconstituted in 100 .mu.L of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.

In Vitro Binding Assays.

[0163] To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution).

[0164] For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25-1000 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide.

In Vitro Displacement Assays to Characterize Antagonists of Peptide-Protein Interactions.

[0165] To assess the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.

[0166] For example, putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.).

[0167] Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.

Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry

[0168] To assess the binding and affinity of test compounds for proteins, an affinity-selection mass spectrometry assay is used, for example. Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system-wide control experiment using 1 .mu.M peptidomimetic macrocycle plus 5 .mu.M hMDM2. A 1 .mu.L DMSO aliquot of a 40 .mu.M stock solution of peptidomimetic macrocycle is dissolved in 19 .mu.L of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl). The resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To a 4 .mu.L aliquot of the resulting supernatant is added 4 .mu.L of 10 .mu.M hMDM2 in PBS. Each 8.0 .mu.L experimental sample thus contains 40 pmol (1.5 .mu.g) of protein at 5.0 .mu.M concentration in PBS plus 1 .mu.M peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4.degree. C. prior to size-exclusion chromatography-LC-MS analysis of 5.0 .mu.L injections. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step. The SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. The (M+3H).sup.3+ ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.

Assay for Protein-Ligand Kd Titration Experiments.

[0169] To assess the binding and affinity of test compounds for proteins, a protein-ligand Kd titration experiment is performed, for example. Protein-ligand K.sub.d titrations experiments are conducted as follows: 2 .mu.L DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared then dissolved in 38 .mu.L of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of the resulting supernatants is added 4.0 .mu.L of 10 .mu.M hMDM2 in PBS. Each 8.0 .mu.L experimental sample thus contains 40 pmol (1.5 .mu.g) of protein at 5.0 .mu.M concentration in PBS, varying concentrations (125, 62.5, . . . , 0.24 .mu.M) of the titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4.degree. C. prior to SEC-LC-MS analysis of 2.0 .mu.L injections. The (M+H).sup.1+, (M+2H).sup.2+, (M+3H).sup.3+, and/or (M+Na).sup.1+ ion is observed by ESI-MS; extracted ion chromatograms are quantified, then fit to equations to derive the binding affinity K.sub.d as described in "A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions" D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-Mass Spectrometry

[0170] To determine the ability of test compounds to bind competitively to proteins, an affinity selection mass spectrometry assay is performed, for example. A mixture of ligands at 40 .mu.M per component is prepared by combining 2 .mu.L aliquots of 400 .mu.M stocks of each of the three compounds with 14 .mu.L of DMSO. Then, 1 .mu.L aliquots of this 40 .mu.M per component mixture are combined with 1 .mu.L at DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078 mM). These 2 .mu.L samples are dissolved in 38 .mu.L of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of the resulting supernatants is added 4.0 .mu.L of 10 .mu.M hMDM2 protein in PBS. Each 8.0 .mu.L experimental sample thus contains 40 pmol (1.5 .mu.g) of protein at 5.0 .mu.M concentration in PBS plus 0.5 .mu.M ligand, 2.5% DMSO, and varying concentrations (125, 62.5, . . . , 0.98 .mu.M) of the titrant peptidomimetic macrocycle. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4.degree. C. prior to SEC-LC-MS analysis of 2.0 .mu.L injections. Additional details on these and other methods are provided in "A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions" D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.

Binding Assays in Intact Cells.

[0171] It is possible to measure binding of peptides or peptidomimetic macrocycles to their natural acceptors in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluoresceinated (FITC-labeled) compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs. Cells are then pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4.degree. C. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 .mu.l goat anti-FITC antibody for 2 hrs, rotating at 4.degree. C. followed by further 2 hrs incubation at 4.degree. C. with protein A/G Sepharose (50 .mu.l of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays.

[0172] A peptidomimetic macrocycle is, for example, more cell penetrable compared to a corresponding uncrosslinked macrocycle. Peptidomimetic macrocycles with optimized linkers possess, for example, cell penetrability that is at least two-fold greater than a corresponding uncrosslinked macrocycle, and often 20% or more of the applied peptidomimetic macrocycle will be observed to have penetrated the cell after 4 hours. To measure the cell penetrability of peptidomimetic macrocycles and corresponding uncrosslinked macrocycle, intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 .mu.M) for 4 hrs in serum free media at 37.degree. C., washed twice with media and incubated with trypsin (0.25%) for 10 min at 37.degree. C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan.RTM. HCS Reader.

Cellular Efficacy Assays.

[0173] The efficacy of certain peptidomimetic macrocycles is determined, for example, in cell-based killing assays using a variety of tumorigenic and non-tumorigenic cell lines and primary cells derived from human or mouse cell populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (0.5 to 50 .mu.M) to identify those that kill at EC50<10 .mu.M. Several standard assays that measure cell viability are commercially available and are optionally used to assess the efficacy of the peptidomimetic macrocycles. In addition, assays that measure Annexin V and caspase activation are optionally used to assess whether the peptidomimetic macrocycles kill cells by activating the apoptotic machinery. For example, the Cell Titer-glo assay is used which determines cell viability as a function of intracellular ATP concentration.

In Vivo Stability Assay.

[0174] To investigate the in vivo stability of the peptidomimetic macrocycles, the compounds are, for example, administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0', 5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 .mu.L of fresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

[0175] To determine the anti-oncogenic activity of peptidomimetic macrocycles of the invention in vivo, the compounds are, for example, given alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In one example, 5.times.10.sup.6 RS4; 11 cells (established from the bone marrow of a patient with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID mice 3 hrs after they have been subjected to total body irradiation. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, Mass.). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocycles alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment. Expired mice are optionally subjected to necropsy at the end of the experiment. Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from human follicular lymphoma, that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical Trials.

[0176] To determine the suitability of the peptidomimetic macrocycles of the invention for treatment of humans, clinical trials are performed. For example, patients diagnosed with cancer and in need of treatment are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle of the invention, while the control groups receive a placebo or a known anti-cancer drug. The treatment safety and efficacy of the peptidomimetic macrocycles of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life. In this example, the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

[0177] The peptidomimetic macrocycles of the invention also include pharmaceutically acceptable derivatives or prodrugs thereof A "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored pharmaceutically acceptable derivatives are those that increase the bioavailability of the compounds of the invention when administered to a mammal (e.g., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.

[0178] In some embodiments, the peptidomimetic macrocycles of the invention are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties. Such modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.

[0179] Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl).sub.4.sup.+ salts.

[0180] For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

[0181] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0182] Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[0183] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0184] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0185] When the compositions of this invention comprise a combination of a peptidomimetic macrocycle and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. In some embodiments, the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition.

Methods of Use

[0186] In one aspect, the present invention provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled. For example, in the p53/HDMX system, labeled peptidomimetic macrocycles based on p53 can be used in a HDMX binding assay along with small molecules that competitively bind to HDMX. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the p53/HDMX system. Such binding studies may be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners.

[0187] The invention further provides for the generation of antibodies against the peptidomimetic macrocycles. In some embodiments, these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as p53, to which the peptidomimetic macrocycles are related. Such antibodies, for example, disrupt the native protein-protein interaction, for example, binding between p53 and HDMX.

[0188] In other aspects, the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant (e.g., insufficient or excessive) expression or activity of the molecules including p53, HDM2 or HDMX.

[0189] In another embodiment, a disorder is caused, at least in part, by an abnormal level of p53 or HDM2 or HDMX, (e.g., over or under expression), or by the presence of p53 or HDM2 or HDMX exhibiting abnormal activity. As such, the reduction in the level and/or activity of p53 or HDM2 or HDMX, or the enhancement of the level and/or activity of p53 or HDM2 or HDMX, by peptidomimetic macrocycles derived from p53, is used, for example, to ameliorate or reduce the adverse symptoms of the disorder.

[0190] In another aspect, the present invention provides methods for treating or preventing a disease including hyperproliferative disease and inflammatory disorder by interfering with the interaction or binding between binding partners, for example, between p53 and HDM2 or p53 and HDMX. These methods comprise administering an effective amount of a compound of the invention to a warm blooded animal, including a human. In some embodiments, the administration of the compounds of the present invention induces cell growth arrest or apoptosis.

[0191] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

[0192] In some embodiments, the peptidomimetics macrocycles of the invention is used to treat, prevent, and/or diagnose cancers and neoplastic conditions. As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of breast, lung, liver, colon and ovarian origin. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. In some embodiments, the peptidomimetics macrocycles are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, lung cancer, metastasis of such cancers and the like.

[0193] Examples of cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

[0194] Examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit Rev. Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.

[0195] Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

[0196] Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

[0197] Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0198] Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

[0199] Examples of cellular proliferative and/or differentiative disorders of the ovary include, but are not limited to, ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.

[0200] In other or further embodiments, the peptidomimetics macrocycles described herein are used to treat, prevent or diagnose conditions characterized by overactive cell death or cellular death due to physiologic insult, etc. Some examples of conditions characterized by premature or unwanted cell death are or alternatively unwanted or excessive cellular proliferation include, but are not limited to hypocellular/hypoplastic, acellular/aplastic, or hypercellular/hyperplastic conditions. Some examples include hematologic disorders including but not limited to fanconi anemia, aplastic anemia, thalaessemia, congenital neutropenia, and myelodysplasia.

[0201] In other or further embodiments, the peptidomimetics macrocycles of the invention that act to decrease apoptosis are used to treat disorders associated with an undesirable level of cell death. Thus, in some embodiments, the anti-apoptotic peptidomimetics macrocycles of the invention are used to treat disorders such as those that lead to cell death associated with viral infection, e.g., infection associated with infection with human immunodeficiency virus (HIV). A wide variety of neurological diseases are characterized by the gradual loss of specific sets of neurons. One example is Alzheimer's disease (AD) Alzheimer's disease is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in gross atrophy of the affected regions. Both amyloid plaques and neurofibrillary tangles are visible in brains of those afflicted by AD Alzheimer's disease has been identified as a protein misfolding disease, due to the accumulation of abnormally folded A-beta and tau proteins in the brain. Plaques are made up of .beta.-amyloid. .beta.-amyloid is a fragment from a larger protein called amyloid precursor protein (APP). APP is critical to neuron growth, survival and post-injury repair. In AD, an unknown process causes APP to be cleaved into smaller fragments by enzymes through proteolysis. One of these fragments is fibrils of .beta.-amyloid, which form clumps that deposit outside neurons in dense formations known as senile plaques. Plaques continue to grow into insoluble twisted fibers within the nerve cell, often called tangles. Disruption of the interaction between .beta.-amyloid and its native receptor is therefore important in the treatment of AD. The anti-apoptotic peptidomimetics macrocycles of the invention are used, in some embodiments, in the treatment of AD and other neurological disorders associated with cell apoptosis. Such neurological disorders include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. The cell loss in these diseases does not induce an inflammatory response, and apoptosis appears to be the mechanism of cell death.

[0202] In addition, a number of hematologic diseases are associated with a decreased production of blood cells. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and the myelodysplastic syndromes. Disorders of blood cell production, such as myelodysplastic syndrome and some forms of aplastic anemia, are associated with increased apoptotic cell death within the bone marrow. These disorders could result from the activation of genes that promote apoptosis, acquired deficiencies in stromal cells or hematopoietic survival factors, or the direct effects of toxins and mediators of immune responses. Two common disorders associated with cell death are myocardial infarctions and stroke. In both disorders, cells within the central area of ischemia, which is produced in the event of acute loss of blood flow, appear to die rapidly as a result of necrosis. However, outside the central ischemic zone, cells die over a more protracted time period and morphologically appear to die by apoptosis. In other or further embodiments, the anti-apoptotic peptidomimetics macrocycles of the invention are used to treat all such disorders associated with undesirable cell death.

[0203] Some examples of neurologic disorders that are treated with the peptidomimetics macrocycles described herein include but are not limited to Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary Cerebral Hemorrhage Amyloidosis, Reactive Amyloidosis, Familial Amyloid Nephropathy with Urticaria and Deafness, Muckle-Wells Syndrome, Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, Isolated Cardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid, Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis, Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis, a prion-mediated disease, and Huntington's Disease.

[0204] In another embodiment, the peptidomimetics macrocycles described herein are used to treat, prevent or diagnose inflammatory disorders. Numerous types of inflammatory disorders exist. Certain inflammatory diseases are associated with the immune system, for example, autoimmune diseases. Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body, i.e. self antigens. In other words, the immune system attacks its own cells. Autoimmune diseases are a major cause of immune-mediated diseases. Rheumatoid arthritis is an example of an autoimmune disease, in which the immune system attacks the joints, where it causes inflammation (i.e. arthritis) and destruction. It can also damage some organs, such as the lungs and skin. Rheumatoid arthritis can lead to substantial loss of functioning and mobility. Rheumatoid arthritis is diagnosed with blood tests especially the rheumatoid factor test. Some examples of autoimmune diseases that are treated with the peptidomimetics macrocycles described herein include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, Bechet's disease, bullous pemphigoid, coeliac disease, Chagas disease, Churg-Strauss syndrome, chronic obstructive pulmonary disease (COPD), Crohn's disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, Hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory bowl disease (IBD), interstitial cystitis, lupus erythematosus, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia, Polymyositis, polymyalgia rheumatica, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, schizophrenia, scleroderma, Sjogren's syndrome, temporal arteritis (also known as "giant cell arteritis"), Takayasu's arteritis, Vasculitis, Vitiligo, and Wegener's granulomatosis.

[0205] Some examples of other types of inflammatory disorders that are treated with the peptidomimetics macrocycles described herein include, but are not limited to, allergy including allergic rhinitis/sinusitis, skin allergies (urticaria/hives, angioedema, atopic dermatitis), food allergies, drug allergies, insect allergies, and rare allergic disorders such as mastocytosis, asthma, arthritis including osteoarthritis, rheumatoid arthritis, and spondyloarthropathies, primary angitis of the CNS, sarcoidosis, organ transplant rejection, fibromyalgia, fibrosis, pancreatitis, and pelvic inflammatory disease.

[0206] Examples of cardiovascular disorders (e.g., inflammatory disorders) that are treated or prevented with the peptidomimetics macrocycles of the invention include, but are not limited to, aortic valve stenosis, atherosclerosis, myocardial infarction, stroke, thrombosis, aneurism, heart failure, ischemic heart disease, angina pectoris, sudden cardiac death, hypertensive heart disease; non-coronary vessel disease, such as arteriolosclerosis, small vessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and chronic pulmonary disease; or a cardiovascular condition associated with interventional procedures ("procedural vascular trauma"), such as restenosis following angioplasty, placement of a shunt, stent, synthetic or natural excision grafts, indwelling catheter, valve or other implantable devices. Preferred cardiovascular disorders include atherosclerosis, myocardial infarction, aneurism, and stroke.

[0207] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

Example 1

Synthesis of 6-Chlorotryptophan Fmoc Amino Acids

##STR00029##

[0209] Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1. To a stirred solution of dry DMF (12 mL) was added dropwise POCl.sub.3 (3.92 mL, 43 mmol, 1.3 equiv) at 0.degree. C. under Argon. The solution was stirred at the same temperature for 20 min before a solution of 6-chloroindole (5.0 g, 33 mmol, 1 eq.) in dry DMF (30 mL) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred for an additional 2.5 h. Water (50 mL) was added and the solution was neutralized with 4M aqueous NaOH (pH .about.8). The resulting solid was filtered off, washed with water and dried under vacuum. This material was directly used in the next step without additional purification. To a stirred solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150 mL) was added successively Boc.sub.2O (7.91 g, 36.3 mmol, 1.1 equiv) and DMAP (0.4 g, 3.3 mmol, 0.1 equiv) at room temperature under N.sub.2. The resulting mixture was stirred at room temperature for 1.5 h and the solvent was evaporated under reduced pressure. The residue was taken up in EtOAc and washed with 1N HCl, dried and concentrated to give the formyl indole 1 (9 g, 98% over 2 steps) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta.: 1.70 (s, Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).

[0210] Tert-butyl 6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To a solution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL) was added NaBH.sub.4 (2.4 g, 63 mmol, 2 eq.). The reaction was stirred for 3 h at room temperature. The reaction mixture was concentrated and the residue was poured into diethyl ether and water. The organic layer was separated, dried over magnesium sulfate and concentrated to give a white solid (8.7 g, 98%). This material was directly used in the next step without additional purification. .sup.1H NMR (CDCl.sub.3) .delta.: 1.65 (s, Boc, 9H); 4.80 (s, 2H, CH.sub.2); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs, 1H).

[0211] Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate, 3. To a solution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in dichloromethane (50 mL) under argon was added a solution of triphenylphosphine (4.59 g, 17.5 mmol, 1.2 eq.) in dichloromethane (50 mL) at -40.degree. C. The reaction solution was stirred an additional 30 min at 40.degree. C. Then NBS (3.38 g, 19 mmol, 1.3 eq.) was added. The resulting mixture was allowed to warm to room temperature and stirred overnight. Dichloromethane was evaporated, Carbon Tetrachloride (100 mL) was added and the mixture was stirred for 1 h and filtrated. The filtrate was concentrated, loaded in a silica plug and quickly eluted with 25% EtOAc in Hexanes. The solution was concentrated to give a white foam (3.84 g, 77%). .sup.1H NMR (CDCl.sub.3) .delta.: 1.66 (s, Boc, 9H); 4.63 (s, 2H, CH.sub.2); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs, 1H); 8.18 (bs, 1H).

[0212] .alpha.Me-6Cl-Trp(Boc)-Ni--S-BPB, 4. To S-Ala-Ni--S-BPB (2.66 g, 5.2 mmol, 1 eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.) was added 50 mL of DMF under argon. The bromide derivative compound 3 (2.68 g, 7.8 mmol, 1.5 eq.) in solution of DMF (5.0 mL) was added via syringe. The reaction mixture was stirred at ambient temperature for 1 h. The solution was then quenched with 5% aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 4 was purified by flash chromatography (solid loading) on normal phase using EtOAc and Hexanes as eluents to give a red solid (1.78 g, 45% yield). .alpha.Me-6Cl-Trp(Boc)-Ni--S-BPB, 4: M+H calc. 775.21, M+H obs. 775.26; .sup.1H NMR (CDCl.sub.3) .delta.: 1.23 (s, 3H, .alpha.Me); 1.56 (m, 11H, Boc+CH.sub.2); 1.82-2.20 (m, 4H, 2CH.sub.2); 3.03 (m, 1H, CH.sub..alpha.); 3.24 (m, 2H, CH.sub.2); 3.57 and 4.29 (AB system, 2H, CH.sub.2 (benzyl), J=12.8 Hz); 6.62 (d, 2H); 6.98 (d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m, 2H); 7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).

[0213] Fmoc-.alpha.Me-6Cl-Trp(Boc)-OH, 6. To a solution of 3N HCl/MeOH (1/3, 15 mL) at 50.degree. C. was added a solution of compound 4 (1.75 g, 2.3 mmol, 1 eq.) in MeOH (5 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0.degree. C. with an ice bath and quenched with an aqueous solution of Na.sub.2CO.sub.3 (1.21 g, 11.5 mmol, 5 eq.). Methanol was removed and 8 more equivalents of Na.sub.2CO.sub.3 (1.95 g, 18.4 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (1.68 g, 4.5 mmol, 2 eq.) was then added and the suspension was stirred for 2 h. A solution of Fmoc-OSu (0.84 g, 2.5 mmol, 1.1 eq.) in acetone (50 mL) was added and the reaction was stirred overnight. Afterwards, the reaction was diluted with diethyl ether and 1N HCl. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 6 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (0.9 g, 70% yield). Fmoc-.alpha.Me-6Cl-Trp(Boc)-OH, 6: M+H calc. 575.19, M+H obs. 575.37; .sup.1H NMR (CDCl.sub.3) 1.59 (s, 9H, Boc); 1.68 (s, 3H, Me); 3.48 (bs, 2H, CH.sub.2); 4.22 (m, 1H, CH); 4.39 (bs, 2H, CH.sub.2); 5.47 (s, 1H, NH); 7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H); 7.75 (d, 2H); 8.12 (bs, 1H).

[0214] 6Cl-Trp(Boc)-Ni--S-BPB, 5. To Gly-Ni--S-BPB (4.6 g, 9.2 mmol, 1 eq.) and KO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95 mL of DMF under argon. The bromide derivative compound 3 (3.5 g, 4.6 mmol, 1.1 eq.) in solution of DMF (10 mL) was added via syringe. The reaction mixture was stirred at ambient temperature for 1 h. The solution was then quenched with 5% aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 5 was purified by flash chromatography (solid loading) on normal phase using EtOAc and Hexanes as eluents to give a red solid (5 g, 71% yield). 6Cl-Trp(Boc)-Ni--S-BPB, 5: M+H calc. 761.20, M+H obs. 761.34; .sup.1H NMR (CDCl.sub.3) .delta.: 1.58 (m, 11H, Boc+CH.sub.2); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m, 2H, CH.sub.2); 3.00 (m, 1H, CH.sub..alpha.); 3.22 (m, 2H, CH.sub.2); 3.45 and 4.25 (AB system, 2H, CH.sub.2 (benzyl), J=12.8 Hz); 4.27 (m, 1H, CH.sub..alpha.); 6.65 (d, 2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m, 3H); 7.35-7.39 (m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m, 2H).

[0215] Fmoc-6Cl-Trp(Boc)-OH, 7. To a solution of 3N HCl/MeOH (1/3, 44 mL) at 50.degree. C. was added a solution of compound 5 (5 g, 6.6 mmol, 1 eq.) in MeOH (10 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0.degree. C. with an ice bath and quenched with an aqueous solution of Na.sub.2CO.sub.3 (3.48 g, 33 mmol, 5 eq.). Methanol was removed and 8 more equivalents of Na.sub.2CO.sub.3 (5.57 g, 52 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (4.89 g, 13.1 mmol, 2 eq.) and the suspension was stirred for 2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in acetone (100 mL) was added and the reaction was stirred overnight. Afterwards, the reaction was diluted with diethyl ether and 1N HCl. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 7 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (2.6 g, 69% yield). Fmoc-6Cl-Trp(Boc)-OH, 7: M+H calc. 561.17, M+H obs. 561.37; .sup.1H NMR (CDCl.sub.3) 1.63 (s, 9H, Boc); 3.26 (m, 2H, CH.sub.2); 4.19 (m, 1H, CH); 4.39 (m, 2H, CH.sub.2); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H); 7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).

Example 2

Peptidomimetic Macrocycles of the Invention

[0216] Peptidomimetic macrocycles were synthesized, purified and analyzed as previously described and as described below (Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions. Peptide synthesis was performed either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.

[0217] Purification of cross-linked compounds was achieved by high performance liquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18 column (Varian) to yield the pure compounds. Chemical composition of the pure products was confirmed by LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).

[0218] Table 4 shows a list of peptidomimetic macrocycles of the invention prepared.

TABLE-US-00004 TABLE 4 SEQ ID Exact Observed NO: SP Seq Mass M + 2 mass (m/e) 38 SP-1 Ac-LSQETF$r8DLWKLL$EN-NH.sub.2 2068.13 1035.07 1035.36 39 SP-2 Ac-LSQETF$r8NLWKLL$QN-NH.sub.2 2066.16 1034.08 1034.31 40 SP-3 Ac-LSQQTF$r8NLWRLL$QN-NH.sub.2 2093.18 1047.59 1047.73 41 SP-4 Ac-QSQQTF$r8NLWKLL$QN-NH.sub.2 2080.15 1041.08 1041.31 42 SP-5 Ac-QSQQTF$r8NLWRLL$QN-NH.sub.2 2108.15 1055.08 1055.32 43 SP-6 Ac-QSQQTA$r8NLWRLL$QN-NH.sub.2 2032.12 1017.06 1017.24 44 SP-7 Ac-QAibQQTF$r8NLWRLL$QN-NH.sub.2 2106.17 1054.09 1054.34 45 SP-8 Ac-QSQQTFSNLWRLLPQN-NH.sub.2 2000.02 1001.01 1001.26 46 SP-9 Ac-QSQQTF$/r8NLWRLL$/QN-NH.sub.2 2136.18 1069.09 1069.37 47 SP-10 Ac-QSQAibTF$r8NLWRLL$QN-NH.sub.2 2065.15 1033.58 1033.71 48 SP-11 Ac-QSQQTF$r8NLWRLL$AN-NH.sub.2 2051.13 1026.57 1026.70 49 SP-12 Ac-ASQQTF$r8NLWRLL$QN-NH.sub.2 2051.13 1026.57 1026.90 50 SP-13 Ac-QSQQTF$r8ALWRLL$QN-NH.sub.2 2065.15 1033.58 1033.41 51 SP-14 Ac-QSQETF$r8NLWRLL$QN-NH.sub.2 2109.14 1055.57 1055.70 52 SP-15 Ac-RSQQTF$r8NLWRLL$QN-NH.sub.2 2136.20 1069.10 1069.17 53 SP-16 Ac-RSQQTF$r8NLWRLL$EN-NH.sub.2 2137.18 1069.59 1069.75 54 SP-17 Ac-LSQETFSDLWKLLPEN-NH.sub.2 1959.99 981.00 981.24 55 SP-18 Ac-QSQ$TFS$LWRLLPQN-NH.sub.2 2008.09 1005.05 1004.97 56 SP-19 Ac-QSQQ$FSN$WRLLPQN-NH.sub.2 2036.06 1019.03 1018.86 57 SP-20 Ac-QSQQT$SNL$RLLPQN-NH.sub.2 1917.04 959.52 959.32 58 SP-21 Ac-QSQQTF$NLW$LLPQN-NH.sub.2 2007.06 1004.53 1004.97 59 SP-22 Ac-RTQATF$r8NQWAibANle$TNAibTR-NH.sub.2 2310.26 1156.13 1156.52 60 SP-23 Ac-QSQQTF$r8NLWRLL$RN-NH.sub.2 2136.20 1069.10 1068.94 61 SP-24 Ac-QSQRTF$r8NLWRLL$QN-NH.sub.2 2136.20 1069.10 1068.94 62 SP-25 Ac-QSQQTF$r8NNleWRLL$QN-NH.sub.2 2108.15 1055.08 1055.44 63 SP-26 Ac-QSQQTF$r8NLWRNleL$QN-NH.sub.2 2108.15 1055.08 1055.84 64 SP-27 Ac-QSQQTF$r8NLWRLNle$QN-NH.sub.2 2108.15 1055.08 1055.12 65 SP-28 Ac-QSQQTY$r8NLWRLL$QN-NH.sub.2 2124.15 1063.08 1062.92 66 SP-29 Ac-RAibQQTF$r8NLWRLL$QN-NH.sub.2 2134.22 1068.11 1068.65 67 SP-30 Ac-MPRFMDYWEGLN-NH.sub.2 1598.70 800.35 800.45 68 SP-31 Ac-RSQQRF$r8NLWRLL$QN-NH.sub.2 2191.25 1096.63 1096.83 69 SP-32 Ac-QSQQRF$r8NLWRLL$QN-NH.sub.2 2163.21 1082.61 1082.87 70 SP-33 Ac-RAibQQRF$r8NLWRLL$QN-NH.sub.2 2189.27 1095.64 1096.37 71 SP-34 Ac-RSQQRF$r8NFWRLL$QN-NH.sub.2 2225.23 1113.62 1114.37 72 SP-35 Ac-RSQQRF$r8NYWRLL$QN-NH.sub.2 2241.23 1121.62 1122.37 73 SP-36 Ac-RSQQTF$r8NLWQLL$QN-NH.sub.2 2108.15 1055.08 1055.29 74 SP-37 Ac-QSQQTF$r8NLWQAmlL$QN-NH.sub.2 2094.13 1048.07 1048.32 75 SP-38 Ac-QSQQTF$r8NAmlWRLL$QN-NH.sub.2 2122.17 1062.09 1062.35 76 SP-39 Ac-NlePRF$r8DYWEGL$QN-NH.sub.2 1869.98 935.99 936.20 77 SP-40 Ac-NlePRF$r8NYWRLL$QN-NH.sub.2 1952.12 977.06 977.35 78 SP-41 Ac-RF$r8NLWRLL$Q-NH.sub.2 1577.96 789.98 790.18 79 SP-42 Ac-QSQQTF$r8N2ffWRLL$QN-NH.sub.2 2160.13 1081.07 1081.40 80 SP-43 Ac-QSQQTF$r8N3ffWRLL$QN-NH.sub.2 2160.13 1081.07 1081.34 81 SP-44 Ac-QSQQTF#r8NLWRLL#QN-NH.sub.2 2080.12 1041.06 1041.34 82 SP-45 Ac-RSQQTA$r8NLWRLL$QN-NH.sub.2 2060.16 1031.08 1031.38 83 SP-46 Ac-QSQQTF%r8NLWRLL%QN-NH.sub.2 2110.17 1056.09 1056.55 84 SP-47 HepQSQ$TFSNLWRLLPQN-NH.sub.2 2051.10 1026.55 1026.82 85 SP-48 HepQSQ$TF$r8NLWRLL$QN-NH.sub.2 2159.23 1080.62 1080.89 86 SP-49 Ac-QSQQTF$r8NL6clWRLL$QN-NH.sub.2 2142.11 1072.06 1072.35 87 SP-50 Ac-QSQQTF$r8NLMe6clwRLL$QN-NH.sub.2 2156.13 1079.07 1079.27 88 SP-51 Ac-LTFEHYWAQLTS-NH.sub.2 1535.74 768.87 768.91 89 SP-52 Ac-LTF$HYW$QLTS-NH.sub.2 1585.83 793.92 794.17 90 SP-53 Ac-LTFE$YWA$LTS-NH.sub.2 1520.79 761.40 761.67 91 SP-54 Ac-LTF$zr8HYWAQL$zS-NH.sub.2 1597.87 799.94 800.06 92 SP-55 Ac-LTF$r8HYWRQL$S-NH.sub.2 1682.93 842.47 842.72 93 SP-56 Ac-QS$QTFStNLWRLL$s8QN-NH.sub.2 2145.21 1073.61 1073.90 94 SP-57 Ac-QSQQTASNLWRLLPQN-NH.sub.2 1923.99 963.00 963.26 95 SP-58 Ac-QSQQTA$/r8NLWRLL$/QN-NH.sub.2 2060.15 1031.08 1031.24 96 SP-59 Ac-ASQQTF$/r8NLWRLL$/QN-NH.sub.2 2079.16 1040.58 1040.89 97 SP-60 Ac-$SQQ$FSNLWRLLAibQN-NH.sub.2 2009.09 1005.55 1005.86 98 SP-61 Ac-QS$QTF$NLWRLLAibQN-NH.sub.2 2023.10 1012.55 1012.79 99 SP-62 Ac-QSQQ$FSN$WRLLAibQN-NH.sub.2 2024.06 1013.03 1013.31 100 SP-63 Ac-QSQQTF$NLW$LLAibQN-NH.sub.2 1995.06 998.53 998.87 101 SP-64 Ac-QSQQTFS$LWR$LAibQN-NH.sub.2 2011.06 1006.53 1006.83 102 SP-65 Ac-QSQQTFSNLW$LLA$N-NH.sub.2 1940.02 971.01 971.29 103 SP-66 Ac-$/SQQ$/FSNLWRLLAibQN-NH.sub.2 2037.12 1019.56 1019.78 104 SP-67 Ac-QS$/QTF$/NLWRLLAibQN-NH.sub.2 2051.13 1026.57 1026.90 105 SP-68 Ac-QSQQ$/FSN$/WRLLAibQN-NH.sub.2 2052.09 1027.05 1027.36 106 SP-69 Ac-QSQQTF$/NLW$/LLAibQN-NH.sub.2 2023.09 1012.55 1013.82 107 SP-70 Ac-QSQ$TFS$LWRLLAibQN-NH.sub.2 1996.09 999.05 999.39 108 SP-71 Ac-QSQ$/TFS$LWRLLAibQN-NH.sub.2 2024.12 1013.06 1013.37 109 SP-72 Ac-QS$/QTFSt//NLWRLL$/s8QN-NH.sub.2 2201.27 1101.64 1102.00 110 SP-73 Ac-$r8SQQTFS$LWRLLAibQN-NH.sub.2 2038.14 1020.07 1020.23 111 SP-74 Ac-QSQ$r8TFSNLW$LLAibQN-NH.sub.2 1996.08 999.04 999.32 112 SP-75 Ac-QSQQTFS$r8LWRLLA$N-NH.sub.2 2024.12 1013.06 1013.37 113 SP-76 Ac-QS$r5QTFStNLW$LLAibQN-NH.sub.2 2032.12 1017.06 1017.39 114 SP-77 Ac-$/r8SOQTFS$/LWRLLAibQN-NH.sub.2 2066.17 1034.09 1034.80 115 SP-78 Ac-QSQ$/r8TFSNLW$/LLAibQN-NH.sub.2 2024.11 1013.06 1014.34 116 SP-79 Ac-QSQQTFS$/rXLWRLLA$/N-NH.sub.2 2052.15 1027.08 1027.16 117 SP-80 Ac-QS$/r5QTFSt/NLW$LLAibQN-NH.sub.2 2088.18 1045.09 1047.10 118 SP-81 Ac-QSQQTFSNLWRLLAibQN-NH.sub.2 1988.02 995.01 995.31 119 SP-82 Hep/QSQv/TF$/r8NLWRLL$/QN-NH.sub.2 2215.29 1108.65 1108.93 120 SP-83 Ac-ASQQTF$r8NLRWLL$QN-NH.sub.2 2051.13 1026.57 1026.90 121 SP-84 Ac-QSQQTF$/r8NLWRLL$/Q-NH.sub.2 2022.14 1012.07 1012.66 122 SP-85 Ac-QSQQTF$r8NLWRLL$Q-NH.sub.2 1994.11 998.06 998.42 123 SP-86 Ac-AAARAA$r8AAARAA$AA-NH.sub.2 1515.90 758.95 759.21 124 SP-87 Ac-LTFEHYWAQLTSA-NH.sub.2 1606.78 804.39 804.59 125 SP-88 Ac-LTF$r8HYWAQL$SA-NH.sub.2 1668.90 835.45 835.67 126 SP-89 Ac-ASQQTFSNLWRLLPQN-NH.sub.2 1943.00 972.50 973.27 127 SP-90 Ac-QS$QTFStNLW$r5LLAibQN-NH.sub.2 2032.12 1017.06 1017.30 128 SP-91 Ac-QSQQTFAibNLWRLLAibQN-NH.sub.2 1986.04 994.02 994.19 129 SP-92 Ac-QSQQTFNleNLWRLLNleQN-NH.sub.2 2042.11 1022.06 1022.23 130 SP-93 Ac-QSQQTF$/r8NLWRLLAibQN-NH.sub.2 2082.14 1042.07 1042.23 131 SP-94 Ac-QSQQTF$/r8NLWRLLNleQN-NH.sub.2 2110.17 1056.09 1056.29 132 SP-95 Ac-QSQQTFAibNLWRLL$/QN-NH.sub.2 2040.09 1021.05 1021.25 133 SP-96 Ac-QSQQTFNleNLWRLL$/QN-NH.sub.2 2068.12 1035.06 1035.31 134 SP-97 Ac-QSQQTF%r8NL6clWRNleL%QN-NH.sub.2 2144.13 1073.07 1073.32 135 SP-98 Ac-QSQQTF%r8NLMe6clWRLL%QN-NH.sub.2 2158.15 1080.08 1080.31 136 SP-101 Ac-FNle$YWE$L-NH.sub.2 1160.63 - 1161.70 137 SP-102 Ac-F$r8AYWELL$A-NH.sub.2 1344.75 - 1345.90 138 SP-103 Ac-F$r8AYWQLL$A-NH.sub.2 1343.76 - 1344.83 139 SP-104 Ac-NlePRF$r8NYWELL$QN-NH.sub.2 1925.06 963.53 963.69 140 SP-105 Ac-NlePRF$r8DYWRLL$QN-NH.sub.2 1953.10 977.55 977.68 141 SP-106 Ac-NlePRF$r8NYWRLL$Q-NH.sub.2 1838.07 920.04 920.18 142 SP-107 Ac-NlePRF$r8NYWRLL$-NH.sub.2 1710.01 856.01 856.13 143 SP-108 Ac-QSQQTF$r8DLWRLL$QN-NH.sub.2 2109.14 1055.57 1055.64 144 SP-109 Ac-QSQQTF$r8NLWRLL$EN-NH.sub.2 2109.14 1055.57 1055.70 145 SP-110 Ac-QSQQTF$r8NLWRLL$QD-NH.sub.2 2109.14 1055.57 1055.64 146 SP-111 Ac-QSQQTF$r8NLWRLL$S-NH.sub.2 1953.08 977.54 977.60 147 SP-112 Ac-ESQQTF$r8NLWRLL$QN-NH.sub.2 2109.14 1055.57 1055.70 148 SP-113 Ac-LTF$r8NLWRNleL$Q-NH.sub.2 1635.99 819.00 819.10 149 SP-114 Ac-LRF$r8NLWRNleL$Q-NH.sub.2 1691.04 846.52 846.68 150 SP-115 Ac-QSQQTF$r8NWWRNleL$QN-NH.sub.2 2181.15 1091.58 1091.64 151 SP-116 Ac-QSQQTF$r8NLWRNleL$Q-NH.sub.2 1994.11 998.06 998.07 152 SP-117 Ac-QTF$r8NLWRNleL$QN-NH.sub.2 1765.00 883.50 883.59 153 SP-118 Ac-NlePRF$r8NWWRLL$QN-NH.sub.2 1975.13 988.57 988.75 154 SP-119 Ac-NlePRF$r8NWWRLL$A-NH.sub.2 1804.07 903.04 903.08 155 SP-120 Ac-TSFAEYWNLLSP-NH.sub.2 1467.70 734.85 734.90 156 SP-121 Ac-QTF$r8HWWSQL$S-NH.sub.2 1651.85 826.93 827.12 157 SP-122 Ac-FM$YWE$L-NH.sub.2 1178.58 - 1179.64 158 SP-123 Ac-QTFEHWWSQLLS-NH.sub.2 1601.76 801.88 801.94 159 SP-124 Ac-QSQQTF$r8NLAmwRLNle$QN-NH.sub.2 2122.17 1062.09 1062.24

160 SP-125 Ac-FMAibY6clWEAc3cL-NH.sub.2 1130.47 - 1131.53 161 SP-126 Ac-FNle$Y6clWE$L-NH.sub.2 1194.59 - 1195.64 162 SP-127 Ac-F$zr8AY6clWEAc3cL$z-NH.sub.2 1277.63 639.82 1278.71 163 SP-128 Ac-F$r8AY6clWEAc3cL$A-NH.sub.2 1348.66 - 1350.72 164 SP-129 Ac-NlePRF$r8NY6clWRLL$QN-NH.sub.2 1986.08 994.04 994.64 165 SP-130 Ac-AF$r8AAWALA$A-NH.sub.2 1223.71 - 1224.71 166 SP-131 Ac-TF$r8AAWRLA$Q-NH.sub.2 1395.80 698.90 399.04 167 SP-132 Pr-TF$r8AAWRLA$Q-NH.sub.2 1409.82 705.91 706.04 168 SP-133 Ac-QSQQTF%r8NLWRNleL%QN-NH.sub.2 2110.17 1056.09 1056.22 169 SP-134 Ac-LTF%r8HYWAQL%SA-NH.sub.2 1670.92 836.46 836.58 170 SP-135 Ac-NlePRF%r8NYWRLL%QN-NH.sub.2 1954.13 978.07 978.19 171 SP-136 Ac-NlePRF%r8NY6clWRLL%QN-NH.sub.2 1988.09 995.05 995.68 172 SP-137 Ac-LTF%r8HY6clWAQL%S-NH.sub.2 1633.84 817.92 817.93 173 SP-138 Ac-QS%QTF%StNLWRLL%s8QN-NH.sub.2 2149.24 1075.62 1075.65 174 SP-139 Ac-LTF%r8HY6clWRQL%S-NH.sub.2 1718.91 860.46 860.54 175 SP-140 Ac-QSQQTF%r8NL6clWRLL%QN-NH.sub.2 2144.13 1073.07 1073.64 176 SP-141 Ac-%r8SQQTFS%LWRLLAibQN-NH.sub.2 2040.15 1021.08 1021.13 177 SP-142 Ac-LTF%r8HYWAQL%S-NH.sub.2 1599.88 800.94 801.09 178 SP-143 Ac-TSF%r8QYWNLL%P-NH.sub.2 1602.88 802.44 802.58 179 SP-147 Ac-LTFEHYWAQLTS-NH2 1535.74 768.87 769.5 180 SP-152 Ac-F$er8AY6clWEAc3cL$e-NH.sub.2 1277.63 639.82 1278.71 181 SP-153 Ac-AF$r8AAWALA$A-NH.sub.2 1277.63 639.82 1277.84 182 SP-154 Ac-TF$r8AAWRLA$Q-NH.sub.2 1395.80 698.90 699.04 183 SP-155 Pr-TF$r8AAWRLA$Q-NH.sub.2 1409.82 705.91 706.04 184 SP-156 Ac-LTF$er8HYWAQL$eS-NH.sub.2 1597.87 799.94 800.44 185 SP-159 Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH.sub.2 2745.30 1373.65 1372.99 186 SP-160 Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-NH.sub.2 2669.27 1335.64 1336.09 187 SP-161 Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH.sub.2 2589.26 1295.63 1296.2 188 SP-162 Ac-LTF$/r8HYWAQL$/S-NH.sub.2 1625.90 813.95 814.18 189 SP-163 Ac-F%r8HY6clWRAc3cL%-NH.sub.2 1372.72 687.36 687.59 190 SP-164 Ac-QTF%r8HWWSQL%S-NH.sub.2 1653.87 827.94 827.94 191 SP-165 Ac-LTA$r8HYWRQL$S-NH.sub.2 1606.90 804.45 804.66 192 SP-166 Ac-Q$r8QQTFSN$WRLLAibQN-NH.sub.2 2080.12 1041.06 1041.61 193 SP-167 Ac-QSQQ$r8FSNLWR$LAibQN-NH.sub.2 2066.11 1034.06 1034.58 194 SP-168 Ac-F$r8AYWEAc3cL$A-NH.sub.2 1314.70 658.35 1315.88 195 SP-169 Ac-F$r8AYWEAc3cL$S-NH.sub.2 1330.70 666.35 1331.87 196 SP-170 Ac-F$r8AYWEAc3cL$Q-NH.sub.2 1371.72 686.86 1372.72 197 SP-171 Ac-F$r8AYWEAibL$S-NH.sub.2 1332.71 667.36 1334.83 198 SP-172 Ac-F$r8AYWEAL$S-NH.sub.2 1318.70 660.35 1319.73 199 SP-173 Ac-F$r8AYWEQL$S-NH.sub.2 1375.72 688.86 1377.53 200 SP-174 Ac-F$r8HYWEQL$S-NH.sub.2 1441.74 721.87 1443.48 201 SP-175 Ac-F$r8HYWAQL$S-NH.sub.2 1383.73 692.87 1385.38 202 SP-176 Ac-F$r8HYWAAc3cL$S-NH.sub.2 1338.71 670.36 1340.82 203 SP-177 Ac-F$r8HYWRAc3cL$S-NH.sub.2 1423.78 712.89 713.04 204 SP-178 Ac-F$r8AYWEAc3cL#A-NH.sub.2 1300.69 651.35 1302.78 205 SP-179 Ac-NlePTF%r8NYWRLL%QN-NH.sub.2 1899.08 950.54 950.56 206 SP-180 Ac-TF$r8AAWRAL$Q-NH.sub.2 1395.80 698.90 699.13 207 SP-181 Ac-TSF%r8HYWAQL%S-NH.sub.2 1573.83 787.92 787.98 208 SP-184 Ac-F%r8AY6clWEAc3cL%A-NH.sub.2 1350.68 676.34 676.91 209 SP-185 Ac-LTF$r8HYWAQI$S-NH.sub.2 1597.87 799.94 800.07 210 SP-186 Ac-LTF$r8HYWAQNle$S-NH.sub.2 1597.87 799.94 800.07 211 SP-187 Ac-LTF$r8HYWAQL$A-NH.sub.2 1581.87 791.94 792.45 212 SP-188 Ac-LTF$r8HYWAQL$Abu-NH.sub.2 1595.89 798.95 799.03 213 SP-189 Ac-LTF$r8HYWAbuQL$S-NH.sub.2 1611.88 806.94 807.47 214 SP-190 Ac-LTF$er8AYWAQL$eS-NH.sub.2 1531.84 766.92 766.96 215 SP-191 Ac-LAF$r8HYWAQL$S-NH.sub.2 1567.86 784.93 785.49 216 SP-192 Ac-LAF$r8AYWAQL$S-NH.sub.2 1501.83 751.92 752.01 217 SP-193 Ac-LTF$er8AYWAQL$eA-NH.sub.2 1515.85 758.93 758.97 218 SP-194 Ac-LAF$r8AYWAQL$A-NH.sub.2 1485.84 743.92 744.05 219 SP-195 Ac-LTF$r8NLWANleL$Q-NH.sub.2 1550.92 776.46 776.61 220 SP-196 Ac-LTF$r8NLWANleL$A-NH.sub.2 1493.90 747.95 1495.6 221 SP-197 Ac-LTF$r8ALWANleL$Q-NH.sub.2 1507.92 754.96 755 222 SP-198 Ac-LAF$r8NLWANleL$Q-NH.sub.2 1520.91 761.46 761.96 223 SP-199 Ac-LAF$r8ALWANleL$A-NH.sub.2 1420.89 711.45 1421.74 224 SP-200 Ac-A$r8AYWEAc3cL$A-NH.sub.2 1238.67 620.34 1239.65 225 SP-201 Ac-F$r8AYWEAc3cL$AA-NH.sub.2 1385.74 693.87 1386.64 226 SP-202 Ac-F$r8AYWEAc3cL$Abu-NH.sub.2 1328.72 665.36 1330.17 227 SP-203 Ac-F$r8AYWEAc3cL$Nle-NH.sub.2 1356.75 679.38 1358.22 228 SP-204 Ac-F$r5AYWEAc3cL$s8A-NH.sub.2 1314.70 658.35 1315.51 229 SP-205 Ac-F$AYWEAc3cL$r8A-NH.sub.2 1314.70 658.35 1315.66 230 SP-206 Ac-F$r8AYWEAc3cI$A-NH.sub.2 1314.70 658.35 1316.18 231 SP-207 Ac-F$r8AYWEAc3cNle$A-NH.sub.2 1314.70 658.35 1315.66 232 SP-208 Ac-F$r8AYWEAmlL$A-NH.sub.2 1358.76 680.38 1360.21 233 SP-209 Ac-F$r8AYWENleL$A-NH.sub.2 1344.75 673.38 1345.71 234 SP-210 Ac-F$r8AYWQAc3cL$A-NH.sub.2 1313.72 657.86 1314.7 235 SP-211 Ac-F$r8AYWAAc3cL$A-NH.sub.2 1256.70 629.35 1257.56 236 SP-212 Ac-F$r8AYWAbuAc3cL$A-NH.sub.2 1270.71 636.36 1272.14 237 SP-213 Ac-F$r8AYWNleAc3cL$A-NH.sub.2 1298.74 650.37 1299.67 238 SP-214 Ac-F$r8AbuYWEAc3cL$A-NH.sub.2 1328.72 665.36 1329.65 239 SP-215 Ac-F$r8NleYWEAc3cL$A-NH.sub.2 1356.75 679.38 1358.66 240 SP-216 5-FAM-BaLTFEHYWAQLTS-NH.sub.2 1922.82 962.41 962.87 241 SP-217 5-FAM-BaLTF%r8HYWAQL%S-NH.sub.2 1986.96 994.48 994.97 242 SP-218 Ac-LTF$r8HYWAQhL$S-NH.sub.2 1611.88 806.94 807 243 SP-219 Ac-LTF$r8HYWAQTle$S-NH.sub.2 1597.87 799.94 799.97 244 SP-220 Ac-LTF$r8HYWAQAdm$S-NH.sub.2 1675.91 838.96 839.09 245 SP-221 Ac-LTF$r8HYWAQhCha$S-NH.sub.2 1651.91 826.96 826.98 246 SP-222 Ac-LTF$r8HYWAQCha$S-NH.sub.2 1637.90 819.95 820.02 247 SP-223 Ac-LTF$r8HYWAc6cQL$S-NH.sub.2 1651.91 826.96 826.98 248 SP-224 Ac-LTF$r8HYWAc5cQL$S-NH.sub.2 1637.90 819.95 820.02 249 SP-225 Ac-LThF$r8HYWAQL$S-NH.sub.2 1611.88 806.94 807 250 SP-226 Ac-LTIgl$r8HYWAQL$S-NH.sub.2 1625.90 813.95 812.99 251 SP-227 Ac-LTF$r8HYWAQChg$S-NH.sub.2 1623.88 812.94 812.99 252 SP-228 Ac-LTF$r8HYWAQF$S-NH.sub.2 1631.85 816.93 816.99 253 SP-229 Ac-LTF$r8HYWAQIgl$S-NH.sub.2 1659.88 830.94 829.94 254 SP-230 Ac-LTF$r8HYWAQCba$S-NH.sub.2 1609.87 805.94 805.96 255 SP-231 Ac-LTF$r8HYWAQCpg$S-NH.sub.2 1609.87 805.94 805.96 256 SP-232 Ac-LTF$r8HhYWAQL$S-NH.sub.2 1611.88 806.94 807 257 SP-233 Ac-F$r8AYWEAc3chL$A-NH.sub.2 1328.72 665.36 665.43 258 SP-234 Ac-F$r8AYWEAc3cTle$A-NH.sub.2 1314.70 658.35 1315.62 259 SP-235 Ac-F$r8AYWEAc3cAdm$A-NH.sub.2 1392.75 697.38 697.47 260 SP-236 Ac-F$r8AYWEAc3chCha$A-NH.sub.2 1368.75 685.38 685.34 261 SP-237 Ac-F$r8AYWEAc3cCha$A-NH.sub.2 1354.73 678.37 678.38 262 SP-238 Ac-F$r8AYWEAc6cL$A-NH.sub.2 1356.75 679.38 679.42 263 SP-239 Ac-F$r8AYWEAc5cL$A-NH.sub.2 1342.73 672.37 672.46 264 SP-240 Ac-hF$r8AYWEAc3cL$A-NH.sub.2 1328.72 665.36 665.43 265 SP-241 Ac-Igl$r8AYWEAc3cL$A-NH.sub.2 1342.73 672.37 671.5 266 SP-243 Ac-F$r8AYWEAc3cF$A-NH.sub.2 1348.69 675.35 675.35 267 SP-244 Ac-F$r8AYWEAc3cIgl$A-NH.sub.2 1376.72 689.36 688.37 268 SP-245 Ac-F$r8AYWEAc3cCba$A-NH.sub.2 1326.70 664.35 664.47 269 SP-246 Ac-F$r8AYWEAc3cCpg$A-NH.sub.2 1326.70 664.35 664.39 270 SP-247 Ac-F$r8AhYWEAc3cL$A-NH.sub.2 1328.72 665.36 665.43 271 SP-248 Ac-F$r8AYWEAc3cL$Q-NH.sub.2 1371.72 686.86 1372.87 272 SP-249 Ac-F$r8AYWEAibL$A-NH.sub.2 1316.72 659.36 1318.18 273 SP-250 Ac-F$r8AYWEAL$A-NH.sub.2 1302.70 652.35 1303.75 274 SP-251 Ac-LAF$r8AYWAAL$A-NH.sub.2 1428.82 715.41 715.49 275 SP-252 Ac-LTF$r8HYWAAc3cL$S-NH.sub.2 1552.84 777.42 777.5 276 SP-253 Ac-NleTF$r8HYWAQL$S-NH.sub.2 1597.87 799.94 800.04 277 SP-254 Ac-VTF$r8HYWAQL$S-NH.sub.2 1583.85 792.93 793.04 278 SP-255 Ac-FTF$r8HYWAQL$S-NH.sub.2 1631.85 816.93 817.02 279 SP-256 Ac-WTF$r8HYWAQL$S-NH.sub.2 1670.86 836.43 836.85 280 SP-257 Ac-RTF$r8HYWAQL$S-NH.sub.2 1640.88 821.44 821.9 281 SP-258 Ac-KTF$r8HYWAQL$S-NH.sub.2 1612.88 807.44 807.91 282 SP-259 Ac-LN1eF$r8HYWAQL$S-NH.sub.2 1609.90 805.95 806.43 283 SP-260 Ac-LVF$r8HYWAQL$S-NH.sub.2 1595.89 798.95 798.93 284 SP-261 Ac-LFF$r8HYWAQL$S-NH.sub.2 1643.89 822.95 823.38 285 SP-262 Ac-LWF$r8HYWAQL$S-NH.sub.2 1682.90 842.45 842.55

286 SP-263 Ac-LRF$r8HYWAQL$S-NH.sub.2 1652.92 827.46 827.52 287 SP-264 Ac-LKF$r8HYWAQL$S-NH.sub.2 1624.91 813.46 813.51 288 SP-265 Ac-LTF$r8NleYWAQL$S-NH.sub.2 1573.89 787.95 788.05 289 SP-266 Ac-LTF$r8VYWAQL$S-NH.sub.2 1559.88 780.94 780.98 290 SP-267 Ac-LTF$r8FYWAQL$S-NH.sub.2 1607.88 804.94 805.32 291 SP-268 Ac-LTF$r8WYWAQL$S-NH.sub.2 1646.89 824.45 824.86 292 SP-269 Ac-LTF$r8RYWAQL$S-NH.sub.2 1616.91 809.46 809.51 293 SP-270 Ac-LTF$r8KYWAQL$S-NH.sub.2 1588.90 795.45 795.48 294 SP-271 Ac-LTF$r8HNleWAQL$S-NH.sub.2 1547.89 774.95 774.98 295 SP-272 Ac-LTF$r8HVWAQL$S-NH.sub.2 1533.87 767.94 767.95 296 SP-273 Ac-LTF$r8HFWAQL$S-NH.sub.2 1581.87 791.94 792.3 297 SP-274 Ac-LTF$r8HWWAQL$S-NH.sub.2 1620.88 811.44 811.54 298 SP-275 Ac-LTF$r8HRWAQL$S-NH.sub.2 1590.90 796.45 796.52 299 SP-276 Ac-LTF$r8HKWAQL$S-NH.sub.2 1562.90 782.45 782.53 300 SP-277 Ac-LTF$r8HYWNleQL$S-NH.sub.2 1639.91 820.96 820.98 301 SP-278 Ac-LTF$r8HYWVQL$S-NH.sub.2 1625.90 813.95 814.03 302 SP-279 Ac-LTF$r8HYWFQL$S-NH.sub.2 1673.90 837.95 838.03 303 SP-280 Ac-LTF$r8HYWWQL$S-NH.sub.2 1712.91 857.46 857.5 304 SP-281 Ac-LTF$r8HYWKQL$S-NH.sub.2 1654.92 828.46 828.49 305 SP-282 Ac-LTF$r8HYWANleL$S-NH.sub.2 1582.89 792.45 792.52 306 SP-283 Ac-LTF$r8HYWAVL$S-NH.sub.2 1568.88 785.44 785.49 307 SP-284 Ac-LTF$r8HYWAFL$S-NH.sub.2 1616.88 809.44 809.47 308 SP-285 Ac-LTF$r8HYWAWL$S-NH.sub.2 1655.89 828.95 829 309 SP-286 Ac-LTF$r8HYWARL$S-NH.sub.2 1625.91 813.96 813.98 310 SP-287 Ac-LTF$r8HYWAQL$Nle-NH.sub.2 1623.92 812.96 813.39 311 SP-288 Ac-LTF$r8HYWAQL$V-NH.sub.2 1609.90 805.95 805.99 312 SP-289 Ac-LTF$r8HYWAQL$F-NH.sub.2 1657.90 829.95 830.26 313 SP-290 Ac-LTF$r8HYWAQL$W-NH.sub.2 1696.91 849.46 849.5 314 SP-291 Ac-LTF$r8HYWAQL$R-NH.sub.2 1666.94 834.47 834.56 315 SP-292 Ac-LTF$r8HYWAQL$K-NH.sub.2 1638.93 820.47 820.49 316 SP-293 Ac-Q$r8QQTFSN$WRLLAibQN-NH.sub.2 2080.12 1041.06 1041.54 317 SP-294 Ac-QSQQ$r8FSNLWR$LAibQN-NH.sub.2 2066.11 1034.06 1034.58 318 SP-295 Ac-LT2Pal$r8HYWAQL$S-NH.sub.2 1598.86 800.43 800.49 319 SP-296 Ac-LT3Pal$r8HYWAQL$S-NH.sub.2 1598.86 800.43 800.49 320 SP-297 Ac-LT4Pal$r8HYWAQL$S-NH.sub.2 1598.86 800.43 800.49 321 SP-298 Ac-LTF2CF3$r8HYWAQL$S-NH.sub.2 1665.85 833.93 834.01 322 SP-299 Ac-LTF2CN$r8HYWAQL$S-NH.sub.2 1622.86 812.43 812.47 323 SP-300 Ac-LTF2Me$r8HYWAQL$S-NH.sub.2 1611.88 806.94 807 324 SP-301 Ac-LTF3Cl$r8HYWAQL$S-NH.sub.2 1631.83 816.92 816.99 325 SP-302 Ac-LTF4CF3$r8HYWAQL$S-NH.sub.2 1665.85 833.93 833.94 326 SP-303 Ac-LTF4tBu$r8HYWAQL$S-NH.sub.2 1653.93 827.97 828.02 327 SP-304 Ac-LTF5F$r8HYWAQL$S-NH.sub.2 1687.82 844.91 844.96 328 SP-305 Ac-LTF$r8HY3BthAAQL$S-NH.sub.2 1614.83 808.42 808.48 329 SP-306 Ac-LTF2Br$r8HYWAQL$S-NH.sub.2 1675.78 838.89 838.97 330 SP-307 Ac-LTF4Br$r8HYWAQL$S-NH.sub.2 1675.78 838.89 839.86 331 SP-308 Ac-LTF2Cl$r8HYWAQL$S-NH.sub.2 1631.83 816.92 816.99 332 SP-309 Ac-LTF4Cl$r8HYWAQL$S-NH.sub.2 1631.83 816.92 817.36 333 SP-310 Ac-LTF3CN$r8HYWAQL$S-NH.sub.2 1622.86 812.43 812.47 334 SP-311 Ac-LTF4CN$r8HYWAQL$S-NH.sub.2 1622.86 812.43 812.47 335 SP-312 Ac-LTF34Cl2$r8HYWAQL$S-NH.sub.2 1665.79 833.90 833.94 336 SP-313 Ac-LTF34F2$r8HYWAQL$S-NH.sub.2 1633.85 817.93 817.95 337 SP-314 Ac-LTF35F2$r8HYWAQL$S-NH.sub.2 1633.85 817.93 817.95 338 SP-315 Ac-LTDip$r8HYWAQL$S-NH.sub.2 1673.90 837.95 838.01 339 SP-316 Ac-LTF2F$r8HYWAQL$S-NH.sub.2 1615.86 808.93 809 340 SP-317 Ac-LTF3F$r8HYWAQL$S-NH.sub.2 1615.86 808.93 809 341 SP-318 Ac-LTF4F$r8HYWAQL$S-NH.sub.2 1615.86 808.93 809 342 SP-319 Ac-LTF4I$r8HYWAQL$S-NH.sub.2 1723.76 862.88 862.94 343 SP-320 Ac-LTF3Me$r8HYWAQL$S-NH.sub.2 1611.88 806.94 807.07 344 SP-321 Ac-LTF4Me$r8HYWAQL$S-NH.sub.2 1611.88 806.94 807 345 SP-322 Ac-LT1Na1$r8HYWAQL$S-NH.sub.2 1647.88 824.94 824.98 346 SP-323 Ac-LT2Na1$r8HYWAQL$S-NH.sub.2 1647.88 824.94 825.06 347 SP-324 Ac-LTF3CF3$r8HYWAQL$S-NH.sub.2 1665.85 833.93 834.01 348 SP-325 Ac-LTF4NO2$r8HYWAQL$S-NH.sub.2 1642.85 822.43 822.46 349 SP-326 Ac-LTF3NO2$r8HYWAQL$S-NH.sub.2 1642.85 822.43 822.46 350 SP-327 Ac-LTF$r82ThiYWAQL$S-NH.sub.2 1613.83 807.92 807.96 351 SP-328 Ac-LTF$r8HBipWAQL$S-NH.sub.2 1657.90 829.95 830.01 352 SP-329 Ac-LTF$r8HF4tBuWAQL$S-NH.sub.2 1637.93 819.97 820.02 353 SP-330 Ac-LTF$r8HF4CF3WAQL$S-NH.sub.2 1649.86 825.93 826.02 354 SP-331 Ac-LTF$r8HF4CIWAQL$S-NH.sub.2 1615.83 808.92 809.37 355 SP-332 Ac-LTF$r8HF4MeWAQL$S-NH.sub.2 1595.89 798.95 799.01 356 SP-333 Ac-LTF$r8HF4BrWAQL$S-NH.sub.2 1659.78 830.89 830.98 357 SP-334 Ac-LTF$r8HF4CNWAQL$S-NH.sub.2 1606.87 804.44 804.56 358 SP-335 Ac-LTF$r8HF4NO2WAQL$S-NH.sub.2 1626.86 814.43 814.55 359 SP-336 Ac-LTF$r8H1NaIWAQL$S-NH.sub.2 1631.89 816.95 817.06 360 SP-337 Ac-LTF$r8H2NaIWAQL$S-NH.sub.2 1631.89 816.95 816.99 361 SP-338 Ac-LTF$r8HWAQL$S-NH.sub.2 1434.80 718.40 718.49 362 SP-339 Ac-LTF$r8HY1NalAQL$S-NH.sub.2 1608.87 805.44 805.52 363 SP-340 Ac-LTF$r8HY2NalAQL$S-NH.sub.2 1608.87 805.44 805.52 364 SP-341 Ac-LTF$r8HYWAQI$S-NH.sub.2 1597.87 799.94 800.07 365 SP-342 Ac-LTF$r8HYWAQNle$S-NH.sub.2 1597.87 799.94 800.44 366 SP-343 Ac-LTF$er8HYWAQL$eA-NH.sub.2 1581.87 791.94 791.98 367 SP-344 Ac-LTF$r8HYWAQL$Abu-NH.sub.2 1595.89 798.95 799.03 368 SP-345 Ac-LTF$r8HYWAbuQL$S-NH.sub.2 1611.88 806.94 804.47 369 SP-346 Ac-LAF$r8HYWAQL$S-NH.sub.2 1567.86 784.93 785.49 370 SP-347 Ac-LTF$r8NLWANleL$Q-NH.sub.2 1550.92 776.46 777.5 371 SP-348 Ac-LTF$r8ALWANleL$Q-NH.sub.2 1507.92 754.96 755.52 372 SP-349 Ac-LAF$r8NLWANleL$Q-NH.sub.2 1520.91 761.46 762.48 373 SP-350 Ac-F$r8AYWAAc3cL$A-NH.sub.2 1256.70 629.35 1257.56 374 SP-351 Ac-LTF$r8AYWAAL$S-NH.sub.2 1474.82 738.41 738.55 375 SP-352 Ac-LVF$r8AYWAQL$S-NH.sub.2 1529.87 765.94 766 376 SP-353 Ac-LTF$r8AYWAbuQL$S-NH.sub.2 1545.86 773.93 773.92 377 SP-354 Ac-LTF$r8AYWNleQL$S-NH.sub.2 1573.89 787.95 788.17 378 SP-355 Ac-LTF$r8AbuYWAQL$S-NH.sub.2 1545.86 773.93 773.99 379 SP-356 Ac-LTF$r8AYWHQL$S-NH.sub.2 1597.87 799.94 799.97 380 SP-357 Ac-LTF$r8AYWKQL$S-NH.sub.2 1588.90 795.45 795.53 381 SP-358 Ac-LTF$r8AYWOQL$S-NH.sub.2 1574.89 788.45 788.5 382 SP-359 Ac-LTF$r8AYWRQL$S-NH.sub.2 1616.91 809.46 809.51 383 SP-360 Ac-LTF$r8AYWSQL$S-NH.sub.2 1547.84 774.92 774.96 384 SP-361 Ac-LTF$r8AYWRAL$S-NH.sub.2 1559.89 780.95 780.95 385 SP-362 Ac-LTF$r8AYWRQL$A-NH.sub.2 1600.91 801.46 801.52 386 SP-363 Ac-LTF$r8AYWRAL$A-NH.sub.2 1543.89 772.95 773.03 387 SP-364 Ac-LTF$r5HYWAQL$s8S-NH.sub.2 1597.87 799.94 799.97 388 SP-365 Ac-LTF$HYWAQL$r8S-NH.sub.2 1597.87 799.94 799.97 389 SP-366 Ac-LTF$r8HYWAAL$S-NH.sub.2 1540.84 771.42 771.48 390 SP-367 Ac-LTF$r8HYWAAbuL$S-NH.sub.2 1554.86 778.43 778.51 391 SP-368 Ac-LTF$r8HYWALL$S-NH.sub.2 1582.89 792.45 792.49 392 SP-369 Ac-F$r8AYWHAL$A-NH.sub.2 1310.72 656.36 656.4 393 SP-370 Ac-F$r8AYWAAL$A-NH.sub.2 1244.70 623.35 1245.61 394 SP-371 Ac-F$r8AYWSAL$A-NH.sub.2 1260.69 631.35 1261.6 395 SP-372 Ac-F$r8AYWRAL$A-NH.sub.2 1329.76 665.88 1330.72 396 SP-373 Ac-F$r8AYWKAL$A-NH.sub.2 1301.75 651.88 1302.67 397 SP-374 Ac-F$r8AYWOAL$A-NH.sub.2 1287.74 644.87 1289.13 398 SP-375 Ac-F$r8VYWEAc3cL$A-NH.sub.2 1342.73 672.37 1343.67 399 SP-376 Ac-F$r8FYWEAc3cL$A-NH.sub.2 1390.73 696.37 1392.14 400 SP-377 Ac-F$r8WYWEAc3cL$A-NH.sub.2 1429.74 715.87 1431.44 401 SP-378 Ac-F$r8RYWEAc3cL$A-NH.sub.2 1399.77 700.89 700.95 402 SP-379 Ac-F$r8KYWEAc3cL$A-NH.sub.2 1371.76 686.88 686.97 403 SP-380 Ac-F$r8ANleWEAc3cL$A-NH.sub.2 1264.72 633.36 1265.59 404 SP-381 Ac-F$r8AVWEAc3cL$A-NH.sub.2 1250.71 626.36 1252.2 405 SP-382 Ac-F$r8AFWEAc3cL$A-NH.sub.2 1298.71 650.36 1299.64 406 SP-383 Ac-F$r8AWWEAc3cL$A-NH.sub.2 1337.72 669.86 1338.64 407 SP-384 Ac-F$r8ARWEAc3cL$A-NH.sub.2 1307.74 654.87 655 408 SP-385 Ac-F$r8AKWEAc3cL$A-NH.sub.2 1279.73 640.87 641.01 409 SP-386 Ac-F$r8AYWVAc3cL$A-NH.sub.2 1284.73 643.37 643.38 410 SP-387 Ac-F$r8AYWFAc3cL$A-NH.sub.2 1332.73 667.37 667.43

411 SP-388 Ac-F$r8AYWWAc3cL$A-NH.sub.2 1371.74 686.87 686.97 412 SP-389 Ac-F$r8AYWRAc3cL$A-NH.sub.2 1341.76 671.88 671.94 413 SP-390 Ac-F$r8AYWKAc3cL$A-NH.sub.2 1313.75 657.88 657.88 414 SP-391 Ac-F$r8AYWEVL$A-NH.sub.2 1330.73 666.37 666.47 415 SP-392 Ac-F$r8AYWEFL$A-NH.sub.2 1378.73 690.37 690.44 416 SP-393 Ac-F$r8AYWEWL$A-NH.sub.2 1417.74 709.87 709.91 417 SP-394 Ac-F$r8AYWERL$A-NH.sub.2 1387.77 694.89 1388.66 418 SP-395 Ac-F$r8AYWEKL$A-NH.sub.2 1359.76 680.88 1361.21 419 SP-396 Ac-F$r8AYWEAc3cL$V-NH.sub.2 1342.73 672.37 1343.59 420 SP-397 Ac-F$r8AYWEAc3cL$F-NH.sub.2 1390.73 696.37 1392.58 421 SP-398 Ac-F$r8AYWEAc3cL$W-NH.sub.2 1429.74 715.87 1431.29 422 SP-399 Ac-F$r8AYWEAc3cL$R-NH.sub.2 1399.77 700.89 700.95 423 SP-400 Ac-F$r8AYWEAc3cL$K-NH.sub.2 1371.76 686.88 686.97 424 SP-401 Ac-F$r8AYWEAc3cL$AV-NH.sub.2 1413.77 707.89 707.91 425 SP-402 Ac-F$r8AYWEAc3cL$AF-NH.sub.2 1461.77 731.89 731.96 426 SP-403 Ac-F$r8AYWEAc3cL$AW-NH.sub.2 1500.78 751.39 751.5 427 SP-404 Ac-F$r8AYWEAc3cL$AR-NH.sub.2 1470.80 736.40 736.47 428 SP-405 Ac-F$r8AYWEAc3cL$AK-NH.sub.2 1442.80 722.40 722.41 429 SP-406 Ac-F$r8AYWEAc3cL$AH-NH.sub.2 1451.76 726.88 726.93 430 SP-407 Ac-LTF2NO2$r8HYWAQL$S-NH.sub.2 1642.85 822.43 822.54 431 SP-408 Ac-LTA$r8HYAAQL$S-NH.sub.2 1406.79 704.40 704.5 432 SP-409 Ac-LTF$r8HYAAQL$S-NH.sub.2 1482.82 742.41 742.47 433 SP-410 Ac-QSQQTF$r8NLWALL$AN-NH.sub.2 1966.07 984.04 984.38 434 SP-411 Ac-QAibQQTF$r8NLWALL$AN-NH.sub.2 1964.09 983.05 983.42 435 SP-412 Ac-QAibQQTF$r8ALWALL$AN-NH.sub.2 1921.08 961.54 961.59 436 SP-413 Ac-AAAATF$r8AAWAAL$AA-NH.sub.2 1608.90 805.45 805.52 437 SP-414 Ac-F$r8AAWRAL$Q-NH.sub.2 1294.76 648.38 648.48 438 SP-415 Ac-TF$r8AAWAAL$Q-NH.sub.2 1310.74 656.37 1311.62 439 SP-416 Ac-TF$r8AAWRAL$A-NH.sub.2 1338.78 670.39 670.46 440 SP-417 Ac-VF$r8AAWRAL$Q-NH.sub.2 1393.82 697.91 697.99 441 SP-418 Ac-AF$r8AAWAAL$A-NH.sub.2 1223.71 612.86 1224.67 442 SP-420 Ac-TF$r8AAWKAL$Q-NH.sub.2 1367.80 684.90 684.97 443 SP-421 Ac-TF$r8AAWOAL$Q-NH.sub.2 1353.78 677.89 678.01 444 SP-422 Ac-TF$r8AAWSAL$Q-NH.sub.2 1326.73 664.37 664.47 445 SP-423 Ac-LTF$r8AAWRAL$Q-NH.sub.2 1508.89 755.45 755.49 446 SP-424 Ac-F$r8AYWAQL$A-NH.sub.2 1301.72 651.86 651.96 447 SP-425 Ac-F$r8AWWAAL$A-NH.sub.2 1267.71 634.86 634.87 448 SP-426 Ac-F$r8AWWAQL$A-NH.sub.2 1324.73 663.37 663.43 449 SP-427 Ac-F$r8AYWEAL$-NH.sub.2 1231.66 616.83 1232.93 450 SP-428 Ac-F$r8AYWAAL$-NH.sub.2 1173.66 587.83 1175.09 451 SP-429 Ac-F$r8AYWKAL$-NH.sub.2 1230.72 616.36 616.44 452 SP-430 Ac-F$r8AYWOAL$-NH.sub.2 1216.70 609.35 609.48 453 SP-431 Ac-F$r8AYWQAL$-NH.sub.2 1230.68 616.34 616.44 454 SP-432 Ac-F$r8AYWAQL$-NH.sub.2 1230.68 616.34 616.37 455 SP-433 Ac-F$r8HYWDQL$S-NH.sub.2 1427.72 714.86 714.86 456 SP-434 Ac-F$r8HFWEQL$S-NH.sub.2 1425.74 713.87 713.98 457 SP-435 Ac-F$r8AYWHQL$S-NH.sub.2 1383.73 692.87 692.96 458 SP-436 Ac-F$r8AYWKQL$S-NH.sub.2 1374.77 688.39 688.45 459 SP-437 Ac-F$r8AYWOQL$S-NH.sub.2 1360.75 681.38 681.49 460 SP-438 Ac-F$r8HYWSQL$S-NH.sub.2 1399.73 700.87 700.95 461 SP-439 Ac-F$r8HWWEQL$S-NH.sub.2 1464.76 733.38 733.44 462 SP-440 Ac-F$r8HWWAQL$S-NH.sub.2 1406.75 704.38 704.43 463 SP-441 Ac-F$r8AWWHQL$S-NH.sub.2 1406.75 704.38 704.43 464 SP-442 Ac-F$r8AWWKQL$S-NH.sub.2 1397.79 699.90 699.92 465 SP-443 Ac-F$r8AWWOQL$S-NH.sub.2 1383.77 692.89 692.96 466 SP-444 Ac-F$r8HWWSQL$S-NH.sub.2 1422.75 712.38 712.42 467 SP-445 Ac-LTF$r8NYWANleL$Q-NH.sub.2 1600.90 801.45 801.52 468 SP-446 Ac-LTF$r8NLWAQL$Q-NH.sub.2 1565.90 783.95 784.06 469 SP-447 Ac-LTF$r8NYWANleL$A-NH.sub.2 1543.88 772.94 773.03 470 SP-448 Ac-LTF$r8NLWAQL$A-NH.sub.2 1508.88 755.44 755.49 471 SP-449 Ac-LTF$r8AYWANleL$Q-NH.sub.2 1557.90 779.95 780.06 472 SP-450 Ac-LTF$r8ALWAQL$Q-NH.sub.2 1522.89 762.45 762.45 473 SP-451 Ac-LAF$r8NYWANleL$Q-NH.sub.2 1570.89 786.45 786.5 474 SP-452 Ac-LAF$r8NLWAQL$Q-NH.sub.2 1535.89 768.95 769.03 475 SP-453 Ac-LAF$r8AYWANleL$A-NH.sub.2 1470.86 736.43 736.47 476 SP-454 Ac-LAF$r8ALWAQL$A-NH.sub.2 1435.86 718.93 719.01 477 SP-455 Ac-LAF$r8AYWAAL$A-NH.sub.2 1428.82 715.41 715.41 478 SP-456 Ac-F$r8AYWEAc3cL$AAib-NH.sub.2 1399.75 700.88 700.95 479 SP-457 Ac-F$r8AYWAQL$AA-NH.sub.2 1372.75 687.38 687.78 480 SP-458 Ac-F$r8AYWAAc3cL$AA-NH.sub.2 1327.73 664.87 664.84 481 SP-459 Ac-F$r8AYWSAc3cL$AA-NH.sub.2 1343.73 672.87 672.9 482 SP-460 Ac-F$r8AYWEAc3cL$AS-NH.sub.2 1401.73 701.87 701.84 483 SP-461 Ac-F$r8AYWEAc3cL$AT-NH.sub.2 1415.75 708.88 708.87 484 SP-462 Ac-F$r8AYWEAc3cL$AL-NH.sub.2 1427.79 714.90 714.94 485 SP-463 Ac-F$r8AYWEAc3cL$AQ-NH.sub.2 1442.76 722.38 722.41 486 SP-464 Ac-F$r8AFWEAc3cL$AA-NH.sub.2 1369.74 685.87 685.93 487 SP-465 Ac-F$r8AWWEAc3cL$AA-NH.sub.2 1408.75 705.38 705.39 488 SP-466 Ac-F$r8AYWEAc3cL$SA-NH.sub.2 1401.73 701.87 701.99 489 SP-467 Ac-F$r8AYWEAL$AA-NH.sub.2 1373.74 687.87 687.93 490 SP-468 Ac-F$r8AYWENleL$AA-NH.sub.2 1415.79 708.90 708.94 491 SP-469 Ac-F$r8AYWEAc3cL$AbuA-NH.sub.2 1399.75 700.88 700.95 492 SP-470 Ac-F$r8AYWEAc3cL$NIeA-NH.sub.2 1427.79 714.90 714.86 493 SP-471 Ac-F$r8AYWEAibL$NIeA-NH.sub.2 1429.80 715.90 715.97 494 SP-472 Ac-F$r8AYWEAL$NleA-NH.sub.2 1415.79 708.90 708.94 495 SP-473 Ac-F$r8AYWENleL$NleA-NH.sub.2 1457.83 729.92 729.96 496 SP-474 Ac-F$r8AYWEAibL$Abu-NH.sub.2 1330.73 666.37 666.39 497 SP-475 Ac-F$r8AYWENleL$Abu-NH.sub.2 1358.76 680.38 680.39 498 SP-476 Ac-F$r8AYWEAL$Abu-NH.sub.2 1316.72 659.36 659.36 499 SP-477 Ac-LTF$r8AFWAQL$S-NH.sub.2 1515.85 758.93 759.12 500 SP-478 Ac-LTF$r8AWWAQL$S-NH.sub.2 1554.86 778.43 778.51 501 SP-479 Ac-LTF$r8AYWAQI$S-NH.sub.2 1531.84 766.92 766.96 502 SP-480 Ac-LTF$r8AYWAQNle$S-NH.sub.2 1531.84 766.92 766.96 503 SP-481 Ac-LTF$r8AYWAQL$SA-NH.sub.2 1602.88 802.44 802.48 504 SP-482 Ac-LTF$r8AWWAQL$A-NH.sub.2 1538.87 770.44 770.89 505 SP-483 Ac-LTF$r8AYWAQI$A-NH.sub.2 1515.85 758.93 759.42 506 SP-484 Ac-LTF$r8AYWAQNle$A-NH.sub.2 1515.85 758.93 759.42 507 SP-485 Ac-LTF$r8AYWAQL$AA-NH.sub.2 1586.89 794.45 794.94 508 SP-486 Ac-LTF$r8HWWAQL$S-NH.sub.2 1620.88 811.44 811.47 509 SP-487 Ac-LTF$r8HRWAQL$S-NH.sub.2 1590.90 796.45 796.52 510 SP-488 Ac-LTF$r8HKWAQL$S-NH.sub.2 1562.90 782.45 782.53 511 SP-489 Ac-LTF$r8HYWAQL$W-NH.sub.2 1696.91 849.46 849.5 512 SP-491 Ac-F$r8AYWAbuAL$A-NH.sub.2 1258.71 630.36 630.5 513 SP-492 Ac-F$r8AbuYWEAL$A-NH.sub.2 1316.72 659.36 659.51 514 SP-493 Ac-N1ePRF%r8NYWRLL%QN-NH.sub.2 1954.13 978.07 978.54 515 SP-494 Ac-TSF%r8HYWAQL%S-NH.sub.2 1573.83 787.92 787.98 516 SP-495 Ac-LTF%r8AYWAQL%S-NH.sub.2 1533.86 767.93 768 517 SP-496 Ac-HTF$r8HYWAQL$S-NH.sub.2 1621.84 811.92 811.96 518 SP-497 Ac-LHF$r8HYWAQL$S-NH.sub.2 1633.88 817.94 818.02 519 SP-498 Ac-LTF$r8HHWAQL$S-NH.sub.2 1571.86 786.93 786.94 520 SP-499 Ac-LTF$r8HYWHQL$S-NH.sub.2 1663.89 832.95 832.38 521 SP-500 Ac-LTF$r8HYWAHL$S-NH.sub.2 1606.87 804.44 804.48 522 SP-501 Ac-LTF$r8HYWAQL$H-NH.sub.2 1647.89 824.95 824.98 523 SP-502 Ac-LTF$r8HYWAQL$S-NHPr 1639.91 820.96 820.98 524 SP-503 Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 525 SP-504 Ac-LTF$r8HYWAQL$S-NHiBu 1653.93 827.97 828.02 526 SP-505 Ac-LTF$r8HYWAQL$S-NHBn 1687.91 844.96 844.44 527 SP-506 Ac-LTF$r8HYWAQL$S-NHPe 1700.92 851.46 851.99 528 SP-507 Ac-LTF$r8HYWAQL$S-NHChx 1679.94 840.97 841.04 529 SP-508 Ac-ETF$r8AYWAQL$S-NH.sub.2 1547.80 774.90 774.96 530 SP-509 Ac-STF$r8AYWAQL$S-NH.sub.2 1505.79 753.90 753.94 531 SP-510 Ac-LEF$r8AYWAQL$S-NH.sub.2 1559.84 780.92 781.25 532 SP-511 Ac-LSF$r8AYWAQL$S-NH.sub.2 1517.83 759.92 759.93 533 SP-512 Ac-LTF$r8EYWAQL$S-NH.sub.2 1589.85 795.93 795.97 534 SP-513 Ac-LTF$r8SYWAQL$S-NH.sub.2 1547.84 774.92 774.96 535 SP-514 Ac-LTF$r8AYWEQL$S-NH.sub.2 1589.85 795.93 795.9 536 SP-515 Ac-LTF$r8AYWAEL$S-NH.sub.2 1532.83 767.42 766.96

537 SP-516 Ac-LTF$r8AYWASL$S-NH.sub.2 1490.82 746.41 746.46 538 SP-517 Ac-LTF$r8AYWAQL$E-NH.sub.2 1573.85 787.93 787.98 539 SP-518 Ac-LTF2CN$r8HYWAQL$S-NH.sub.2 1622.86 812.43 812.47 540 SP-519 Ac-LTF3Cl$r8HYWAQL$S-NH.sub.2 1631.83 816.92 816.99 541 SP-520 Ac-LTDip$r8HYWAQL$S-NH.sub.2 1673.90 837.95 838.01 542 SP-521 Ac-LTF$r8HYWAQTle$S-NH.sub.2 1597.87 799.94 800.04 543 SP-522 Ac-F$r8AY6clWEAL$A-NH.sub.2 1336.66 669.33 1338.56 544 SP-523 Ac-F$r8AYdl6brWEAL$A-NH.sub.2 1380.61 691.31 692.2 545 SP-524 Ac-F$r8AYdl6fWEAL$A-NH.sub.2 1320.69 661.35 1321.61 546 SP-525 Ac-F$r8AYdl4mWEAL$A-NH.sub.2 1316.72 659.36 659.36 547 SP-526 Ac-F$r8AYdl5clWEAL$A-NH.sub.2 1336.66 669.33 669.35 548 SP-527 Ac-F$r8AYd17mWEAL$A-NH.sub.2 1316.72 659.36 659.36 549 SP-528 Ac-LTF%r8HYWAQL%A-NH.sub.2 1583.89 792.95 793.01 550 SP-529 Ac-LTF$r8HCouWAQL$S-NH.sub.2 1679.87 840.94 841.38 551 SP-530 Ac-LTFEHCouWAQLTS-NH.sub.2 1617.75 809.88 809.96 552 SP-531 Ac-LTA$r8HCouWAQL$S-NH.sub.2 1603.84 802.92 803.36 553 SP-532 Ac-F$r8AYWEAL$AbuA-NH.sub.2 1387.75 694.88 694.88 554 SP-533 Ac-F$r8AYWEAl$AA-NH.sub.2 1373.74 687.87 687.93 555 SP-534 Ac-F$r8AYWEANle$AA-NH.sub.2 1373.74 687.87 687.93 556 SP-535 Ac-F$r8AYWEAmlL$AA-NH.sub.2 1429.80 715.90 715.97 557 SP-536 Ac-F$r8AYWQAL$AA-NH.sub.2 1372.75 687.38 687.48 558 SP-537 Ac-F$r8AYWAAL$AA-NH.sub.2 1315.73 658.87 658.92 559 SP-538 Ac-F$r8AYWAbuAL$AA-NH.sub.2 1329.75 665.88 665.95 560 SP-539 Ac-F$r8AYWNleAL$AA-NH.sub.2 1357.78 679.89 679.94 561 SP-540 Ac-F$r8AbuYWEAL$AA-NH.sub.2 1387.75 694.88 694.96 562 SP-541 Ac-F$r8NleYWEAL$AA-NH.sub.2 1415.79 708.90 708.94 563 SP-542 Ac-F$r8FYWEAL$AA-NH.sub.2 1449.77 725.89 725.97 564 SP-543 Ac-LTF$r8HYWAQhL$S-NH.sub.2 1611.88 806.94 807 565 SP-544 Ac-LTF$r8HYWAQAdm$S-NH.sub.2 1675.91 838.96 839.04 566 SP-545 Ac-LTF$r8HYWAQIgl$S-NH.sub.2 1659.88 830.94 829.94 567 SP-546 Ac-F$r8AYWAQL$AA-NH.sub.2 1372.75 687.38 687.48 568 SP-547 Ac-LTF$r8ALWAQL$Q-NH.sub.2 1522.89 762.45 762.52 569 SP-548 Ac-F$r8AYWEAL$AA-NH.sub.2 1373.74 687.87 687.93 570 SP-549 Ac-F$r8AYWENleL$AA-NH.sub.2 1415.79 708.90 708.94 571 SP-550 Ac-F$r8AYWEAibL$Abu-NH.sub.2 1330.73 666.37 666.39 572 SP-551 Ac-F$r8AYWENleL$Abu-NH.sub.2 1358.76 680.38 680.38 573 SP-552 Ac-F$r8AYWEAL$Abu-NH.sub.2 1316.72 659.36 659.36 574 SP-553 Ac-F$r8AYWEAc3cL$AbuA-NH.sub.2 1399.75 700.88 700.95 575 SP-554 Ac-F$r8AYWEAc3cL$NleA-NH.sub.2 1427.79 714.90 715.01 576 SP-555 H-LTF$r8AYWAQL$S-NH.sub.2 1489.83 745.92 745.95 577 SP-556 mdPEG3-LTF$r8AYWAQL$S-NH.sub.2 1679.92 840.96 840.97 578 SP-557 mdPEG7-LTF$r8AYWAQL$S-NH.sub.2 1856.02 929.01 929.03 579 SP-558 Ac-F$r8ApmpEt6clWEAL$A-NH.sub.2 1470.71 736.36 788.17 580 SP-559 Ac-LTF3Cl$r8AYWAQL$S-NH.sub.2 1565.81 783.91 809.18 581 SP-560 Ac-LTF3Cl$r8HYWAQL$A-NH.sub.2 1615.83 808.92 875.24 582 SP-561 Ac-LTF3Cl$r8HYWWQL$S-NH.sub.2 1746.87 874.44 841.65 583 SP-562 Ac-LTF3Cl$r8AYWWQL$S-NH.sub.2 1680.85 841.43 824.63 584 SP-563 Ac-LTF$r8AYWWQL$S-NH.sub.2 1646.89 824.45 849.98 585 SP-564 Ac-LTF$r8HYWWQL$A-NH.sub.2 1696.91 849.46 816.67 586 SP-565 Ac-LTF$r8AYWWQL$A-NH.sub.2 1630.89 816.45 776.15 587 SP-566 Ac-LTF4F$r8AYWAQL$S-NH.sub.2 1549.83 775.92 776.15 588 SP-567 Ac-LTF2F$r8AYWAQL$S-NH.sub.2 1549.83 775.92 776.15 589 SP-568 Ac-LTF3F$r8AYWAQL$S-NH.sub.2 1549.83 775.92 785.12 590 SP-569 Ac-LTF34F2$r8AYWAQL$S-NH.sub.2 1567.83 784.92 785.12 591 SP-570 Ac-LTF35F2$r8AYWAQL$S-NH.sub.2 1567.83 784.92 1338.74 592 SP-571 Ac-F3Cl$r8AYWEAL$A-NH.sub.2 1336.66 669.33 705.28 593 SP-572 Ac-F3Cl$r8AYWEAL$AA-NH.sub.2 1407.70 704.85 680.11 594 SP-573 Ac-F$r8AY6clWEAL$AA-NH.sub.2 1407.70 704.85 736.83 595 SP-574 Ac-F$r8AY6clWEAL$-NH.sub.2 1265.63 633.82 784.1 596 SP-575 Ac-LTF$r8HYWAQLSt/S-NH.sub.2 16.03 9.02 826.98 597 SP-576 Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 598 SP-577 Ac-STF$r8AYWAQL$S-NH.sub.2 1505.79 753.90 753.94 599 SP-578 Ac-LTF$r8AYWAEL$S-NH.sub.2 1532.83 767.42 767.41 600 SP-579 Ac-LTF$r8AYWAQL$E-NH.sub.2 1573.85 787.93 787.98 601 SP-580 mdPEG3-LTF$r8AYWAQL$S-NH.sub.2 1679.92 840.96 840.97 602 SP-581 Ac-LTF$r8AYWAQhL$S-NH.sub.2 1545.86 773.93 774.31 603 SP-583 Ac-LTF$r8AYWAQCha$S-NH.sub.2 1571.88 786.94 787.3 604 SP-584 Ac-LTF$r8AYWAQChg$S-NH.sub.2 1557.86 779.93 780.4 605 SP-585 Ac-LTF$r8AYWAQCba$S-NH.sub.2 1543.84 772.92 780.13 606 SP-586 Ac-LTF$r8AYWAQF$S-NH.sub.2 1565.83 783.92 784.2 607 SP-587 Ac-LTF4F$r8HYWAQhL$S-NH.sub.2 1629.87 815.94 815.36 608 SP-588 Ac-LTF4F$r8HYWAQCha$S-NH.sub.2 1655.89 828.95 828.39 609 SP-589 Ac-LTF4F$r8HYWAQChg$S-NH.sub.2 1641.87 821.94 821.35 610 SP-590 Ac-LTF4F$r8HYWAQCba$S-NH.sub.2 1627.86 814.93 814.32 611 SP-591 Ac-LTF4F$r8AYWAQhL$S-NH.sub.2 1563.85 782.93 782.36 612 SP-592 Ac-LTF4F$r8AYWAQCha$S-NH.sub.2 1589.87 795.94 795.38 613 SP-593 Ac-LTF4F$r8AYWAQChg$S-NH.sub.2 1575.85 788.93 788.35 614 SP-594 Ac-LTF4F$r8AYWAQCba$S-NH.sub.2 1561.83 781.92 781.39 615 SP-595 Ac-LTF3Cl$r8AYWAQhL$S-NH.sub.2 1579.82 790.91 790.35 616 SP-596 Ac-LTF3Cl$r8AYWAQCha$S-NH.sub.2 1605.84 803.92 803.67 617 SP-597 Ac-LTF3Cl$r8AYWAQChg$S-NH.sub.2 1591.82 796.91 796.34 618 SP-598 Ac-LTF3Cl$r8AYWAQCba$S-NH.sub.2 1577.81 789.91 789.39 619 SP-599 Ac-LTF$r8AYWAQhF$S-NH.sub.2 1579.84 790.92 791.14 620 SP-600 Ac-LTF$r8AYWAQF3CF3$S-NH.sub.2 1633.82 817.91 818.15 621 SP-601 Ac-LTF$r8AYWAQF3Me$S-NH.sub.2 1581.86 791.93 791.32 622 SP-602 Ac-LTF$r8AYWAQlNa1$S-NH.sub.2 1615.84 808.92 809.18 623 SP-603 Ac-LTF$r8AYWAQBip$S-NH.sub.2 1641.86 821.93 822.13 624 SP-604 Ac-LTF$r8FYWAQL$A-NH.sub.2 1591.88 796.94 797.33 625 SP-605 Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.92 626 SP-606 Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.55 627 SP-607 Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.79 628 SP-608 Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.49 629 SP-610 Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.58 630 SP-611 Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 631 SP-612 Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 632 SP-613 Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 818.35 633 SP-614 Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.3 634 SP-615 Ac-LTF$r8AYWAQL$A-NHmdPeg4 1705.97 853.99 854.33 635 SP-616 Ac-F$r8AYdl4mWEAL$A-NH.sub.2 1316.72 659.36 659.44 636 SP-617 Ac-F$r8AYdl5clWEAL$A-NH.sub.2 1336.66 669.33 669.43 637 SP-618 Ac-LThF$r8AYWAQL$S-NH.sub.2 1545.86 773.93 774.11 638 SP-619 Ac-LT2Na1$r8AYWAQL$S-NH.sub.2 1581.86 791.93 792.43 639 SP-620 Ac-LTA$r8AYWAQL$S-NH.sub.2 1455.81 728.91 729.15 640 SP-621 Ac-LTF$r8AYWVQL$S-NH.sub.2 1559.88 780.94 781.24 641 SP-622 Ac-LTF$r8HYWAAL$A-NH.sub.2 1524.85 763.43 763.86 642 SP-623 Ac-LTF$r8VYWAQL$A-NH.sub.2 1543.88 772.94 773.37 643 SP-624 Ac-LTF$r8IYWAQL$S-NH.sub.2 1573.89 787.95 788.17 644 SP-625 Ac-FTF$r8VYWSQL$S-NH.sub.2 1609.85 805.93 806.22 645 SP-626 Ac-ITF$r8FYWAQL$S-NH.sub.2 1607.88 804.94 805.2 646 SP-627 Ac-2Na1TF$r8VYWSQL$S-NH.sub.2 1659.87 830.94 831.2 647 SP-628 Ac-ITF$r8LYWSQL$S-NH.sub.2 1589.89 795.95 796.13 648 SP-629 Ac-FTF$r8FYWAQL$S-NH.sub.2 1641.86 821.93 822.13 649 SP-630 Ac-WTF$r8VYWAQL$S-NH.sub.2 1632.87 817.44 817.69 650 SP-631 Ac-WTF$r8WYWAQL$S-NH.sub.2 1719.88 860.94 861.36 651 SP-632 Ac-VTF$r8AYWSQL$S-NH.sub.2 1533.82 767.91 768.19 652 SP-633 Ac-WTF$r8FYWSQL$S-NH.sub.2 1696.87 849.44 849.7 653 SP-634 Ac-FTF$r8IYWAQL$S-NH.sub.2 1607.88 804.94 805.2 654 SP-635 Ac-WTF$r8VYWSQL$S-NH.sub.2 1648.87 825.44 824.8 655 SP-636 Ac-FTF$r8LYWSQL$S-NH.sub.2 1623.87 812.94 812.8 656 SP-637 Ac-YTF$r8FYWSQL$S-NH.sub.2 1673.85 837.93 837.8 657 SP-638 Ac-LTF$r8AY6clWEAL$A-NH.sub.2 1550.79 776.40 776.14 658 SP-639 Ac-LTF$r8AY6clWSQL$S-NH.sub.2 1581.80 791.90 791.68 659 SP-640 Ac-F$r8AY6clWSAL$A-NH.sub.2 1294.65 648.33 647.67 660 SP-641 Ac-F$r8AY6clWQAL$AA-NH.sub.2 1406.72 704.36 703.84 661 SP-642 Ac-LHF$r8AYWAQL$S-NH.sub.2 1567.86 784.93 785.21

662 SP-643 Ac-LTF$r8AYWAQL$S-NH.sub.2 1531.84 766.92 767.17 663 SP-644 Ac-LTF$r8AHWAQL$S-NH.sub.2 1505.84 753.92 754.13 664 SP-645 Ac-LTF$r8AYWAHL$S-NH.sub.2 1540.84 771.42 771.61 665 SP-646 Ac-LTF$r8AYWAQL$H-NH.sub.2 1581.87 791.94 792.15 666 SP-647 H-LTF$r8AYWAQL$A-NH.sub.2 1473.84 737.92 737.29 667 SP-648 Ac-HHF$r8AYWAQL$S-NH.sub.2 1591.83 796.92 797.35 668 SP-649 Ac-aAibWTF$r8VYWSQL$S-NH.sub.2 1804.96 903.48 903.64 669 SP-650 Ac-AibWTF$r8HYWAQL$S-NH.sub.2 1755.91 878.96 879.4 670 SP-651 Ac-AibAWTF$r8HYWAQL$S-NH.sub.2 1826.95 914.48 914.7 671 SP-652 Ac-fWTF$r8HYWAQL$S-NH.sub.2 1817.93 909.97 910.1 672 SP-653 Ac-AibWWTF$r8HYWAQL$S-NH.sub.2 1941.99 972.00 972.2 673 SP-654 Ac-WTF$r8LYWSQL$S-NH.sub.2 1662.88 832.44 832.8 674 SP-655 Ac-WTF$r8NleYWSQL$S-NH.sub.2 1662.88 832.44 832.6 675 SP-656 Ac-LTF$r8AYWSQL$a-NH.sub.2 1531.84 766.92 767.2 676 SP-657 Ac-LTF$r8EYWARL$A-NH.sub.2 1601.90 801.95 802.1 677 SP-658 Ac-LTF$r8EYWAHL$A-NH.sub.2 1582.86 792.43 792.6 678 SP-659 Ac-aTF$r8AYWAQL$S-NH.sub.2 1489.80 745.90 746.08 679 SP-660 Ac-AibTF$r8AYWAQL$S-NH.sub.2 1503.81 752.91 753.11 680 SP-661 Ac-AmfTF$r8AYWAQL$S-NH.sub.2 1579.84 790.92 791.14 681 SP-662 Ac-AmwTF$r8AYWAQL$S-NH.sub.2 1618.86 810.43 810.66 682 SP-663 Ac-NmLTF$r8AYWAQL$S-NH.sub.2 1545.86 773.93 774.11 683 SP-664 Ac-LNmTF$r8AYWAQL$S-NH.sub.2 1545.86 773.93 774.11 684 SP-665 Ac-LSarF$r8AYWAQL$S-NH.sub.2 1501.83 751.92 752.18 685 SP-667 Ac-LGF$r8AYWAQL$S-NH.sub.2 1487.82 744.91 745.15 686 SP-668 Ac-LTNmF$r8AYWAQL$S-NH.sub.2 1545.86 773.93 774.2 687 SP-669 Ac-TF$r8AYWAQL$S-NH.sub.2 1418.76 710.38 710.64 688 SP-670 Ac-ETF$r8AYWAQL$A-NH.sub.2 1531.81 766.91 767.2 689 SP-671 Ac-LTF$r8EYWAQL$A-NH.sub.2 1573.85 787.93 788.1 690 SP-672 Ac-LT2Nal$r8AYWSQL$S-NH.sub.2 1597.85 799.93 800.4 691 SP-673 Ac-LTF$r8AYWAAL$S-NH.sub.2 1474.82 738.41 738.68 692 SP-674 Ac-LTF$r8AYWAQhCha$S-NH.sub.2 1585.89 793.95 794.19 693 SP-675 Ac-LTF$r8AYWAQChg$S-NH.sub.2 1557.86 779.93 780.97 694 SP-676 Ac-LTF$r8AYWAQCba$S-NH.sub.2 1543.84 772.92 773.19 695 SP-677 Ac-LTF$r8AYWAQF3CF3$S-NH.sub.2 1633.82 817.91 818.15 696 SP-678 Ac-LTF$r8AYWAQlNal$S-NH.sub.2 1615.84 808.92 809.18 697 SP-679 Ac-LTF$r8AYWAQBip$S-NH.sub.2 1641.86 821.93 822.32 698 SP-680 Ac-LT2Na1$r8AYWAQL$S-NH.sub.2 1581.86 791.93 792.15 699 SP-681 Ac-LTF$r8AYWVQL$S-NH.sub.2 1559.88 780.94 781.62 700 SP-682 Ac-LTF$r8AWWAQL$S-NH.sub.2 1554.86 778.43 778.65 701 SP-683 Ac-FTF$r8VYWSQL$S-NH.sub.2 1609.85 805.93 806.12 702 SP-684 Ac-ITF$r8FYWAQL$S-NH.sub.2 1607.88 804.94 805.2 703 SP-685 Ac-ITF$r8LYWSQL$S-NH.sub.2 1589.89 795.95 796.22 704 SP-686 Ac-FTF$r8FYWAQL$S-NH.sub.2 1641.86 821.93 822.41 705 SP-687 Ac-VTF$r8AYWSQL$S-NH.sub.2 1533.82 767.91 768.19 706 SP-688 Ac-LTF$r8AHWAQL$S-NH.sub.2 1505.84 753.92 754.31 707 SP-689 Ac-LTF$r8AYWAQL$H-NH.sub.2 1581.87 791.94 791.94 708 SP-690 Ac-LTF$r8AYWAHL$S-NH.sub.2 1540.84 771.42 771.61 709 SP-691 Ac-aAibWTF$r8VYWSQL$S-NH.sub.2 1804.96 903.48 903.9 710 SP-692 Ac-AibWTF$r8HYWAQL$S-NH.sub.2 1755.91 878.96 879.5 711 SP-693 Ac-AibAWTF$r8HYWAQL$S-NH.sub.2 1826.95 914.48 914.7 712 SP-694 Ac-fWTF$r8HYWAQL$S-NH.sub.2 1817.93 909.97 910.2 713 SP-695 Ac-AibWWTF$r8HYWAQL$S-NH.sub.2 1941.99 972.00 972.7 714 SP-696 Ac-WTF$r8LYWSQL$S-NH.sub.2 1662.88 832.44 832.7 715 SP-697 Ac-WTF$r8NleYWSQL$S-NH.sub.2 1662.88 832.44 832.7 716 SP-698 Ac-LTF$r8AYWSQL$a-NH.sub.2 1531.84 766.92 767.2 717 SP-699 Ac-LTF$r8EYWARL$A-NH.sub.2 1601.90 801.95 802.2 718 SP-700 Ac-LTF$r8EYWAHL$A-NH.sub.2 1582.86 792.43 792.6 719 SP-701 Ac-aTF$r8AYWAQL$S-NH.sub.2 1489.80 745.90 746.1 720 SP-702 Ac-AibTF$r8AYWAQL$S-NH.sub.2 1503.81 752.91 753.2 721 SP-703 Ac-AmfTF$r8AYWAQL$S-NH.sub.2 1579.84 790.92 791.2 722 SP-704 Ac-AmwTF$r8AYWAQL$S-NH.sub.2 1618.86 810.43 810.7 723 SP-705 Ac-NmLTF$r8AYWAQL$S-NH.sub.2 1545.86 773.93 774.1 724 SP-706 Ac-LNmTF$r8AYWAQL$S-NH.sub.2 1545.86 773.93 774.4 725 SP-707 Ac-LSarF$r8AYWAQL$S-NH.sub.2 1501.83 751.92 752.1 726 SP-708 Ac-TF$r8AYWAQL$S-NH.sub.2 1418.76 710.38 710.8 727 SP-709 Ac-ETF$r8AYWAQL$A-NH.sub.2 1531.81 766.91 767.4 728 SP-710 Ac-LTF$r8EYWAQL$A-NH.sub.2 1573.85 787.93 788.2 729 SP-711 Ac-WTF$r8VYWSQL$S-NH.sub.2 1648.87 825.44 825.2 730 SP-713 Ac-YTF$r8FYWSQL$S-NH.sub.2 1673.85 837.93 837.3 731 SP-714 Ac-F$r8AY6cIWSAL$A-NH.sub.2 1294.65 648.33 647.74 732 SP-715 Ac-ETF$r8EYWVQL$S-NH.sub.2 1633.84 817.92 817.36 733 SP-716 Ac-ETF$r8EHWAQL$A-NH.sub.2 1563.81 782.91 782.36 734 SP-717 Ac-ITF$r8EYWAQL$S-NH.sub.2 1589.85 795.93 795.38 735 SP-718 Ac-ITF$r8EHWVQL$A-NH.sub.2 1575.88 788.94 788.42 736 SP-719 Ac-ITF$r8EHWAQL$S-NH.sub.2 1563.85 782.93 782.43 737 SP-720 Ac-LTF4F$r8AYWAQCba$S-NH.sub.2 1561.83 781.92 781.32 738 SP-721 Ac-LTF3Cl$r8AYWAQhL$S-NH.sub.2 1579.82 790.91 790.64 739 SP-722 Ac-LTF3Cl$r8AYWAQCha$S-NH.sub.2 1605.84 803.92 803.37 740 SP-723 Ac-LTF3Cl$r8AYWAQChg$S-NH.sub.2 1591.82 796.91 796.27 741 SP-724 Ac-LTF3Cl$r8AYWAQCba$S-NH.sub.2 1577.81 789.91 789.83 742 SP-725 Ac-LTF$r8AY6clWSQL$S-NH.sub.2 1581.80 791.90 791.75 743 SP-726 Ac-LTF4F$r8HYWAQhL$S-NH.sub.2 1629.87 815.94 815.36 744 SP-727 Ac-LTF4F$r8HYWAQCba$S-NH.sub.2 1627.86 814.93 814.32 745 SP-728 Ac-LTF4F$r8AYWAQhL$S-NH.sub.2 1563.85 782.93 782.36 746 SP-729 Ac-LTF4F$r8AYWAQChg$S-NH.sub.2 1575.85 788.93 788.35 747 SP-730 Ac-ETF$r8EYWVAL$S-NH.sub.2 1576.82 789.41 788.79 748 SP-731 Ac-ETF$r8EHWAAL$A-NH.sub.2 1506.79 754.40 754.8 749 SP-732 Ac-ITF$r8EYWAAL$S-NH.sub.2 1532.83 767.42 767.75 750 SP-733 Ac-ITF$r8EHWVAL$A-NH.sub.2 1518.86 760.43 760.81 751 SP-734 Ac-ITF$r8EHWAAL$S-NH.sub.2 1506.82 754.41 754.8 752 SP-735 Pam-LTF$r8EYWAQL$S-NH.sub.2 1786.07 894.04 894.48 753 SP-736 Pam-ETF$r8EYWAQL$S-NH.sub.2 1802.03 902.02 902.34 754 SP-737 Ac-LTF$r8AYWLQL$S-NH.sub.2 1573.89 787.95 787.39 755 SP-738 Ac-LTF$r8EYWLQL$S-NH.sub.2 1631.90 816.95 817.33 756 SP-739 Ac-LTF$r8EHWLQL$S-NH.sub.2 1605.89 803.95 804.29 757 SP-740 Ac-LTF$r8VYWAQL$S-NH.sub.2 1559.88 780.94 781.34 758 SP-741 Ac-LTF$r8AYWSQL$S-NH.sub.2 1547.84 774.92 775.33 759 SP-742 Ac-ETF$r8AYWAQL$S-NH.sub.2 1547.80 774.90 775.7 760 SP-743 Ac-LTF$r8EYWAQL$S-NH.sub.2 1589.85 795.93 796.33 761 SP-744 Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.37 762 SP-745 Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.27 763 SP-746 Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.42 764 SP-747 Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.67 765 SP-748 Ac-LTF$r8HYWAQL$S-NHnBu 1653.93 827.97 828.24 766 SP-749 Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.31 767 SP-750 Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 768 SP-751 Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 769 SP-752 Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 855.35 770 SP-753 Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.58 771 SP-754 Ac-LTF$r5AYWAAL$s8S-NH.sub.2 1474.82 738.41 738.79 772 SP-755 Ac-LTF$r8AYWCouQL$S-NH.sub.2 1705.88 853.94 854.61 773 SP-756 Ac-LTF$r8CouYWAQL$S-NH.sub.2 1705.88 853.94 854.7 774 SP-757 Ac-CouTF$r8AYWAQL$S-NH.sub.2 1663.83 832.92 833.33 775 SP-758 H-LTF$r8AYWAQL$A-NH.sub.2 1473.84 737.92 737.29 776 SP-759 Ac-HHF$r8AYWAQL$S-NH.sub.2 1591.83 796.92 797.72 777 SP-760 Ac-LT2Nal$r8AYWSQL$S-NH.sub.2 1597.85 799.93 800.68 778 SP-761 Ac-LTF$r8HCouWAQL$S-NH.sub.2 1679.87 840.94 841.38 779 SP-762 Ac-LTF$r8AYWCou2QL$S-NH.sub.2 1789.94 895.97 896.51 780 SP-763 Ac-LTF$r8Cou2YWAQL$S-NH.sub.2 1789.94 895.97 896.5 781 SP-764 Ac-Cou2TF$r8AYWAQL$S-NH.sub.2 1747.90 874.95 875.42 782 SP-765 Ac-LTF$r8ACou2WAQL$S-NH.sub.2 1697.92 849.96 850.82 783 SP-766 Dmaac-LTF$r8AYWAQL$S-NH.sub.2 1574.89 788.45 788.82 784 SP-767 Hexac-LTF$r8AYWAQL$S-NH.sub.2 1587.91 794.96 795.11 785 SP-768 Napac-LTF$r8AYWAQL$S-NH.sub.2 1657.89 829.95 830.36 786 SP-769 Pam-LTF$r8AYWAQL$S-NH.sub.2 1728.06 865.03 865.45 787 SP-770 Ac-LT2Nal$r8HYAAQL$S-NH.sub.2 1532.84 767.42 767.61

788 SP-771 Ac-LT2Nal$/r8HYWAQL$/S-NH.sub.2 1675.91 838.96 839.1 789 SP-772 Ac-LT2Nal$r8HYFAQL$S-NH.sub.2 1608.87 805.44 805.9 790 SP-773 Ac-LT2Nal$r8HWAAQL$S-NH.sub.2 1555.86 778.93 779.08 791 SP-774 Ac-LT2Nal$r8HYAWQL$S-NH.sub.2 1647.88 824.94 825.04 792 SP-775 Ac-LT2Nal$r8HYAAQW$S-NH.sub.2 1605.83 803.92 804.05 793 SP-776 Ac-LTW$r8HYWAQL$S-NH.sub.2 1636.88 819.44 819.95 794 SP-777 Ac-LT1Nal$r8HYWAQL$S-NH.sub.2 1647.88 824.94 825.41

[0219] In the sequences shown above and elsewhere, the following abbreviations are used: "Nle" represents norleucine, "Aib" represents 2-aminoisobutyric acid, "Ac" represents acetyl, and "Pr" represents propionyl Amino acids represented as "$" are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond Amino acids represented as "$r5" are alpha-Me R5-pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond Amino acids represented as "$s8" are alpha-Me S8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond Amino acids represented as "$r8" are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. "Ahx" represents an aminocyclohexyl linker. The crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid Amino acids represented as "$/" are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker Amino acids represented as "$/r5" are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker Amino acids represented as "$/s8" are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/r8" are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker Amino acids represented as "Amw" are alpha-Me tryptophan amino acids Amino acids represented as "Aml" are alpha-Me leucine amino acids Amino acids represented as "2ff" are 2-fluoro-phenylalanine amino acids. Amino acids represented as "3ff" are 3-fluoro-phenylalanine amino acids. Amino acids represented as "St" are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated Amino acids represented as "St//" are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked Amino acids represented as "% St" are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks.

[0220] For example, the compounds represented as SP-72, SP-56 and SP-138 have the following structures (SEQ ID NOS 109, 93 and 173, respectively, in order of appearance):

##STR00030##

[0221] For example, additional compounds have the following structures (SEQ ID NOS 83, 177, 303, 163, 225, 273, 366, 217, 214, 387 and 184, respectively, in order of appearance):

##STR00031## ##STR00032## ##STR00033## ##STR00034##

Example 3

Competition Binding ELISA (HDM2 & HDMX)

[0222] p53-His6 ("His6" disclosed as SEQ ID NO: 796) protein (30 nM/well) is coated overnight at room temperature in the wells of a 96-well Immulon plates. On the day of the experiment, plates are washed with 1.times.PBS-Tween 20 (0.05%) using an automated ELISA plate washer, blocked with ELISA Micro well Blocking for 30 minutes at room temperature; excess blocking agent is washed off by washing plates with 1.times.PBS-Tween 20 (0.05%). Peptides are diluted from 10 mM DMSO stocks to 500 .mu.M working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The peptides are added to wells at 2.times. desired concentrations in 50 .mu.l volumes, followed by addition of diluted GST-HDM2 or GST-HMDX protein (final concentration: 10 nM). Samples are incubated at room temperature for 2 h, plates are washed with PBS-Tween 20 (0.05%) prior to adding 100 .mu.l of HRP-conjugated anti-GST antibody [Hypromatrix, INC] diluted to 0.5 .mu.g/ml in HRP-stabilizing buffer. Post 30 min incubation with detection antibody, plates are washed and incubated with 100 .mu.l per well of TMB-E Substrate solution up to 30 minutes; reactions are stopped using 1M HCL and absorbance measured at 450 nm on micro plate reader. Data is analyzed using Graph Pad PRISM software.

Example 4

SJSA-1 Cell Viability Assay

[0223] SJSA1 cells are seeded at the density of 5000 cells/100 .mu.l/well in 96-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 90 .mu.L of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 .mu.M working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final concentration range .mu.M will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0 .mu.M in 100 .mu.L final volume per well for peptides. Final highest DMSO concentration is 0.5% and will be used as the negative control. Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides 10 .mu.l of 10.times. desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 20-24 h at 37.degree. C. in humidified 5% CO2 atmosphere. Post-incubation period, cell viability is measured using Promega Cell Titer-Glo reagents according to manufacturer' instructions.

Example 5

SJSA-1 p21 Up-Regulation Assay

[0224] SJSA1 cells are seeded at the density of 0.8 million cells/2 ml/well in 6-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 1350 .mu.L of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 .mu.M working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as positive control. Nutlin is diluted using the same dilution scheme as peptides 150 .mu.l of 10.times. desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 18-20 h at 37.degree. C. in humidified 5% CO2 atmosphere. Post-incubation period, cells are harvested, washed with 1.times.PBS (without Ca++/Mg++) and lysed in 1.times. Cell lysis buffer (Cell Signaling technologies 10.times. buffer diluted to 1.times. and supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min. Lysates are centrifuged at 13000 rpm speed in a microfuge at 40 C for 8 min; clear supernatants are collected and stored at -800 C till further use. Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. 25 .mu.g of the total protein is used for p21 detection ELISA assay. Each condition is set in triplicate for ELISA plate. The ELISA assay protocol is followed as per the manufacturer's instructions. 25 .mu.g total protein used for each well, and each well is set up in triplicate. Data is analyzed using Graph Pad PRISM software.

Example 6

p53 GRIP Assay

[0225] Thermo Scientific* BioImage p53-Hdm2 Redistribution Assay monitors the protein interaction with Hdm2 and cellular translocation of GFP-tagged p53 in response to drug compounds or other stimuli. Recombinant CHO-hIR cells stably express human p53(1-312) fused to the C-terminus of enhanced green fluorescent protein (EGFP) and PDE4A4-Hdm2(1-124), a fusion protein between PDE4A4 and Hdm2(1-124). They provide a ready-to-use assay system for measuring the effects of experimental conditions on the interaction of p53 and Hdm2. Imaging and analysis is performed with a HCS platform.

[0226] CHO-hIR cells are regularly maintained in Ham's F12 media supplemented with 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin and 10% FBS. Cells seeded into 96-well plates at the density of 7000 cells/100 .mu.l per well 18-24 hours prior to running the assay using culture media. The next day, media is refreshed and PD177 is added to cells to the final concentration of 3 .mu.M to activate foci formation. Control wells are kept without PD-177 solution. 24 h post stimulation with PD177, cells are washed once with Opti-MEM Media and 50 .mu.L of the Opti-MEM Media supplemented with PD-177(6 .mu.M) is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 .mu.M working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides. 50 .mu.l of 2.times. desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 6 h at 37.degree. C. in humidified 5% CO2 atmosphere. Post-incubation period, cells are fixed by gently aspirating out the media and adding 150 .mu.l of fixing solution per well for 20 minutes at room temperature. Fixed cells are washed 4 times with 200 .mu.l PBS per well each time. At the end of last wash, 100 .mu.l of 1 .mu.M Hoechst staining solution is added. Sealed plates incubated for at least 30 min in dark, washed with PBS to remove excess stain and PBS is added to each well. Plates can be stored at 4.degree. C. in dark up to 3 days. The translocation of p53/HDM2 is imaged using Molecular translocation module on Cellomics Arrayscan instrument using 10.times. objective, XF-100 filter sets for Hoechst and GFP. The output parameters was Mean--CircRINGAveIntenRatio (the ratio of average fluorescence intensities of nucleus and cytoplasm, (well average)). The minimally acceptable number of cells per well used for image analysis was set to 500 cells.

Example 7

Direct Binding Assay hDM2 with Fluorescence Polarization (FP)

[0227] The assay was performed according to the following general protocol:

1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 .mu.M working stock solution. 2. Add 30 .mu.l of 10 .mu.M of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices). 3. Fill in 30 .mu.l of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12. 4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM concentration at the last dilution point. 5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water (dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 6. Add 10 .mu.l of 10 nM of FAM labeled peptide into each well and incubate, and read at different time points. Kd with 5-FAM-BaLTFEHYWAQLTS-NH.sub.2 (SEQ ID NO: 795) is .about.13.38 nM.

Example 8

Competitive Fluorescence Polarization Assay for hDM2

[0228] The assay was performed according to the following general protocol:

1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 84 nM (2.times.) working stock solution. 2. Add 20 .mu.l of 84 nM (2.times.) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices) 3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water (dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 4. Make unlabeled peptide dose plate with FP buffer starting with 1 .mu.M (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 .mu.M with H.sub.2O (dilution 1:10) and then dilute with FP buffer from 500 .mu.M to 20 .mu.M (dilution 1:25). Making 5 fold serial dilutions from 4 .mu.M (4.times.) for 6 points. 5. Transfer 10 .mu.l of serial diluted unlabeled peptides to each well which is filled with 20 .mu.l of 84 nM of protein. 6. Add 10 .mu.l of 10 nM (4.times.) of FAM labeled peptide into each well and incubate for 3 hr to read. [0229] Results of Examples 7 and 8 are provided in HDM2 data in FIGS. 7A-D.

Example 9

Direct Binding Assay hDMX with Fluorescence Polarization (FP)

[0230] The assay was performed according to the following general protocol:

1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 .mu.M working stock solution. 2. Add 30 .mu.l of 10 .mu.M of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices). 3. Fill in 30 .mu.l of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12. 4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM concentration at the last dilution point. 5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water (dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 6. Add 10 .mu.l of 10 nM of FAM labeled peptide into each well and incubate, and read at different time points. Kd with 5-FAM-BaLTFEHYWAQLTS-NH.sub.2 (SEQ ID NO: 795) is .about.51 nM.

Example 10

Competitive Fluorescence Polarization Assay for hDMX

[0231] The assay was performed according to the following general protocol:

1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 300 nM (2.times.) working stock solution. 2. Add 20 .mu.l of 300 nM (2.times.) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices) 3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water (dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 4. Make unlabeled peptide dose plate with FP buffer starting with 5 .mu.M (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme. 5. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 .mu.M with H.sub.2O (dilution 1:10) and then dilute with FP buffer from 500 .mu.M to 20 .mu.M (dilution 1:25). Making 5 fold serial dilutions from 20 .mu.M (4.times.) for 6 points. 6. Transfer 10 .mu.l of serial diluted unlabeled peptides to each well which is filled with 20 .mu.l of 300 nM of protein. 7. Add 10 .mu.l of 10 nM (4.times.) of FAM labeled peptide into each well and incubate for 3 hr to read. [0232] Results of Examples 9 and 10 are provided in HDMX data in FIGS. 7A-D.

Example 11

Cell Viability Assay

[0233] The assay was performed according to the following general protocol:

[0234] Cell Plating: Trypsinize, count and seed cells at the pre-determined densities in 96-well plates a day prior to assay. Following cell densities are used for each cell line in use:

[0235] SJSA-1: 7500 cells/well

[0236] RKO: 5000 cells/well

[0237] RKO-E6: 5000 cells/well

[0238] HCT-116: 5000 cells/well

[0239] SW-480: 2000 cells/well

[0240] MCF-7: 5000 cells/well

[0241] On the day of study, replace media with fresh media with 11% FBS (assay media) at room temperature. Add 180 .mu.L of the assay media per well. Control wells with no cells, receive 200 .mu.l media.

[0242] Peptide dilution: all dilutions are made at room temperature and added to cells at room temperature. [0243] Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water. This gives range of 10.times. working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells. [0244] Thus the working stocks concentration range .mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at each dilution step using multichannel. [0245] Row H has controls. H1-H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells. [0246] Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.

[0247] Addition of working stocks to cells: [0248] Add 20 .mu.l of 10.times. desired concentration to appropriate well to achieve the final concentrations in total 200 .mu.l volume in well. (20 .mu.l of 300 .mu.M peptide+180 .mu.l of cells in media=30 .mu.M final concentration in 200 .mu.l volume in wells). Mix gently a few times using pipette. Thus final concentration range used will be 30, 10, 3, 1, 0.3, 0.1, 0.03 & 0 .mu.M (for potent peptides further dilutions are included). [0249] Controls include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS. [0250] Incubate for 72 hours at 37.degree. C. in humidified 5% CO.sub.2 atmosphere. [0251] The viability of cells is determined using MTT reagent from Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116 cells is determined on day 3, MCF-7 cells on day 5 and SW-480 cells on day 6. At the end of designated incubation time, allow the plates to come to room temperature. Remove 80 .mu.l of assay media from each well. Add 15 .mu.l of thawed MTT reagent to each well. [0252] Allow plate to incubate for 2 h at 37.degree. C. in humidified 5% CO.sub.2 atmosphere and add 100 .mu.l solubilization reagent as per manufacturer's protocol. Incubate with agitation for 1 h at room temperature and read on Synergy Biotek multiplate reader for absorbance at 570 nM. [0253] Analyze the cell viability against the DMSO controls using GraphPad PRISM analysis tools.

[0254] Reagents: [0255] Invitrogen cell culture Media [0256] i. Falcon 96-well clear cell culture treated plates (Nunc 353072) [0257] DMSO (Sigma D 2650) [0258] RPMI 1640 (Invitrogen 72400) [0259] MTT (Promega G4000)

[0260] Instruments:

[0261] Multiplate Reader for Absorbance readout (Synergy 2)

[0262] Results of Example 11 are provided in SJSA-1 EC50 data in FIGS. 7A-D.

Example 12

P21 ELISA Assay

[0263] The assay was performed according to the following general protocol:

[0264] Cell Platimg: [0265] Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100 .mu.l/well in 96-well plates a day prior to assay. [0266] On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 90 .mu.L of the assay media per well. Control wells with no cells, receive 100 .mu.l media.

[0267] Peptide Dilution: [0268] Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10.times. working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells. [0269] Thus the working stocks concentration range .mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at each dilution step using multichannel. [0270] Row H has controls. H1-H3 will receive 10 ul of assay media. H4-H9 will receive 10 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells. [0271] Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control.

[0272] Nutlin was diluted using the same dilution scheme as peptides.

[0273] Addition of Working Stocks to Cells: [0274] Add 10 .mu.l of 10.times. desired concentration to appropriate well to achieve the final concentrations in total 100 .mu.l volume in well. (10 .mu.l of 300 .mu.M peptide+90 .mu.l of cells in media=30 .mu.M final concentration in 100 .mu.l volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3& 0 .mu.M. [0275] Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS. [0276] 20 h-post incubation, aspirate the media; wash cells with 1.times.PBS (without Ca.sup.++/Mg.sup.++) and lyse in 60 .mu.l of 1.times. Cell lysis buffer (Cell Signaling technologies 10.times. buffer diluted to 1.times. and supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min. [0277] Centrifuge plates in at 5000 rpm speed in at 4.degree. C. for 8 min; collect clear supernatants and freeze at -80.degree. C. till further use.

[0278] Protein Estimation: [0279] Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. Typically about 6-7 .mu.g protein is expected per well. [0280] Use 50 .mu.L of the lysate per well to set up p21 ELISA.

[0281] Human Total p21 ELISA:

[0282] The ELISA assay protocol is followed as per the manufacturer's instructions. 50 .mu.l lysate is used for each well, and each well is set up in triplicate.

[0283] Reagents: [0284] Cell-Based Assay (-)-Nutlin-3 (10 mM): Cayman Chemicals, catalog #600034 [0285] OptiMEM, Invitrogen catalog #51985 [0286] Cell Signaling Lysis Buffer (10.times.), Cell signaling technology, Catalog #9803 [0287] Protease inhibitor Cocktail tablets (mini), Roche Chemicals, catalog #04693124001 [0288] Phosphatase inhibitor Cocktail tablet, Roche Chemicals, catalog #04906837001 [0289] Human total p21 ELISA kit, R&D Systems, DYC1047-5 [0290] STOP Solution (1M HCL), Cell Signaling Technologies, Catalog #7002

[0291] Instruments: Micro centrifuge-Eppendorf 5415D and Multiplate Reader for Absorbance readout (Synergy 2)

[0292] Results of Example 12 are provided in p21 data in FIGS. 7A-D.

Example 13

Caspase 3 Detection Assay

[0293] The assay was performed according to the following general protocol:

[0294] Cell Platimg:

[0295] Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100 .mu.l/well in 96-well plates a day prior to assay. On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 180 .mu.L of the assay media per well. Control wells with no cells, receive 200 .mu.l media.

[0296] Peptide Dilution: [0297] Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10.times. working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells. [0298] Thus the working stocks concentration range .mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at each dilution step using multichannel. Add 20 ul of 10.times. working stocks to appropriate wells. [0299] Row H has controls. H1-H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells. [0300] Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control.

[0301] Nutlin was diluted using the same dilution scheme as peptides.

[0302] Addition of Working Stocks to Cells: [0303] Add 10 .mu.l of 10.times. desired concentration to appropriate well to achieve the final concentrations in total 100 .mu.l volume in well. (10 .mu.l of 300 .mu.M peptide+90 .mu.l of cells in media=30 .mu.M final concentration in 100 .mu.l volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3& 0 .mu.M. [0304] Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS. [0305] 48 h-post incubation, aspirate 80 .mu.l media from each well; add 100 .mu.l Caspase3/7Glo assay reagent (Promega Caspase 3/7 glo assay system, G8092) per well, incubate with gentle shaking for 1 h at room temperature. [0306] read on Synergy Biotek multiplate reader for luminescence. [0307] Data is analyzed as Caspase 3 activation over DMSO-treated cells. [0308] Results of Example 13 are provided in p21 data in FIGS. 7A-D.

Sequence CWU 0 SQTB SEQUENCE LISTING The patent application contains a lengthy "Sequence Listing" section. A copy of the "Sequence Listing" is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140378390A1). An electronic copy of the "Sequence Listing" will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

0 SQTB SEQUENCE LISTING The patent application contains a lengthy "Sequence Listing" section. A copy of the "Sequence Listing" is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140378390A1). An electronic copy of the "Sequence Listing" will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Claims

1. A peptidomimetic macrocycle comprising an amino acid sequence which is at least 60% identical to the amino acid sequence of: SEQ ID NO. 254, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 374, SEQ ID NO. 375, SEQ ID NO. 507, SEQ ID NO. 533, SEQ ID NO. 587, SEQ ID NO. 605, SEQ ID NO. 624, SEQ ID NO. 642, SEQ ID NO. 699, SEQ ID NO. 702, SEQ ID NO. 703, SEQ ID NO. 704, SEQ ID NO. 714, SEQ ID NO. 734, or SEQ ID NO.

2. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least 80% identical to the amino acid sequence of: SEQ ID NO. 254, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 374, SEQ ID NO. 375, SEQ ID NO. 507, SEQ ID NO. 533, SEQ ID NO. 587, SEQ ID NO. 605, SEQ ID NO. 624, SEQ ID NO. 642, SEQ ID NO. 699, SEQ ID NO. 702, SEQ ID NO. 703, SEQ ID NO. 704, SEQ ID NO. 714, SEQ ID NO. 734, or SEQ ID NO. 742.

3. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of: SEQ ID NO. 254, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 374, SEQ ID NO. 375, SEQ ID NO. 507, SEQ ID NO. 533, SEQ ID NO. 587, SEQ ID NO. 605, SEQ ID NO. 624, SEQ ID NO. 642, SEQ ID NO. 699, SEQ ID NO. 702, SEQ ID NO. 703, SEQ ID NO. 704, SEQ ID NO. 714, SEQ ID NO. 734, or SEQ ID NO. 742.

4. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of: SEQ ID NO. 254, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 374, SEQ ID NO. 375, SEQ ID NO. 507, SEQ ID NO. 533, SEQ ID NO. 587, SEQ ID NO. 605, SEQ ID NO. 624, SEQ ID NO. 642, SEQ ID NO. 699, SEQ ID NO. 702, SEQ ID NO. 703, SEQ ID NO. 704, SEQ ID NO. 714, SEQ ID NO. 734, or SEQ ID NO. 742.

5. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence which is: SEQ ID NO. 254, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 374, SEQ ID NO. 375, SEQ ID NO. 507, SEQ ID NO. 533, SEQ ID NO. 587, SEQ ID NO. 605, SEQ ID NO. 624, SEQ ID NO. 642, SEQ ID NO. 699, SEQ ID NO. 702, SEQ ID NO. 703, SEQ ID NO. 704, SEQ ID NO. 714, SEQ ID NO. 734, or SEQ ID NO. 742.

6. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises a helix.

7. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an .alpha.-helix.

8. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises an .alpha.,.alpha.-disubstituted amino acid.

9. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises a crosslinker linking the .alpha.-positions of at least two amino acids within the peptidomimetic macrocycle.

10. The peptidomimetic macrocycle of claim 9, wherein at least one of the two amino acids is an .alpha.,.alpha.-disubstituted amino acid.

11. The peptidomimetic macrocycle of any one of claims 1-10, wherein the peptidomimetic macrocycle has the formula: ##STR00035## wherein: each A, C, D, and E is independently a natural or non-natural amino acid, and each D and E independently optionally includes a capping group; each B is independently a natural or non-natural amino acid, amino acid analog, or ##STR00036## ; each R.sub.1 and R.sub.2 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; each R.sub.3 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5; each L is independently a macrocycle-forming linker of the formula -L.sub.1-L.sub.2-; each L.sub.1 and L.sub.2 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each optionally substituted with R.sub.5; each R.sub.4 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each R.sub.5 is independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a therapeutic agent; each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each optionally substituted with R.sub.5, or forms part of a cyclic structure with a D residue; each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each optionally substituted with R.sub.5, or forms part of a cyclic structure with an E residue; each v and w is independently an integer from 1-1000; u is an integer from 1-10; each x, y and z is independently an integer from 0-10; and each n is independently an integer from 1-5.

12. The peptidomimetic macrocycle of claim 11, wherein L does not include a thioether or a triazole.

13. The peptidomimetic macrocycle of claim 1, wherein the peptidomimetic macrocycle comprises a cross linker linking a backbone amino group of a first amino acid within the peptidomimetic macrocycle to a second amino acid within the peptidomimetic macrocycle.

14. The peptidomimetic macrocycle of claim 13, wherein the peptidomimetic macrocycle has the formula (IV) or (IVa): ##STR00037## wherein: each A, C, D, and E is independently a natural or non-natural amino acid, and each D and E independently optionally include a capping group; each B is independently a natural or non-natural amino acid, amino acid analog, or ##STR00038## ; each R.sub.1 and R.sub.2 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue; each R.sub.3 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5; each L.sub.1 and L.sub.2 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each optionally substituted with R.sub.5; each R.sub.4 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each R.sub.5 is independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a therapeutic agent; each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5; each v and w is independently an integer from 1-1000; u is an integer from 1-10; each x, y and z is independently an integer from 0-10; and each n is independently an integer from 1-5.

15. The peptidomimetic macrocycle of claim 14, wherein L.sub.1 and L.sub.2 either alone or in combination do not include a thioether or a triazole.

16. The peptidomimetic macrocycle of claim 11, wherein L.sub.1 and L.sub.2 are independently alkylene, alkenylene or alkynylene.

17. The peptidomimetic macrocycle of claim 11, wherein L.sub.1 and L.sub.2 are independently C.sub.3-C.sub.10 alkylene or C.sub.3-C.sub.10 alkenylene.

18. The peptidomimetic macrocycle of claim 17, wherein L.sub.1 and L.sub.2 are independently C.sub.3-C.sub.6 alkylene or C.sub.3-C.sub.6 alkenylene.

19. The peptidomimetic macrocycle of claim 11, wherein R.sub.1 and R.sub.2 are H.

20. The peptidomimetic macrocycle of claim 11, wherein R.sub.1 and R.sub.2 are independently alkyl.

21. The peptidomimetic macrocycle of claim 11, wherein R.sub.1 and R.sub.2 are methyl.

22. A method of treating cancer in a subject comprising administering to the subject a peptidomimetic macrocycle of claims 1.

23. A method of modulating the activity of a p53 protein, a HDM2 protein, a HDMX protein, or a combination thereof, in a subject comprising administering to the subject a peptidomimetic macrocycle of claim 1.

24. A method of antagonizing the interaction between a p53 protein and a HDM2 protein, between a p53 protein and a HDMX protein, or a combination thereof, in a subject comprising administering to the subject a peptidomimetic macrocycle of claim 1.