AU2012203906A1 - Triazole macrocycle systems - Google Patents

Abstract

The present invention provides novel peptidomimetic macrocycles and methods for their preparation and use, as well as amino acid analogs and macrocycle-forming linkers, and kits useful in their production.

Description

AUSTRALIA Regulation 3.2 Patents Act 1990 Complete Specification Standard Patent APPLICANT: Aileron Therapeutics, Inc. Invention Title: TRIAZOLE MACROCYCLE SYSTEMS The following statement is a full description of this invention, including the best method of performing it known to me: WO 2008/104000 PCT/US2008/054922 TRIAZOLE MACROCYCLE SYSTEMS CROSS-REFERENCE 10001] This application claims the benefit of U.S. Provisional Application No. 60/903,073, filed February 23, 2007, which application is incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] Peptides are becoming increasingly important in pharmaceutical applications. Unmodified peptides often suffer from poor metabolic stability, poor cell penetrability, and promiscuous binding due to conformational flexibility. To improve these properties, researchers have generated cyclic peptides and peptidomimetics by a variety of methods, including disulfide bond formation, aide bond formation, and carbon-carbon bond formation (Jackson et aL. (1991), J. Am. Chem. Soc. 113:9391-9392; Phelan et al. (1997), J Am. Chem. Soc. 119:455-460; Taylor (2002), Biopolymers 66: 49-75; Brunel et at. (2005), Chem. Commun. (20):2552-2554; Hiroshige et aL. (1995), J. Am. Chem. Soc. 117: 11590-11591; Blackwell et al. (1998), Angew. Chem. Int. Ed. 37:3281-3284; Schafineister et al. (2000), J Am. Chem. Soc. 122:5891 5892). Limitations of these methods include poor metabolic stability (disulfide and amide bonds), poor cell penetrability (disulfide and amide bonds), and the use of potentially toxic metals (for carbon-carbon bond formation). Thus, there is a significant need for improved methods to produce peptides or peptidomimetics that possess increased conformational rigidity, metabolic stability and cell penetrability. The present invention addresses these and other needs in the art. SUMMARY OF THE INVENTION [0003] Provided herein are peptidomimetic macrocycles of Formula I, wherein: 0 0

R

7

R

8

N

[D], AvBy[h [E]w

R

1 R2 L (Formula I) each A, C, D, and E is independently a natural or non-natural amino acid;

R

3 HN B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L-CO-], [-NH-Lr-SO-], or [-NH-Ly-];

R

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

R

3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with Rs;

I

WO 20J081104000PTJO00/542 L is a macrocycie-forming linker of the formula PCT/US2008/054922 )NH

L

1 , L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4

-K-R

4 -],, each being optionally substituted with Rs; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, C0 2 , or CONR 3 ; each R, is independently halogen, alkyl, -OR 6 , -N(R 6

)

2 , -S& 6 , -SOR 6 , -SO2R 6 , -C0 2 &6, a fluorescent moiety, a radioisotope or a therapeutic agent; each R, is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

R

7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue; R& is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5. [00041 In some embodiments, L is N-N or N In other embodiments, L is N or N . In further embodiments, at least one of R 1 and R 2 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. In related embodiments, R 1 and 1 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. For example, at least one of R 1 and R 2 may be alkyl, unsubstituted or substituted with halo-. In some embodiments, both R 1 and R 2 are independently ailcyl, unsubstituted or substituted with halo- In other embodiments, at least one of R 1 and R 2 is methyl. In still other embodiments, both R 1 and R 2 are methyl. [0005] In some embodiments, at least one of D and E is a natural or unnatural amino acid substituted with a high molecular weight lipid or hydrocarbon. In other embodiments, at least one of D and E is attached to an additional macrocycle-forming linker. In still other embodiments, a secondary structure of the peptidomimetic macrocycle is more stable than a corresponding secondary structure of a corresponding non-macrocyclic polypeptide. In some embodiments, the peptidomimetic macrocycle comprises an a-helix. The a-helix may comprise, for example, from 1 turn to 5 turns. In other embodiments, the a-helix is more stable than an a-helix of a corresponding non-macrocyclic polypeptide. The macrocycle-forming linker 2 WO 2008/104000 PCT/US2008/054922 may span, for example, from 1 turn to 5 turns of the a-helix, such as approximately 1, 2, 3, 4 or 5 turns of the a-helix. The length of the macrocycle-forming liner may be, for example, about 5 A to about 9 A per turn of the a-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn of the a-helix. In such embodiments, the length of the macrocycle-forming linker may be approximately equal to the length of from about 6 carbon-carbon bonds to about 14 carbon-carbon bonds, or may be equal to the length of from about 8 carbon-carbon bonds to about 12 carbon-carbon bonds. In related embodiments, the the macrocycle may comprise a ring of about 18 atoms to 26 atoms. [00061 In other embodiments, the macrocycle-forming linker spans approximately 2 turns of the a-helix. In some embodiments, the length of the macrocycle-forming linker may be approximately equal to the length of from about 8 carbon-carbon bonds to about 16 carbon-carbon bonds, or may be approximately equal to the length of from about 10 carbon-carbon bonds to about 13 carbon-carbon bonds. In related embodiments, the macrocycle may comprise a ring of about 29 atoms to about 37 atoms. [00071 In some embodiments, an amino acid useful in the synthesis of the peptidomimetic macrocycle of the invention is a compound of Formula Ila or Ilb:

N

3 TR T9 Qh R1 Qh R2 Ry~ R 8 a N C0 2

R

11 N C0 2

R

11

R

10 Re Ia IIb wherein

R

1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; each Q and T is independently selected from the group consisting of alkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and [-R 4

-K-R

4 -],, each of which is unsubstituted or substituted with RS; K is 0, S, SO, SO 2 , CO, CO 2 , or CONR 3 ;

R

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

R

4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each Rs is independently halogen, alkyl, -OR 6 , -N(R 6

)

2 , -SR 6 , -SOR6, -S0 2 R, -CO 2 R6, a fluorescent moiety, a radioisotope or a therapeutic agent; each R is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

R

7 and R are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl; RIO and R, 1 are independently -H or any protecting group suitable for peptide synthesis; 3 WO 2008/104000 PCT/US2008/054922 g and h are each independently an integer from 0 to 5, wherein g+h is greater than I; and n is an integer from I to S. [00081 In some embodiments, the compound is a compound of Formula Ila and R 1 is alkyl, unsubstituted or substituted with halo-. In other embodiments, the compound is a compound of Formula Ilb and R 2 is alkyl, unsubstituted or substituted with halo-. In yet other embodiments, the compound is a compound of Formula lIla and R, is unsubstituted alkyl. For example, R 1 may be methyl. In still other embodiments, the compound is a compound of Formula Ilb and R2 is unsubstituted alkyl. For example, R2 may be methyl. In some embodiments of the compounds of the invention, at least one of R, and Rio is a protected group suitable for peptide synthesis. In another aspect, the present invention further provides kits comprising compounds of the invention or other amino acid analogs useful in the preparation of the peptidomimetic macrocycles of the invention along with macrocyclization reagents as described herein. [00091 In some embodiments, the invention provides a kit comprising: a) at least one compound selected from the group consisting of compounds of Formulas Ila and Ilb:

N

3 TgT Qh R1 Oh R2

R

7

R

8 N C0 2

R

11 N C0 2

R

1

R

10

R

10 Ila Ilb wherein R, and R2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo--; each Q and T is independently selected from the group consisting of alkylene, cycloalkylene, heterocycloalkylene, arylene, beteroarylene and [-14-K-R 4 -], each of which is unsubstituted or substituted with R5; K is 0, S, SO, SO 2 , CO, C0 2 , or CONR 3 ;

R

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

R

4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each R 5 is independently halogen, alkyl, -O&, -N(16) 2 , -S1(, -SOR, -S021,6, -CO2R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

R

7 and R are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl;

R

10 and RI are independently -H or any protecting group suitable for peptide synthesis; g and h are each independently an integer from 0 to 5; 4 WO 2008/104000 PCT/US2008/054922 n is an integer trom I to 5; and b) a macrocyclization reagent. [0010] In some embodiments,the kit comprises a compound of Formula Ila and R 1 is alkyl, unsubstituted or substituted with halo-. In related embodiments, R 1 is unsubstituted alkyl. For example, R 1 may be methyl. In other embodiments, the kit comprises a compound of Formula Ilb and R 2 is alkyl, unsubstituted or substituted with halo- In related embodiments, R 2 is unsubstituted alkyl. For example, R 2 may be methyl. 100111 In some embodiments, a kit comprises at least one compound of Formula Ila and at least one compound of Formula Ilb. A kit of the invention may also comprise a compound of Formula Ila or Formula Ilb wherein at least one of R 9 and RIO is a protected group suitable for peptide synthesis. In specific embodiments of the kit of the invention, the macrocyclization reagent is a Cu reagent. In yet other embodiments of the kit of the invention, the macrocyclization reagent is a Ru reagent. 10012] The present invention also provides a method for synthesizing a peptidomimetic macrocycle, the method comprising the steps of contacting a peptidomimetic precursor of Formula III or Formula IV: 0 [D~v/ [A]X-[B]y-[C]z[) [DLV zEI

R

1 l2 R2 N3 R12 (Formula III) 0 [DL z IV 7 [E]w

R

1 L 2 R2

N

3 || R12 (Formula IV) with a macrocyclization reagent; herein v, w, x, y, z, A, B, C, D, E, R 1 , R2, R 7 , 1&, Li and L 2 are as defined above; R 12 is -H when the macrocyclization reagent is a Cu reagent and R 12 is -H or alkyl when the macrocyclization reagent is a Ru reagent; and further wherein said contacting step results in a covalent linkage being formed between the alkyne and azide moiety in Formula III or Formula IV. For example, R12 may be methyl when the macrocyclization reagent is a Ru reagent. [0013] In some embodiments of the method of the invention, at least one of R1 and R 2 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. In related embodiments, R, and R2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. [00141 For instance, at least one of R1 and R 2 may be alkyl, unsubstituted or substituted with halo-. In another example, both R 1 and R2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R, and R 2 is methyl. In other embodiments, R1 and R2 are methyl. The 5 WO 20081104000 PCT/US2008/054922 macrocyclization reagent may be a Cu reagent. Alternatively, the macrocyclization reagent may De a Ru reagent. [00151 In some embodiments, the peptidomimetic precursor is purified prior to the contacting step. In other embodiments, the peptidomimetic macrocycle is purified after the contacting step. In still other embodiments, the peptidomimetic macrocycle is refolded after the contacting step. The method may be performed in solution, or, alternatively, the method may be performed on a solid support. [00161 Also envisioned herein is performing the method of the invention in the presence of a target macromolecule that binds to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding. In some embodiments, the method is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding. The method may also be applied to synthesize a library of peptidomimetic macrocycles. [0017] The peptidomimetic macrocycle resulting from a method of the invention may comprise an a-helix in aqueous solution. For example, the peptidomimetic macrocycle may exhibit increased a-helical structure in aqueous solution compared to a corresponding non-macrocyclic polypeptide. In some embodiments, the peptidomimetic macrocycle exhibits increased thermal stability compared to a corresponding non macrocyclic polypeptide. In other embodiments, the peptidomimetic macrocycle exhibits increased biological activity compared to a corresponding non-macrocyclic polypeptide. In still other embodiments, the peptidomimetic macrocycle exhibits increased resistance to proteolytic degradation compared to a corresponding non-macrocyclic polypeptide. In yet other embodiments, the the peptidomimetic macrocycle exhibits increased ability to penetrate living cells compared to a corresponding non-macrocyclic polypeptide. [00181 In some embodiments, the alkyne moiety of the peptidomimetic precursor of Formula III or Formula IV is a sidechain of an amino acid selected from the group consisting of L-propargylglycine, D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5 hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2 amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7 octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, (R)-2-amino-2-methyl-8-nonynoic acid and the azide moiety of the peptidomimetic precursor of Formula III or Formula IV is a sidechain of an amino acid selected from the group consisting of e-azido-L-lysine, e-azido-D-lysine, e-azido-alpha-methyl-L-lysine, e azido-alpha-methyl-D-lysine, 5-azido-alpha-methyl-L-ornithine, and 5-azido-alpha-methyl-D-ornithine. [00191 In some embodiments, x+y+z is 3, and and A, B and C are independently natural or non-natural amino acids. In other embodiments, x+y+z is 6, and and A, B and C are independently natural or non-natural amino acids. [00201 In some embodiments, the macrocyclization reagent is a Cu reagent and the contacting step is performed in a solvent selected from the group consisting of protic solvent, aqueous solvent, organic solvent, and mixtures thereof. For example, the solvent may be chosen from the group consisting of H 2 0, THF/H 2 0, tBuOH/H 2 O, DMF, DIPEA, CH 3 CN, CH 2 C1 2 , CICHzCH 2 CI or a mixture thereof. In other embodiments, the macrocyclization reagent is a Ru reagent and the contacting step is performed in an organic solvent. For example, the solvent may be DMF, THF, CH 3 CN, CH 2

CI

2 or CICH 2

CH

2 Cl. The solvent may be a solvent which favors helix formation. [00211 In some embodiments, the peptidomimetic macrocycle resulting from performing the method of the invention has the Formula (I): 6 WO 2008/104000 PCT/US2008/054922 0 0 R7 R 8 N .-- N [DIV [E]w R1 R2 L (Formula 1) wherein v, w, x, y, z, A, B, C, D, E, R1, R 2 , R 7 , 1R, and L are as defined above. INCORPORATION BY REFERENCE [00221 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. DETAILED DESCRIPTION OF THE INVENTION Definitions [00231 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. [00241 As used herein, the term "peptidominetic macrocycle" 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 the a carbon of one naturally-occurring amino acid residue or non naturally-occurring amino acid residue or amino acid analog residue and the a carbon of another naturally occurring amino acid residue or non-naturally-occurring amino acid residue or amino acid analog residue. 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. 100251 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 a-helices, P-turns, and P-pleated sheets. [00261 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. 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 a-helicity as determined by circular dichroism compared to a corresponding non-macrocyclic polypeptide. [00271 The term "a-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 a-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. 7 WO 20081104000 PCT/US2008/054922 [0028 1ie term "naturally occurring amino acid" refers to any one of the twenty ammo acias commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, 1, L, K, M, F, P, S, T, W, Y and V. [00291 The term "amino acid analog" 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., a-amino p-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 or the carboxy group with an ester). [00301 A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide (e.g., a BH3 domain or the p53 MDM2 binding domain) 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. 100311 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, 1) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a BH3 polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family. [00321 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. [00331 The symbol " " when used as part of a molecular structure refers to a single bond or a trans or cis double bond. [00341 The term "amino acid side chain" refers to a moiety attached to the a-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 a,a di-substituted amino acid). 10035} 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). [0036] The term "macrocyclization 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 an azide and alkyne. Such reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, 8 WO 2008/104000 PCT/US2008/054922 sucn as Cutr, GuI or CuOTf, as well as Cu(II) salts such as Cu(CO 2

CH

3

)

2 , Cu6, ana cuti, mat 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 additionally include, for example, Ru reagents known in the art such as Cp*RuCI(PPh 3

)

2 , [Cp*RuCl] 4 or other Ru reagents which may provide a reactive Ru(II) species. [00371 The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine or a radical thereof. [00381 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 1

-C

1 0 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. [00391 The term "alkylene" refers to a divalent alkyl (i.e., -R-). [00401 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 2 -Cio indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term "lower alkenyl" refers to a C 2

-C

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. [00411 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 2

-C

10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The terni "lower alkynyl" refers to a C 2

-C

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. [0042] 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. [0043] "Arylalkyl" refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C-C 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. [00441 "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 2 groups. Representative examples of an arylamido group include 2-C(O)NH2-phenyl, 3-C(O)NH 2 -phenyl, 4-C(O)NH 2 -phenyl, 2-C(O)NH 2 -pyridyl, 3-C(O)NH 2 -pyridyl, and 4-C(O)NH 2 -pyridyl, 100451 "Alkylheterocycle" refers to a C-C 5 alkyl group, as defined above, wherein one of the C-C 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 2 CH,-morpholine, -CH 2

CH

2 -piperidine, -CH 2

CH

2

CH

2 morpholine, and -CH 2

CH

2

CH

2 -imidazole. 10046] "Alkylamido" refers to a C-Cs alkyl group, as defined above, wherein one of the C-Cs alkyl group's hydrogen atoms has been replaced with a -C(O)NH 2 group. Representative examples of an alkylamido 9 WO 2008/104000 PCT/US2008/054922 group meluae, Nut are not limited to, -CH 2

-C(O)NH

2 , -CH 2

CH

2

-C(O)NH

2 , -CH 2 UIt 2

IIH

2 C(U)N H2, CH 2

CH

2

CH

2

CH

2

C(O)NH

2 , -CH 2

CH

2

CH

2

CH

2

CH

2

C(O)NH

2 , -CH 2

CH(C(O)NH

2

)CH

3 , CH 2

CH(C(O)NH

2

)CH

2

CH

3 , -CH(C(O)NH 2

)CH

2

CH

3 and -C(CH 3

)

2

CH

2

C(O)NH

2 100471 "Alkanol" refers to a C-Cs alkyl group, as defined above, wherein one of the C-Cs alkyl group's hydrogen atoms has been replaced with a hydroxyl group. Representative examples of an alkanol group include, but are not limited to, -CHOH, -CH 2

CH

2 OH, -CH 2

CH

2

CH

2 OH, -CH 2

CH

2

CH

2

CH

2 OH, -CH 2

CH

2

CH

2

CH

2

CH

2 OH, -CH 2

CH(OH)CH

3 , -CH 2

CH(OH)CH

2

CH

3 , -CH(OH)CH 3 and -C(CH 3

)

2

CH

2 OH. [00481 "Alkylcarboxy" refers to a C-C 5 alkyl group, as defined above, wherein one of the Cj-C 5 alcyl group's hydrogen atoms has been replaced with a --COOH group. Representative examples of an alkylcarboxy group include, but are not limited to, -CH 2 COOH, -CH 2

CH

2 COOH, -CH 2

CH

2

CH

2 COOH, CH 2

CH

2

CH

2

CH

2 COOH, -CH 2

CH(COOH)CH

3 , -CH 2

CH

2

CH

2

CH

2

CH

2 COOH, -CH 2

CH(COOH)CH

2

CH

3 , CH(COOH)CH 2

CH

3 and -C(CH 3

)

2

CH

2 COOH. [0049] 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. [00501 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 0, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of 0, 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. [0051] 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. 10052] 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. [00531 The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or I 1-14 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 0, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of 0, 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. [0054] The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of that group. 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. 10055] 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 10 WO 20081104000 PCT/US2008/054922 in multiple tautomeric forms, in such instances, the invention includes all tautomeric torms or trie 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. [00561 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%. [00571 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 > 0 and 2 if the variable is inherently continuous. 100581 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." [00591 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. Peptidomimetic Macrocycles of the Invention [0060] The present invention provides a peptidomimetic macrocycle of Formula (I): 0 0

R

7 R 8 N .- N [D]v A-BC- [E]w R1 R2 L (Formula I) wherein: each A, C, D, and E is independently a natural or non-natural amino acid;

R

3 N ,N { B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L 3 -CO-], [-NH-L-SO-1, or [-NH-L 3 -1; Ri and R2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; 11 WO 2008/104000 PCT/US2008/054922 13 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with Rs; L is a macrocycle-forming linker of the formula L, L 2 NH N: --- N; LI, L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4

-K-R

4 -],, each being optionally substituted with Rs; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, C0 2 , or CONR 3 ; each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6

)

2 , -SR6, -SOR6, -SO216, -C0 2 R6, a fluorescent moiety, a radioisotope or a therapeutic agent; each & 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

R

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

R

8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R, or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5. 10061] 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],, 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 ofx, y, or z in the indicated ranges. [00621 In some embodiments, the peptidomimetic macrocycle of the invention comprises a secondary structure which is an a-helix and R is -H, allowing intrahelical hydrogen bonding. [0063] In other embodiments, the length of the macrocycle-forming linker L as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca. 10064] In some embodiments, the peptidomimetic macrocycle comprises at least one cc-helix motif. For example, A, B and/or C in the compound of Formula I include one or more a-helices. As a general matter, a-helices include between 3 and 4 amino acid residues per turn. In some embodiments, the a-helix of the peptidomimetic macrocycle includes I to 5 turns and, therefore, 3 to 20 amino acid residues. In specific 12 WO 20081104000 PCT/US20081054922 embodiments, the a-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. in some embodiments, the macrocycle-forming linker stabilizes an a-helix motif included within the peptidomimetic macrocycle. Thus, in some embodiments, the length of the macrocycle-forming linker L from a first Ca to a second Ca is selected to increase the stability of an a-helix. In some embodiments, the macrocycle-forming linker spans from I turn to 5 turns of the a-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the a-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 A to 9 A per turn of the a-helix, or approximately 6 A to 8 A per turn of the a-helix. Where the macrocycle-forming linker spans approximately I turn of an a-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 a-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 a-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 a-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 a-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 I turn of an a-helix, the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming tinker spans approximately 2 turns of the a-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 a-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 a 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 a-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 I turn of the a-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 a-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 a-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 a 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 a-helix, the resulting macrocycle forms a ring containing 13 WO 2008/104000 PCT/US2008/054922 approximatety$ 4 members to 82 members, approximately 76 members to 80 members, or approximately 78 members. [00651 Exemplary embodiments of the macrocycle-forming linker L are shown below: N/ N-N N-N N N N=N N N NN N= NN NNNN N-N NN N N -N NN NNN N N NNN NNN NNN N .k7 NNNN N N N N-N N-N NN NN N -N=N NNN NN N N N=NN N=NN N NN N=NN N-N NN-N N N NN NN 1 N4N N -NN 7-< NAN- - N-N NN= NN=N N---- - N- N-N Ii14 WO 20081104000 PCT/US2008/054922 N N N NN N-N NN N-N N-N N " N N NN N-N N NN N NN N N 'N N ' N-N N-N N=N NN K'-N N N N-N N N=N N NN N=N NN N NN N N N N N N N-N NN N N N N=N N-N N='N > NN N=N N-N N NN NN NN NN [0066] In other embodiments, D and/or E 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. [0067] In one embodiment, a peptidomimetic macrocycle exhibits improved biological properties such as increased structural stability, increased affinity for a target, increased resistance to proteolytic degradation and/or increased cell penetrability when compared to a corresponding non-macrocyclic polypeptide. In another embodiment, a peptidomimetic macrocycle comprises one or more a-helices in aqueous solutions and/or exhibits an increased degree of a-helicity in comparison to a corresponding non-macrocyclic polypeptide. In some embodiments, a macrocycle-forming linker increases cell permeability of the peptidomimetic macrocycle. Without wishing to be bound by theory, it is hypothesized that the 15 WO 20081104000 PCT/US2008/054922 macrocycle-torming linker may increase the overall hydrophobicity of the peptidomimetic macrocycle relative to a corresponding non-macrocyclic polypeptide. [00681 Any protein or polypeptide with a known primary amino acid sequence which contains a secondary structure believed to impart biological activity is the subject of the present invention. For example, the sequence of the polypeptide can be analyzed and azide-containing and alkyne-containing amino acid analogs of the invention can be substituted at the appropriate positions. The appropriate positions are determined by ascertaining which molecular surface(s) of the secondary structure is (are) required for biological activity and, therefore, across which other surface(s) the macrocycle forming linkers of the invention can form a macrocycle without sterically blocking the surface(s) required for biological activity. Such determinations are made using methods such as X-ray crystallography of complexes between the secondary structure and a natural binding partner to visualize residues (and surfaces) critical for activity; by sequential mutagenesis of residues in the secondary structure to functionally identify residues (and surfaces) critical for activity; or by other methods. By such determinations, the appropriate amino acids are substituted with the amino acids analogs and macrocycle-forming linkers of the invention. For example, for an a-helical secondary structure, one surface of the helix (e.g., a molecular surface extending longitudinally along the axis of the helix and radially 45-135' about the axis of the helix) may be required to make contact with another biomolecule in vivo or in vitro for biological activity. In such a case, a macrocycle-forming linker is designed to link two a-carbons of the helix while extending longitudinally along the surface of the helix in the portion of that surface not directly required for activity. [0069] In some embodiments of the invention, the peptide sequence is derived from the BCL-2 family of proteins. The BCL-2 family is defined by the presence of up to four conserved BCL-2 homology (BH) domains designated BH1, BH2, BH3, and BH4, all of which include a-helical segments (Chittenden et al. (1995), EMBO 14:5589; Wang et al. (1996), Genes Dev. 10:2859). Anti-apoptotic proteins, such as BCL-2 and BCL-XL, display sequence conservation in all BH domains. Pro-apoptotic proteins are divided into "multidomain" family members (e.g., BAK, BAX), which possess homology in the BH1, BH2, and BH3 domains, and "BH3-domain only" family members (e.g., BID, BAD, BIM, BIK, NOXA, PUMA), that contain sequence homology exclusively in the BH3 amphipathic a-helical segment. BCL-2 family members have the capacity to form homo- and heterodimers, suggesting that competitive binding and the ratio between pro- and anti-apoptotic protein levels dictates susceptibility to death stimuli. Anti-apoptotic proteins function to protect cells from pro-apoptotic excess, i.e., excessive programmed cell death. Additional "security" measures include regulating transcription of pro-apoptotic proteins and maintaining them as inactive conformers, requiring either proteolytic activation, dephosphorylation, or ligand-induced conformational change to activate pro-death functions. In certain cell types, death signals received at the plasma membrane trigger apoptosis via a mitochondrial pathway. The mitochondria can serve as a gatekeeper of cell death by sequestering cytochrome c, a critical component of a cytosolic complex which activates caspase 9, leading to fatal downstream proteolytic events. Multidomain proteins such as BCL 2/BCL-XL and BAK/BAX play dueling roles of guardian and executioner at the mitochondrial membrane, with their activities further regulated by upstream BH3-only members of the BCL-2 family. For example, BID is a member of the BH3-domain only family of pro-apoptotic proteins, and transmits death signals received at the plasma membrane to effector pro-apoptotic proteins at the mitochondrial membrane. BID has the capability of interacting with both pro- and anti-apoptotic proteins, and upon activation by caspase 8, triggers cytochrome c release and mitochondrial apoptosis. Deletion and mutagenesis studies determined 16 WO 2008/104000 PCT/US2008/054922 tnat tne ampnipathic a-helical BH3 segment of pro-apoptotic family members may function as a aeath domain and thus may represent a critical structural motif for interacting with multidomain apoptotic proteins. Structural studies have shown that the BH3 helix can interact with anti-apoptotic proteins by inserting into a hydrophobic groove formed by the interface of BH1, 2 and 3 domains. Activated BID can be bound and sequestered by anti-apoptotic proteins (e.g., BCL-2 and BCL-XL) and can trigger activation of the pro-apoptotic proteins BAX and BAK, leading to cytochrome c release and a mitochondrial apoptosis program. BAD is also a BH3 -domain only pro-apoptotic family member whose expression triggers the activation of BAX/BAK. In contrast to BID, however, BAD displays preferential binding to anti-apoptotic family members, BCL-2 and BCL-XL. Whereas the BAD BH3 domain exhibits high affinity binding to BCL-2, BAD BH3 peptide is unable to activate cytochrome c release from mitochondria in vitro, suggesting that BAD is not a direct activator of BAX/BAK. Mitochondria that over-express BCL-2 are resistant to BID-induced cytochrome c release, but co-treatment with BAD can restore BID sensitivity. Induction of mitochondrial apoptosis by BAD appears to result from either: (1) displacement of BAX/BAK activators, such as BID and BID-like proteins, from the BCL-2/BCL-XL binding pocket, or (2) selective occupation of the BCL-2/BCL-XL binding pocket by BAD to prevent sequestration of BID-like proteins by anti-apoptotic proteins. Thus, two classes of BH3-domain only proteins have emerged, BID-like proteins that directly activate mitochondrial apoptosis, and BAD-like proteins, that have the capacity to sensitize mitochondria to BID-like pro-apoptotics by occupying the binding pockets of multidomain anti-apoptotic proteins. Various a-helical domains of BCL-2 family member proteins amendable to the methodology disclosed herein have been disclosed (Walensky el at. (2004), Science 305:1466; and Walensky et al., U.S. Patent Publication No. 2005/0250680, the entire disclosures of which are incorporated herein by reference). 10070] In other embodiments, the peptide sequence is derived from the tumor suppressor p53 protein which binds to the oncogene protein MDM2. The MDM2 binding site is localized within a region of the p53 tumor suppressor that forms an a helix. In U.S. Pat. No. 7,083,983, the entire contents of which are incorporated herein by reference, Lane et al. disclose that the region of p53 responsible for binding to MDM2 is represented approximately by amino acids 13-31 (PLSQETFSDLWKLLPENNV) of mature human P53 protein. Other modified sequences disclosed by Lane are also contemplated in the instant invention. Furthermore, the interaction of p53 and MDM2 has been discussed by Shair et al. (1997), Chem. & Biol. 4:791, the entire contents of which are incorporated herein by reference, and mutations in the p53 gene have been identified in virtually half of all reported cancer cases. As stresses are imposed on a cell, p 5 3 is believed to orchestrate a response that leads to either cell-cycle arrest and DNA repair, or programmed cell death. As well as mutations in the p53 gene that alter the function of the p53 protein directly, p53 can be altered by changes in M.DM2. The MDM2 protein has been shown to bind to p53 and disrupt transcriptional activation by associating with the transactivation domain of p53. For example, an I I amino acid peptide derived from the transactivation domain of p53 forms an amphipathic a-helix of 2.5 turns that inserts into the MDM2 crevice. Thus, in some embodiments, novel a-helix structures generated by the method of the present invention are engineered to generate structures that bind tightly to the helix acceptor and disrupt native protein-protein interactions. These structures are then screened using high throughput techniques to identify optimal small molecule peptides. The novel structures that disrupt the MDM2 interaction are useful for many applications, including, but not limited to, control of soft tissue sarcomas (which over-expresses MDM2 in the presence of wild type p53). These cancers are then, in some embodiments, held in check with small molecules that intercept MDM2, thereby preventing suppression of 17 WO 2008/104000. PCT/US2008/054922 p.). AaaCitionally, m some embodiments, small molecules disrupters of MDM2-p3 interactions are used as adjuvant therapy to help control and modulate the extent of the p53 dependent apoptosis response in conventional chemotherapy. [0071] A non-limiting exemplary list of suitable peptide sequences for use in the present invention is given below: TABLE I Name Sequence (bold = critical residues) Cross-linked Sequence (X = x-link residue) BH3 peptides BID-BH3 QEDIIRNIARHLAQVGDSMDRSIPP QEDIIRNIARHLAXVGDXMDRSIPP BIM-BH3 DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIAQELRXIGDXFNAYYAR BAD-BH3 NLWAAQRYGRELRRMSDEFVDSFKK NLWAAQRYGRELRXMSDXFVDSFKK PUMA-BH3 EEQWAREIGAQLRRMADDLNAQYER EEQWAREIGAQLRXMADXLNAQYER Hrk-BH3 RSSAAQLTAARLKALGDELHQRTM RSSAAQLTAARLKXLGDXLHQRTM NOXAA-BH3 AELPPEFAAQLRKIGDKVYCTW AELPPEFAAQLRXIGDXVYCTW NOXAB-BH3 VPADLKDECAQLRRIGDKVNLRQKL VPADLKDECAQLRXIGDXVNLRQKL BMF-BH3 QHRAEVQIARKLQCIADQFHRLHT QHRAEVQIARKLQXIADXFHRLHT BLK-BH3 SSAAQLTAARLKALGDELHQRT SSAAQLTAARLKXLGDXLHQRT BIK-BH3 CMEGSDALALRLACIGDEMDVSLRA CMEGSDALALRLAXIGDXMDVSLRA Bnip3 DIERRKEVESILKKNSDWIWDWSS DIERRKEVESILKXNSDXIWDWSS BOK-BH3 GRLAEVCAVLLRLGDELEMIRP GRLAEVCAVLLXLGDXLEMIRP BAX-BH3 PQDASTKKSECLKRIGDELDSNMEL PQDASTKKSECLKXIGDXLDSNMEL BAK-BH3 PSSTMGQVGRQLAIIGDDINRR PSSTMGQVGRQLAXIGDXINRR BCL2LI-BH3 KQALREAGDEFELR KQALRXAGDXFELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRR LSPPVVHLALALRXAGDXFSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY EVIPMAAVKQALRXAGDXFELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF PADPLHQAMRXAGDXFETRF MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA ATSRKLETLRXVGDXVQRNHETA MTD-BH3 LAEVCTVLLRLGDELEQIR LAEVCTVLLXLGDXLEQIR MAP-1-BH3 MTVGELSRALGHENGSLDP MTVGELSRALGXENGXLDP NIX-BH3 VVEGEKEVEALKKSADWVSDWS VVEGEKEVEALKXSADXVSDWS 41CD(ERBB4)-BH3 SMARDPQRYLVIQGDDRMKL SMARDPQRYLVXQGDXRMKL Table 1 lists human sequences which target the BH3 binding site and are implicated in cancers, autoimmune disorders, metabolic diseases and other human disease conditions. 18 WO 20081104000 PCT[US2008/054922 TABLE 2 Name Sequence (bold =critical residues) Cross-linked Sequence (X x-link residue) BH3 peptides BID-BH3 QEDIIRNIARI-LAQVGDSMDRSIPP QEIJRNIXRHLXQVGDSMIJRSIPP BIM-BH3 DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIXOELXRIGDEFNAYYAR BAD-BH3 NLWAAQRYCTRELRRMSDEFVDSFKK NLWAAQRYXRELXRMSDEFVDSFKK PUMA-BH3 EEQWAREIGAQLRiRMADDLNAQYER EEQWAREIXAQLXRMADDLNAQYER Hrk-BH3 RSSAAQLTAARLKALGDELHQRTM RSSAAQLTXAR!LXALGDELHQRTM NOXAA-BH3 AELPPEFAAQLRKIGDKVYCTW AELPPEFXAQLXKIGDKVYCTW NOXAB-BH3 VPADLKDECAQLRRIGDKVNLRQKL VPADIKDEXAQLXRIGDKVNLRQKL BMF-BH3 QHRAEVQIARKLQCTAIDQFHRIHT QH1PAEVQIXRKLXCIADQFHRLHT BLK-BH3 SSAAQLTAARLKALGDELHQRT SSAAQLTXARLXALGDELHQRT BIK-BH3 CMEGSDALALRIACIGDEMDVSLRA CMEGSDALXLRLXCIGDEMDVSLRA Bnip3 DIERRKEVESILKKNSDWIWDWSS DIERRKEVXSILXKNSDWTWDWSS BOK-BH13 GRLAEVCAVLLRILGDELEMIRP GRLAFVXAVLXRLGDELEMIRP BAX-BH3 PQDASTKKSECLKRJGDELDSNMEL PQDASTKKXECLXRIGDELDSNMEL BAK-BH3 PSSTMGQVGRQLAIIGDDINRR PSSTMGQV2 RQLXllGDDINRR BCL2LI -BH3 KQALREAGDEFELR 2jQALXEAGDEFELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRR LSPPVVHLXLALXQAGDDFSRR BCL-XL-BH43 EVIPMAAVKQALREAGDEFELRY EVIPMAAVXQALXEAGDEFELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF PADPLXQAMXIAAGDEFETR-F MCLI-B113 ATFSRKLETLRRVGDGVQRNHETA ATSRKXETLXRVGDGVQRN14ETA MTD-BH3 LAEVCTVLLRLGDELEQIR LAEVXTVLXRLGDELEQIR MAP-I -BH3 MTVGELSRALGHENGSLDP MTVGELXRALXHENGSLDP NIX-BH3 VVEGEKEVEALKKSADWVSDWS VVEGEKEXEALXKSADWVSDWS 4ICD(ERBB4)-BH3 SMARDPQRYLVIQGDDR-MKL SM\ARDPXRYLXIQGDDRMKL Table 2 lists human sequences which target the BH3 binding site and are implicated in cancers, autoirnmune disorders, metabolic diseases and other human disease conditions. 19 WO 20081104000 TABLE3 PCT/US2008/054922 Name Sequence (bold critical residues) Cross-linked Sequence (X= x-link residue) P53 peptides hp53 peptide veryshort LSQETFSDLWKLLPEN XSQEXFSDLWKLLPEN hp53 peptide short PPLSQETFSDLWKLLPENN PPXSQELCFSDLWKLLPENN hp53-P27S peptide-short PPLSQETFSDLWKLLSENN PPXSQEXFSDLWKLLSENN hp53 peptide_ Long DPSVEPPLSQETFSDLWKLLPENNVLSPLP DPSVEPPXSQEXFSDLWKLLPENNVLSPLP hp53-P27S peptide_ Long DPSVEPPLSQETFSDLWKLLSENNVLSPLP DPSVEPPXSQEXFSDLWKLLSENNVLSPLP hp53 peptide veryshort LSQETFSDLWKLLPEN LSQETFSDLWXLLPXN hp53 peptide short PPLSQETFSDLWKLLPENN PPLSQETFSDLWXLLPXNN hp53-P27S peptide-short PPLSQETFSDLWKLLSENN PPLSQETFSDLWXLLSXNN hp53 peptide_ Long DPSVEPPLSQETFSDLWKLLPENNVLSPLP DPSVEPPLSQETFSDLWXLLPXNNVLSPLP hp53-P27S peptide_ Long DPSVEPPLSQETFSDLWKLLSENNVLSPLP DPSVEPPLSQETFSDLWXLLSXNNVLSPLP Table 3 lists human sequences which target the p53 binding site of MDM2/X and are implicated in cancers. TABLE 4 Name Sequence (bold = critical residues) Cross-linked Sequence (X x-link residue) GPCR peptide ligands Angiotensin II DRVYIHPF DRXYXHPF Bombesin EQRLGNQWAVGHLM EQRLGNXWAVGHLX Bradykinin RPPGFSPFR RPPXFSPFRX C5a ISHKDMQLGR ISHKDMXLGRX C3a ARASHLGLAR ARASHLXLARX a-melanocyte stimulating hormone SYSMEHFRWGKPV SYSMXHFRWXKPV Table 4 lists sequences which target human G protein-coupled receptors and are implicated in numerous human disease conditions (Tyndall et al. (2005), Chem. Rev. 105:793-826). Methods of Preparing the Peptidomimetic Macrocycles of the Invention [0072] Methods of synthesizing the peptidomimetic macrocycles of the invention are disclosed herein. In some embodiments, the synthesis of these peptidomimetic macrocycles involves a multi-step process that features the synthesis of a peptidomimetic precursor containing an azide moiety and an alkyne moiety; followed by contacting the peptidomimetic precursor with a macrocyclization reagent to generate a triazole-linked peptidomimetic macrocycle. Macrocycles or macrocycle precursors are synthesized, for example, by solution phase or solid-phase methods, and can contain both naturally-occurring and non naturally-occurring amino acids. See, for example, Hunt, "The Non-Protein Amino Acids" in Chemistry and Biochemistry of the Amino Acids, edited by G.C. Barrett, Chapman and Hall, 1985. [0073] In some embodiments, an azide is linked to the a-carbon of a residue and an alkyne is attached to the a carbon of another residue. In some embodiments, the azide moieties are azido-analogs of amino acids L lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine, L-ornithine, D-ornithine, alpha-methyl-L ornithine or alpha-methyl-D-omithine. In another embodiment, the alkyne moiety is L-propargylglycine. In yet other embodiments, the alkyne moiety is an amino acid selected from the group consisting of L 20 WO 20081104000 PCT/US2008/054922 propargylglycme, D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acia, (Kj-z-amno-z-methyl-4 pentynoic acid, (S)-2-amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2 amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7 octynoic acid, (R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid and (R)-2 amino-2-methyl-8-nonynoic acid. [0074] In some embodiments, the invention provides a method for synthesizing a peptidomimetic macrocycle, the method comprising the steps of contacting a peptidomimetic precursor of Formula III or Formula IV: 0 [DL [A].-[B~y-[C1. z~ IY

R

1 1 2 R2 N3

R

1 2 (Formula III) 0 [D,/[A],-[B~y-[C]zM [D]v tm

R

1 112 R 2 N3 I R12 (Formula IV) with a macrocyclization reagent; wherein v, w, x, y, z, A, B, C, D, E, R 1 , R 2 , R 7 , Rs, L, and L 2 are as defined above; R 1 2 is -H when the macrocyclization reagent is a Cu reagent and R1, is -H or alkyl when the macrocyclization reagent is a Ru reagent; and further wherein said contacting step results in a covalent linkage being formed between the alkyne and azide moiety in Formula III or Formula IV. For example, R12 may be methyl when the macrocyclization reagent is a Ru reagent. 100751 In some embodiments of the method of the invention, at least one of R 1 and R2 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. In related embodiments, R, and R2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. [0076] For instance, at least one of R, and R2 may be alkyl, unsubstituted or substituted with halo- In another example, both R, and R2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R 1 and R2 is methyl, In other embodiments, R, and R 2 are methyl. The macrocyclization reagent may be a Cu reagent or a Ru reagent. [0077] In some embodiments, the peptidomimetic precursor is purified prior to the contacting step. In other embodiments, the peptidomimetic macrocycle is purified after the contacting step. In still other embodiments, the peptidomimetic macrocycle is refolded after the contacting step. The method may be performed in solution, or, alternatively, the method may be performed on a solid support. [0078] Also envisioned herein is performing the method of the invention in the presence of a target macromolecule that binds to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said 21 WO 20081104000 PCT/US2008/054922 Dmomag. in some embodiments, the method is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding. The method may also be applied to synthesize a library of peptidomimetic macrocycles. [00791 The peptidomimetic macrocycle resulting from a method of the invention may comprise an a-helix in aqueous solution. For example, the peptidomimetic macrocycle may exhibit increased a-helical structure in aqueous solution compared to a corresponding non-macrocyclic polypeptide. In some embodiments, the peptidomimetic macrocycle exhibits increased thermal stability compared to a corresponding non macrocyclic polypeptide. In other embodiments, the peptidomimetic macrocycle exhibits increased biological activity compared to a corresponding non-macrocyclic polypeptide. In still other embodiments, the peptidomimetic macrocycle exhibits increased resistance to proteolytic degradation compared to a corresponding non-macrocyclic polypeptide. In yet other embodiments, the the peptidomimetic macrocycle exhibits increased ability to penetrate living cells compared to a corresponding non-macrocyclic polypeptide. [00801 In some embodiments, the alkyne moiety of the peptidomimetic precursor of Formula III or Formula IV is a sidechain of an amino acid selected from the group consisting of L-propargylglycine, D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5 hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2 amino-2-nethyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7 octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, and (R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, the azide moiety of the peptidomimetic precursor of Formula III or Formula IV is a sidechain of an amino acid selected from the group consisting of e-azido-L-lysine, 6-azido-D-lysine, E azido-a-methyl-L-lysine, e-azido-a -methyl-D-lysine, 6-azido-a-methyl-L-omithine, and 5-azido-a methyl-D-ornithine. [00811 In some embodiments, x+y+z is 3, and and A, B and C are independently natural or non-natural amino acids. In other embodiments, x+y-z is 6, and and A, B and C are independently natural or non-natural amino acids. [00821 In some embodiments, the contacting step is performed in a solvent selected from the group consisting of protic solvent, aqueous solvent, organic solvent, and mixtures thereof For example, the solvent may be chosen from the group consisting of H 2 0, THF, THF/H 2 0, tBuOH/H 2 0, DMF, DIPEA, CH 3 CN or CH 2 C1 2 ,

CICH

2

CH

2 C1 or a mixture thereof. The solvent may be a solvent which favors helix formation. [00831 In some embodiments, the peptidomimetic macrocycle resulting from performing the method of the invention has the Formula (I): 0 0 R7 R [ - N [D]v [E]w

R

1 R2 L (Formula 1) wherein v, w, x, y, z, A, B, C, D, E, R 1 , R 2 , R 7 , R8, and L are as defined above. {0084] Alternative but equivalent protecting groups, leaving groups or reagents are substituted, and certain of the synthetic steps are performed in alternative sequences or orders to produce the desired compounds. 22 WO 20081104000 PCT/US2008/054922 Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein include, for example, those such as described in Larock, Comprehensive Organic Transformations, VCH Publishers (1989); Greene and Wuts, Protective Groups in Organic Synthesis, 2d. Ed. , John Wiley and Sons (1991); Fieser and Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. [00851 The peptidomimetic macrocycles of the invention are made, for example, by chemical synthesis methods, such as described in Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N. Y., 1992, p. 77. Hence, for example, peptides are synthesized using the automated Merrifield techniques of solid phase synthesis with the amine protected by either tBoc or Fmoc chemistry using side chain protected amino acids on, for example, an automated peptide synthesizer (e.g., Applied Biosystems (Foster City, CA), Model 430A, 431, or 433). [00861 One manner of producing the peptidomimetic precursors and peptidomimetic macrocycles described herein uses solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Side chain functional groups are protected as necessary with base stable, acid labile groups. [00871 Longer peptidomimetic precursors are produced, for example, by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides are biosynthesized by well known recombinant DNA and protein expression techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptidomimetic precursor of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods. [00881 The peptidomimetic precursors are made, for example, in a high-throughput, combinatorial fashion using, for example, a high-throughput polychannel combinatorial synthesizer (e.g., Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville, KY). 100891 The following synthetic schemes are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein. To simplify the drawings, the illustrative schemes depict azido amino acid analogs e-azido-a-methyl-L-lysine and E-azido-a -methyl-D-lysine, and alkyne amino acid analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, and (S)-2-amino-2 methyl-6-heptynoic acid. Thus, in the following synthetic schemes, each RI, R 2 , R 7 and R% is -H; each L, is

-(CH

2

)

4 -; and each L 2 is -(CH 2 )-. However, as noted throughout the detailed description above, many other amino acid analogs can be employed in which RI, R 2 , R 7 , R 8 , Li and L 2 can be independently selected from the various structures disclosed herein. 23 [0090] WO 20081104000 PCT/US2008/054922 [0091 netic ucfeme 1: N 0 X --- N3 |o I H N Ni S-ANi?-B _ halogen N N3 FmocsN CO 2 H 0 R =H, CH3 0 H b \R =H,CH3 S-AA-Ni-BPB X N R,, ".' N 3

-

N Ni H X halogen NN N D O R =H, CHR F R-j, R/R =H, CH3 R-AA-Ni-BPB SX = halo N Fmc cO 2 H R =H, CH 3 H R =H, CH 3 S-AA-Ni-BPB Hl ONi N "' -IN--I\> 02H 0 =H, CH 3 Y . H R-AA-Ni-BPB [00911 Synthetic Scheme 1 describes the preparation of several compounds of the invention. Ni(Il) complexes of Schiff bases derived from the chiral auxiliary (S)-2-[N-(N'-benzylprolyl)amino]benzophenone (BPB) and amino acids such as glycine or alanine are prepared as described in Belokon et al. (1998), Tetrahedron Asymm. 9:4249-4252. The resulting complexes are subsequently reacted with alkylating reagents comprising an azido or alkynyt moiety to yield enantiomerically enriched compounds of the invention. If desired, the resulting compounds can be protected for use in peptide synthesis. 24 WO 2008/104000 PCT/US2008/054922 100921 Synthetic Scheme 2:

N

3

N

3

CH

3

H

3 C C Fmoc CO Fm oc ' CO2 N COH N COH H H N--mcC--eh[H H N-a-Fmoc--ethl N-ac-Fmoc-C--mnethyl a-azido-L-tysine E-azido-D-ysine [AAl [AA] [AAIo S,S n R=HorMe N3 lmoc. ~'H moc.. .. CH N COZH N COH H H N-a-Fmoc-L- N-a-Fmoc-(S)-2-amino- H H propargyiglycine 2-methy-4-pentynoic N clav f d [AA]n [AA[ [AA]O ,S n R=HorMe N:, Fmoe. HO Fmoc- CC'H 3 2 H N 2 H H N-a-Fmoc-(S)-2-amlno- N-a-Fmnoc-(S).2-amnino- IDe protect 6-heptynoic acid 2-methyl-6-heptynoic I& cleave from acid solid support H 0 0 H 0 H 0 H H H H NN [ N NJ~ [A~ [A [A[AAAA [AA], [AA]. [AAln [Mian R )R (R)R Rn R=HorMe R ,S n R=HorMe N, N N 3 N Cu () H H H H NNAA ~N N"% [AA3, J. [An zj [AA]. [AA]n [AA]rm z . A~ R R'S R I (z)R h R = HorMe R,S R R= Hor Me NN [00931 In the general method for the synthesis of peptidomimetic macrocycles shown in Synthetic Scheme 2, the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solution phase or solid-phase peptide synthesis (SPPS) using the commercially available amino acid N-a-Fmoc-L propargyiglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl-c-azido-L-lysine, and N-methyl-e-azido-D-lysine. The peptidomimetic precursor is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA). The peptidomimetic precursor is reacted as a crude mixture or is purified prior to reaction with a macrocyclization reagent such as a Cu(I) in organic or aqueous solutions (Rostovtsev el al. (2002), Angew. Chem. Int Ed 41:2596-2599; Tornoe et a. (2002), J Org. Chem. 67:3057-3064; Deiters et al. (2003), J Am. Chem. Soc. 125:11782 11783; Punna et aL (2005), Angew. Chem. Int. Ed 44:2215-2220). In one embodiment, the triazole forming reaction is performed under conditions that favor a-helix formation. In one embodiment, the macrocyclization step is performed in a solvent chosen from the group consisting of H20, THF, CH 3 CN, 25 WO 20081104000 PCT/US2008/054922 DMF , DIPEA, tBuOH or a mixture thereof. In another embodiment, the macrocyclization step is performed in DMF. In some embodiments, the macrocyclization step is performed in a buffered aqueous or partially aqueous solvent. [0094] Synthetic Scheme 3:

N

3 N 3 H3 H 3 C , F mnoc. FmocY N CO 2 H N C0 2 H H H N-a-Fmoc-C-a-methyl N-a-Fmoc-C-ao- methyl H c-azido4--lysine s-azido-D-lysine [AA]n [AAb, [AA]O S n R= H or Me Fmoc.. 'H Fmoc-. O 3 N N CO 2 H N CO 2 H H H N-a-Fmoc-L- N-a-Fmoc-(S)-2-amino- H H propargyiglycine 2-methyl-4-pentynoic N N acid [AAln [AAV, [AA]O JR ( I R R,S n R=HorMe

N

3 Fmoc, 4H Fmoc. "COH 3 N C0 2 H N C0 2 H H H N4x-Fmoc-(S)-2-amino- N-a-Fmoc-(S)-2-amino 6-heptynoic acid 2-methyl-6-heptynoic Cu (I) acid H 0 H 0 H 0 H 0 N A] N _ __ [AA]n [AAJm [AA[A [AA] [AA, 2 L""Jo i"''iAAn R'm[A nR R=HorMe R R R=HorMe N ,N Deprotect N, CN & cleave from N solid support H A H H H LAAh -N EA 2 fA] [AA]. JN AA]O [AA.A]AJ R R,S -'R R=HorMe R R,S )R R= H or Me N, N N, N N N [00951 In the general method for the synthesis of peptidomimetic macrocycles shown in Synthetic Scheme 3, the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solid-phase peptide synthesis (SPPS) using the commercially available amino acid N-a-Fmoc-L-propargylglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6 heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl-s-azido-L-lysine, and N-methyl-s-azido D-lysine. The peptidomimetic precursor is reacted with a macrocyclization reagent such as a Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al. (2002), Angew. Chem. Int. Ed 41:2596-2599; Tornoe et al. (2002), J. Org. Chem. 67:3057-3064; Deiters et al. (2003), J Am. Chem. Soc. 125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed 44:2215-2220). The resultant triazole-containing peptidomimetic 26 WO 20081104000 PCT/US2008/054922 macrocycle is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA). In some embodiments, the macrocyclization step is performed in a solvent chosen from the group consisting of CH 2 Cl2, CICH 2 CH2C, DMF, THF, NMP, DIPEA, 2,6-lutidine, pyridine, DMSO, H20 or a mixture thereof. In some embodiments, the macrocyclization step is performed in a buffered aqueous or partially aqueous solvent. [00961 Synthetic Scheme 4:

N

3 N 3 .Ha H 3 0C Fmqoc,-. Fmoc.. N C0 2 H N CO2H H H N-a-Fmoc-C4-mnethyl N-a-Fmoc-C-a-methyl HH e-azido-L-lysine c-azido-D-lysine [AA]n X [AAI(N - [AA]O S,S n R=HorMe F moC. ' H Fmoc. ',,CH N CO 2 H N CO 2 H SPPS H H N-a-Fmoc-L. N-a-Fmoc-(S)-2-amino- H 0 H propargylglycine 2-mriethyl-4-pentynoic N H acid [AA]n [AAI [AA]O R,S n R=HorMe N3 Fmoc-. HO Fac- OH 3 N CO 2 H N CO 2 H H H N-a-Fmoc-(S)-2-amino- N-a-Fmoc-(S)-2-amino- Deprotect 6-hoptynoic acid 2-methyl-6-heptynoic & cleave from acid solid support H H H H 0 N N N [AA]n [AA [AA]O [AA], [AA]m [AA]. R ' R R (ZR R , R R=HorMe SS n R=HorMe

N

3 N- N Ru (11) H 0 H H 0 H_ R , )R R ( 2 )R R R, R H orMe RS 11(z RH or Me Wz N [0097] In the general method for the synthesis of peptidomimetic macrocycles shown in Synthetic Scheme 4, the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solution phase or solid-phase peptide synthesis (SPPS) using the commercially available amino acid N-a-Fmoc-L propargyiglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl-E-azido-L-lysine, and N-methyl-e-azido-D-lysine. The peptidomimetic precursor is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA). The peptidomimetic precursor is reacted as a crude mixture or is purified prior to reaction with a macrocyclization reagent such as a Ru(II) reagents, for example Cp*RuC(PPh 3

)

2 or [Cp*RuCl] 4 (Rasmussen et al. (2007), Org. Lett. 27 WO 20081104000 PCT/US2008/054922 9:~35-339; Lfiang et al. (2005), J Am. Chem. Soc. 127:15998-15999). In some emnomlments, me macrocyclization step is performed in a solvent chosen from the group consisting of DMF, CH 3 CN and THE. 10098] Synthetic Scheme 5:

N

3 CH3 H3 N 3 Fqmoc,.. Fmoc )K.C N CO 2 H F N 0 2 H H H N-a-Fmoc-C-a-methyl N-c-Fmoc-C-a-methyl N E-azildo-L-lysine &-azldo-D-lyslne [AA1n [AAh [AA]I SS n R=HorMe \__ N3 FmocN OH Fmoc 'N CH 3

C

2 H N C0 2 H SP H H N-a-Fmoc-L- N-a-Fmoc-(S)-2-amino- H H propargylglycine 2-methyl-4-pentynolc N N acid [AAn [AA]m N [AA] 0 R R,S n R=HorMe N3 Fmoc 'N H Fmoc sN 'CH H N 0 2 H N C0 2 H H H N-a-Fmoc-(S)-2-amino- N-a-Fmoc-(S)-2-amino 6-heptynoic acid 2-methyl4-heptynolic Ru (11) acid H 0 H 0 HX.0 AJ' ,N [AAlfl, W, [AAn AA].N [AAbO [AA ~ [AA][AA] 0 R R RH orMe R R=HorMe N-N Deprotect N-N & cleave from A0 H H solid support H 0 H N N [AA]r N ,.N R R R= H or Me R R'S l R R = H orMe N~ NzN N N NN [0099] In the general method for the synthesis of peptidomimetic macrocycles shown in Synthetic Scheme 5, the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solid-phase peptide synthesis (SPPS) using the commercially available amino acidN-a-Fmoc-L-propargylglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6 heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl-s-azido-L-lysine, and N-methyl-s-azido D-lysine. The peptidomimetic precursor is reacted with a macrocyclization reagent such as a Ru(II) reagent on the resin as a crude mixture. For example, the reagent can be Cp*RuC(PPh 3

)

2 or [Cp*RuC] 4 (Rasmussen et al. (2007), Org. Lett. 9:5337-5339; Zhang el al. (2005), J. Am. Chem. Soc. 127:15998 15999). In some embodiments, the macrocyclization step is performed in a solvent chosen from the group consisting of CH 2

CI

2 , ClCH 2

CH

2 CI, CH 3 CN, DMF, and THF. [00100] Several exemplary peptidomimetic macrocycles are shown in Table 5. For these macrocycles, a corresponding non-macrocyclic polypeptide is the BID BH3 polypeptide sequence fragment 28 WO 2008/104000 PCT/US2008/054922 . muIoU-iVuiLAQVGDSMDRSL "Nle" represents norleucine and replaces a menione resimue. It is envisioned that similar linkers are used to synthesize peptidomimetic macrocycles based on the polypeptide sequences disclosed in Table 1 through Table 4. TABLE 5 H-OllD N4eDRSI-NH264 Ac-DIlRNL'ARHLA e VGD NieDRSI-NH 2 MW =2464 CH 3

CH

3

H

3 CH /N N, NNN NN N Ac-DilRNIARHLA- VGD' NeDRSI-NH z MW = 2464 Ac-DIRNIARHLA VGD NIeDRS-NH 2 MW-2464 cH 3 CH,

CH

3 N eN N NH H H H ~Ac-ollRNIARHLA VGD' N' NWeRSMNH2 MW=2478 N H Ac-DIlRNIARHL VGDN NieRSINH 2 MW 2478 Ac-DIIRNLARHILA D NDRSINH 2 MW=2476 CH 3

CH

3 ~cH 3

CH

3 - N NN N N H Ac-IRNIARHLAN VGD1 NN RSNH MW=2492 NoN N Ac-D VGD <NieDRS1-NH 2 MW = 2478 AC-DIlRNIARHLK a VGD , NDRS-NH2 MW = 2476 CH 3 C1 ' H% CH 3 N, NN

N

HH H N H -H~ VGDN-~~DR1N MWz26 ADhRIRL( VG N C-DIIRNIARHLA eRIN, MV29 AcDINA 1IA' 1) \NeRS.NH 2 MW= 2492 ~CH3 CH, N N~ H Hh NH ~ .%A-IIHAHK~VD NleDRSI-NH 2 MW =2492 Ac-DIRNIARHLk< _X VGD' :j, DRSI-NH 2 MW =2492 AcD1NAHk H VD CH 3 2 CH, ~CH 3 Table 5 shows exemplary peptidommimetic macrocycles of the invention. "Nle" represents norleucine. Amino Acid Analogs [00101] The present invention contemplates the use of non-naturally-occurring amino acids and amino acid analogs in the synthesis of the peptidomimetic macrocycles described herein. Any amino acid or amino acid analog amenable to the synthetic methods employed for the synthesis of stable triazole containing peptidomimetic macrocycles can be used in the present invention. For example, L-propargylglycine is contemplated as a useful amino acid in the present invention. However, other alkyne-containing amino acids that contain a different amino acid side chain are also useful in the invention. For example, L-propargylglycine contains one methylene unit between the a-carbon of the amino acid and the alkyne of the amino acid side chain. The invention also contemplates the use of amino acids with multiple methylene units between the a carbon and the alkyne. Also, the azido-analogs of amino acids L-lysine, D-lysine, alpha-methyl-L-lysine, and alpha-methyl-D-lysine are contemplated as useful amino acids in the present invention. However, other terminal azide amino acids that contain a different amino acid side chain are also useful in the invention. For example, the azido-analog of L-lysine contains four methylene units between the a-carbon 29 WO 2008/104000 PCT/US2008/054922 01 me ammo acid and the terminal azide of the amino acid side chain. The invention aiso contemplates the use of amino acids with fewer than or greater than four methylene units between the a-carbon and the terminal azide. Table 6 shows some amino acids useful in the preparation of peptidomimetic macrocycles of the invention. TABLE 6 FmocsN CO2H FmocsN XC2H H H N-a-Fmoc-L-propargyl glycine N-aFmoc-D-propargyl glycine

N

3 N 3 KCH3 HaC Fmo. Fmoc-. X. moc'N COH N CO 2 H H H H N-a-Fmoc-(S)-2-amino-2- N-a-Fmoc-(R)-2-amino-2- FmocsN CO 2 H Fmoc N C0 2 H methyl-4-pentynole acid methyl-4-pentynolc acid H H N-a-Fmoc-e-azido- N-a-Fmoc-s-azido L-lysine D-lysine CH_ H 3 C f N 3 N3 FmocsN CO2H FmocsN CO2H H H N-a-Fmoc-(S)-2-amino-2- N-ca-Fmoc-(R)-2-amino-2-

CH

3

H

3 C methyl-5-hexynoic acid methyl-5-hexynolc acid Fmoc. mocs N CO 2 H F N CO 2 H H H N-a-Fmoc-s-azido- N-a-Fmoc-c-azido ta-methyl-L-ys ine a-methyl-D-lyslne

CH

3 HmC Fmoc.N CO 2 H FmocsN CO 2 H H H

N

3 N 3 N-a-Fmoc-(S)-2-amlno-2- N-a-Fmoc-(R)-2-amlno-2 methyl--heptynolc acid methyl-$ -heptynoic acid ,H Ha FmNc ' CO 2 H Fmoc CO 2 H H- H3C HI

CH

3

H

3 C Fmoc,N Fmoc X N-a-Fmoc-&azIdo- N-a-Fmoc-6-azido N C02H N CO 2 H L-omithine D-ornithine H H N-a-Fmoc-S)-2-amino-2- N-a-Fmoc-(R)-2-amino-2 methyl-7-octynoic acid methyl-7-octynolc acid N 3 N 3

-CH

3 H 3 C H H 3 C FmocN C2 FmocN FNocsN 2 Fmo N CO2HHH H H N-a-Fmoc-s-azido- N-a-Fmoc-c-azido N-a-Fmoc-(S)-2-amlno-2- N-a-Fmoc-(R)-2-amino-2- a-nethyl-L- ac-methyl-D methyl-8-nonynolc acid methyl-8-nonynoic acid ornithine ornithine Table 6 shows exemplary amino acids useful in the preparation of peptidomimetic macrocycles of the invention. [001021 In some embodiments the amino acids and amino acid analogs are of the D-configuration. In other embodiments they are of the L-configuration. In some embodiments, some of the amino acids and amino acid analogs contained in the peptidomimetic are of the D-configuration while some of the amino acids and amino acid analogs are of the L-configuration. In some embodiments the amino acid analogs are a,a disubstituted, such as cE-methyl-L-propargylglycine, a-methyl-D-propargylglycine, E-azido-alpha-methyl-L 30 WO 2008/104000 PCT/US2008/054922 lysine, and e-azido-alpha-methyl-D-lysine. In some embodiments the amino acid analogs are N-alkylated, e.g, N-methyl-L-propargylglycine, N-methyl-D-propargylglycine, N-methyl-s-azido-L-lysine, and N methyl-6-azido-D-lysine. [001031 In some embodiments, the -NH moiety of the amino acid is protected using a protecting group, including without limitation -Fmoc and -Boc. In other embodiments, the amino acid is not protected prior to synthesis of the peptidomimetic macrocycle. [001041 In some embodiments, an amino acid useful in the synthesis of the peptidomimetic macrocycle of the invention is a compound of Formula Ila or Ilb:

N

3 TgQ

R

1 TO R2 N x C0 2

R

11 N C0 2

R

11 Ia Ib wherein

R

1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; each Q and T is independently selected from the group consisting of alkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and [-R 4

-K-R

4 -]a, each of which is unsubstituted or substituted with R 5 ; K is 0, S, SO, S02, CO, CO 2 , or CONR 3 ;

R

3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with RSj;

R

4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each R 5 is independently halogen, alkyl, -OR 6 , -N(R)2, -SR&, -SOR6, -SO 2

R

6 , -C0 2

R

6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each 16 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

R

7 and R 8 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl; RIO and R 1 are independently -H or any protecting group suitable for peptide synthesis; g and h are each independently an integer from 0 to 5, wherein g+h is greater than 1; and n is an integer from I to 5. [00105] In some embodiments, the compound is a compound of Formula Ila and R, is alkyl, unsubstituted or substituted with halo-. In other embodiments, the compound is a compound of Formula Ilb and R 2 is alkyl, unsubstituted or substituted with halo-. In yet other embodiments, the compound is a compound of 31 WO 2008/104000 PCT/US2008/054922 1-ormula na ana R 1 is unsubstituted alkyl. For example, R 1 may be methyl. In stit other emDoiments, the compound is a compound of Formula IlIb and R 2 is unsubstituted alkyl. For example, R 2 may be methyl. [001061 In some embodiments of the compounds of the invention, at least one of R 9 and RIO is a protected group suitable for peptide synthesis. Kits [00107] In another aspect, the present invention further provides kits comprising compounds of the invention or other amino acid analogs useful in the preparation of the peptidomimetic macrocycles of the invention along with macrocyclization reagents as described herein. [001081 In some embodiments, the invention provides a kit comprising: a) at least one compound selected from the group consisting of compounds of Formulas Ila and Ib:

N

3 Tg Tg Qh R1 Qh R2 R R 8 N C0 2

R

11 N C0 2

R

11 I I R1o RIO Ila lIb wherein

R

1 and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; each Q and T is independently selected from the group consisting of alkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and [-R 4

-K-R

4 -1h, each of which is unsubstituted or substituted with R 5 ; K is 0, S, SO, SO 2 , CO, C0 2 , or CONR 3 ;

R

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

R

4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6

)

2 , -SR 6 , -SO& 6 , -SO 2

R

6 , -CO21 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

R

7 and R are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl; RIO and R, 1 are independently -H or any protecting group suitable for peptide synthesis; g and h are each independently an integer from 0 to 5; n is an integer from 1 to 5; and b) a macrocyclization reagent. 32 WO 20081104000 PCT/US2008/054922 [00109] m some emooaments,the kit comprises a compound of Formula Ila and R, is airyi, unsuosutuTea or substituted with halo-. In related embodiments, R is unsubstituted alkyl. For example, R, may be methyl. In other embodiments, the kit comprises a compound of Formula Ilb and R 2 is alkyl, unsubstituted or substituted with halo-. In related embodiments, R 2 is unsubstituted alkyl. For example, R 2 may be methyl. [001101 In some embodiments, a kit comprises at least one compound of Formula Ila and at least one compound of Formula Ilb. A kit of the invention may also comprise a compound of Formula Ila or Formula Ilb wherein at least one of R 9 and Rio is a protected group suitable for peptide synthesis. In specific embodiments of the kit of the invention, the macrocyclization reagent is a Cu reagent or a Ru reagent. In some embodiments, the kit contains a plurality of compounds of Formula Ila and/or Formula Ilb. In some embodiments, the kit comprises one or more containers holding one or more amino acid analogs as described herein. In other embodiments, the kit comprises one or more containers holding one or more macrocyclization reagents as described herein. In yet other embodiments, the kit comprises one or more containers holding one or more amino acid analogs as described herein, as well as one or more containers holding one or more macrocyclization reagents as described herein. [00111] For example, in some embodiments, the kit comprises a container holding at least two amino acid analogs, as described above, at least one having a side-chain alkyne and at least one having a side-chain terminal azide moiety, the amino acid analog optionally protected and suitable for the syntheses described herein. In some embodiments, the amino acid analog is selected from the group consisting of L-propargylglycine, D propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2 amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6 heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2 amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, (R)-2-amino-2-methyl-8 nonynoic acid, e-azido-L-lysine, e-azido-D-lysine, e-azido-a-methyl-L-lysine, and E-azido-a -methyl-D lysine, S-azido-a-methyl-L-ornithine, and 5-azido-a -methyl-D-ornithine and all forms suitably protected for liquid or solid phase peptide synthesis. [00112] In some embodiments, the kit comprises a container holding at least one non-naturally-occurring amino acid, or amino acid analog, bound to a solid support compatible with the syntheses described herein for peptidomimetic macrocycles. In some embodiments, the kit comprises one container holding an amino acid analog of the invention including a terminal alkyne moiety in combination with a container holding an amino acid analog of the invention including a terminal azide moiety in combination with a macrocyclization reagent of the invention. Assays [00113] The properties of the peptidomimetic macrocycles of the invention are assayed, for example, by using the methods described below. In some embodiments, a macrocycle of the invention has enhanced properties relative to a corresponding non-macrocyclic polypeptide. A corresponding non-macrocyclic polypeptide is, for example, a precursor of a peptidomimetic macrocycle, such as a compound of Formula III or IV which is converted into said macrocycle. Alternatively, a corresponding non-macrocyclic polypeptide is a polypeptide sequence, such as a natural polypeptide sequence which has substantial sequence overlap with the macrocycle of the invention. Numerous examples of natural polypeptides corresponding to the macrocyclic polypeptide are shown in Tables 1, 2, 3 and 4. 33 WO 2008/104000 PCT/US2008/054922 [00114] m general, a corresponding non-macrocyclic polypeptide can also be a labelea natural poiypepune or peptidomimetic precursor. Such labeling, for example by fluorescent or radioactive labeling, is used if necessary in some of the assays described below. In such assays, both the macrocycle and the corresponding non-macrocyclic polypeptide are typically labeled by similar or functionally equivalent methods. Assay to Determine a-helicity. [00115] In solution, the secondary structure of polypeptides with a-helical domains will reach a dynamic equilibrium between random coil structures and a-helical structures, often expressed as a "percent helicity". Thus, for example, unmodified pro-apoptotic BH3 domains ate predominantly random coils in solution, with a-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 non-macrocyclic polypeptide. In some embodiments, macrocycles of the invention will possess an alpha helicity of greater than 50%. To assay the helicity of peptidomimetic macrocyles of the invention, such as BH3 domain-based macrocycles, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H 2 0, to concentrations of 25-50 pM). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g, Jasco J-710) using standard measurement parameters (e.g. temperature, 20'C; wavelength, 190-260 nm; step resolution, 0.5 in; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The a-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [CD]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)). Assay to Determine Melting Temperature (Tm). [001161 A peptidomimetic macrocycle of the invention comprising a secondary structure such as an a-helix exhibits, for example, a higher melting temperature than a corresponding non-macrocyclic polypeptide. Typically peptidomimetic macrocycles of the invention exhibit Tm of > 60*C representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on meltine temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H 2 0 (e.g. at a final concentration of 50 pM) and the Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95 *C) on a spectropolarimeter (e.g., Jasco J-710) using standard parameters (e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 un; temperature increase rate: 1 0 C/min; path length, 0.1 cm). Protease Resistance Assay. [001171 The amid 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 non-macrocyclic polypeptide. For example, the peptidomimetic macrocycle and a corresponding non-macrocyclic 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 rnm. Briefly, the peptidomimetic 34 WO 20081104000 . PCT/US2008/054922 macrocycle ana peptidomimetic precursor (5 mcg) are incubated with trypsm agarose trierce) (N/E ~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 In[S] versus time (k=-1Xslope). Ex Vivo Stability Assay. [001181 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 non-macrocyclic polypeptide peptide, 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 non-macrocyclic polypeptide (in a specific example, the corresponding natural polypeptide) (2 mcg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37 0 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 pl of sera to 2 ml centrifuge tubes followed by the addition of 10 iL of 50 % formic acid and 500pL acetonitrile and centrifugation at 14,000 RPM for 10 min at 4 +1 2*C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N 2 < 10 psi, 37*C. The samples are reconstituted in 100 pL of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis. In vitro Binding Assays. [00119] To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) isused, 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). [001201 For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25- 10OOnM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity ismeasured, 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, CA). A peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding non-macrocyclic polypeptide. [001211 Acceptor proteins for BH3-peptides such as BCL-2, BCL-XL, BAX or MCL1 may, for example, be used in this assay. Acceptor proteins for p53 peptides such as MDM2 or MDMX may also be used in this assay. In vitro Displacement Assays To Characterize Antagonists of Peptide-Protein Interactions. 100122] To assess the binding and affinity of compounds that antagonize the interaction between a peptide (e.g. a BH3 peptide or a p53 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 35 WO 20081104000 PCT/US2008/054922 nuorescent tracer. When excited with polarized light, fluorescent tracers (e.g., r iLL) auacnea 1o 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. [001231 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 (140mM NaC, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity ismeasured, 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, CA). [00124] Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay. Acceptor proteins for BH3-peptides such as BCL2, BCL XL, BAX or MCL1 can be used in this assay. Acceptor proteins for p53 peptides such as MDM2 or MDMX can be used in this assay. Binding Assays in Intact Cells. [00125] 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 (50mM Tris [pH 7.6], 150 mM NaCI, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4*C. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 pl goat anti-FITC antibody for 2 hrs, rotating at 4'C followed by further 2 hrs incubation at 4'C with protein A/G Sepharose (50 pl 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, including BCL2, MCL 1, BCL-XL, A l, BAX, BAK, MDM2 or MDMX. Cellular Permeability Assays. [001261 A peptidomimetic macrocycle is, for example, more cell permeable compared to a corresponding non macrocyclic polypeptide. In some embodiments, the peptidomimetic macrocycles are more cell permeable than a corresponding non-macrocyclic polypeptides. Peptidomimetic macrocycles with optimized linkers possess, for example, cell permeability that is at least two-fold greater than a corresponding non macrocyclic polypeptide, and often 20% or more of the applied peptide will be observed to have penetrated the cell after 4 hours.To measure the cell permeability of peptidomimetic macrocycles and corresponding non-macrocyclic polypeptides, intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding non-macrocyclic polypeptides (10 pM) for 4 hrs in serum free media at 37oC, washed 36 WO 2008/104000 PCT/US2008/054922 twice wim memta and incubated with trypsin (0.25%) for 10 min at 374C. The ceits are wasned again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan @ HCS Reader, Cellular Efficacy Assays. [00127] 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 peptidominetic macrocycles (0.5 to 50 pM) to identify those that kill at EC50<l0 pM. 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. In Vivo Stability Assay. [00128] 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 pL of fresh serum are then measured by LC-MS/MS as above. In vivo Efficacy in Animal Models. [00129] 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 x 10' 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, MA). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, MA). Peptidoinimetic 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 0.1mg/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. 37 WO 20081104000 PCT/US2008/054922 [001301 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 peptidomiinetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo. Pharmaceutical Compositions and Routes of Administration [00131] 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. [00132] 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. [001331 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) 4 ' salts. [001341 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. 38 WO 2008/104000 PCT/US2008/054922 [00135] In powders, the carrier is a finely divided solid, which is in a mixture with the Imely 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. [00136] Suitable solid recipients 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. [001371 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. [00138] 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. [001391 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 regimeni, 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 [001401 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 MDM2 system, labeled stabilized peptidomimetic macrocyles based on the p53 is used in an MDM2 binding assay along with small molecules that competitively bind to MDM2. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the p53/MDM2 system. Likewise in the BH3/BCL-XL anti-apoptotic system labeled peptidomimetic macrocycles based on BH13 can be used in a BCL-XL binding assay along with small molecules that competitively bind to BCL-XL. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the BH3/BCL-XL system, 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 p53 or BH3 peptidomimetic precursors upon which the peptidomimetic macrocycles are derived. Such antibodies, for example, disrupt the p53/MDM2 or BH3/BCL-XL systems, respectively. [00141] 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., 39 WO 2008/104000 PCT/US2008/054922 isurncient or excessive) BCL-2 family member expression or activity (e.g., extrinsic or intrinsic apoptotic pathway abnormalities). It is believed that some BCL-2 type disorders are caused, at least in part, by an abnormal level of one or more BCL-2 family members (e.g., over or under expression), or by the presence of one or more BCL-2 family members exhibiting abnormal activity. As such, the reduction in the level and/or activity of the BCL-2 family member or the enhancement of the level and/or activity of the BCL-2 family member, is used, for example, to ameliorate or reduce the adverse symptoms of the disorder. [001421 In another aspect, the present invention provides methods for treating or preventing hyperproliferative disease by interfering with the interaction or binding between p53 and MDM2 in tumor cells. These methods comprise administering an effective amount of a compound of the invention to a warm blooded animal, including a human, or to tumor cells containing wild type p 5 3 . In some embodiments, the administration of the compounds of the present invention induce cell growth arrest or apoptosis. In other or further embodiments, the present invention is used to treat disease and/or tumor cells comprising elevated MDM2 levels. Elevated levels of MDM2 as used herein refers to MDM2 levels greater than those found in cells containing more than the normal copy number (2) of indm2 or above about 10,000 molecules of MDM2 per cell as measured by ELISA and similar assays (Picksley et al. (1994), Oncogene 9, 2523 2529). [00143] 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. [001441 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. [001451 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 40 WO 20081104000 PCT/US2008/054922 auct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's mmor, 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. [00146] 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, lympboid 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. [001471 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. [001481 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. [001491 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. [00150] 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. [001511 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 41 WO 2008/104000 PCT/US2008/054922 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. [001521 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, myelodysplasia [001531 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, and the anti-apoptotic peptidomimetics macrocycles of the invention are used, in some embodiments, in the treatment of these disorders. Such 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. 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. [00154] Some examples of immunologic disorders that are treated with the peptidomimetics macrocycles described herein include but are not limited to organ transplant rejection, arthritis, lupus, IBD, Crohn's disease, asthma, multiple sclerosis, diabetes, etc. [001551 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 42 WO 2008/104000 PCT/US2008/054922 Carcinoma or the Thyroid, Familial Arnyloidosis, Hereditary Cerebral Hemorrnage With Amyloidosis, Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis, a prion-mediated disease, and Huntington's Disease. [001561 Some examples of endocrinologic disorders that are treated with the peptidomimetics macrocycles described herein include but are not limited to diabetes, hypothyroidism, hypopituitarism, hypoparathyroidism, hypogonadism, etc. [00157] 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, 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. EXAMPLES 1001581 The following section provides illustrative examples of the present invention. [001591 Example 1. Preparation of AlphaAlpha-Disubstituted Amino Acids. Compound 1 | N 3 [00160] Compound 1 was prepared by a two step sequence according to Yao et al. (2004) J Org. Chem, 69:1720 1722. To 1-iodo-4-chloro-butane (1 ml, 8.2 mmol) in dimethyl formamide (10 ml) was added sodium azide (0.53 g, 8.2 mmol) and the reaction was stirred at ambient temperature overnight. The reaction was then diluted with ethyl acetate and water. The organic layer was washed with water (3 times), dried over magnesium sulfate and concentrated in vacuo to give I-azido-4-chloro-butane in 80% yield. The azide was diluted with acetone (38 ml) and sodium iodide (1.98 g, 13.00 mmol) was added and the reaction was heated at reflux overnight. Afterwards, the reaction was diluted with water and the product was extracted with ether (three times). The combined organic extracts were washed with sodium bicarbonate and brine. The organic extracts were dried over magnesium sulfate and concentrated in vacuo. The product 1 was purified by passing it through a plug of neutral alumina. The yield was 89%. 43 WO 20081104000 Compound 2 PCT/US2008/054922 <X N NiN H 0% S-Ala-Ni-BPB 1001611 Compound 2 was prepared by a three step sequence according to Belokon et aL. (1998), Tetrahedron Asymm. 9:4249-4252. A solution of S-proline (100 g, 0.869 mol) and KOH (172 g, 2.61 mol) in isopropanol (600 ml) was prepared with stirring at 40C. As soon as the solution became transparent, benzyl chloride (156 ml, 1,34 mol) was added at the same temperature. After the addition was complete (3.5 h), the reaction was stirred overnight at 40C. The reaction was neutralized with conc. HCl (110 ml) until pH 5, then chloroform (400 ml) was added to the reaction mixture and the mixture was left stirring overnight. The mixture was then filtered and the precipitate washed with CHCL. The CFICL3 solutions were combined and evaporated, the residue was treated with acetone and the precipitate of the crude product filtered and additionally washed with acetone. The benzyl proline product was isolated in 75% yield. [00162] To a solution of benzyl proline (41 g, 0.2 mol) in methylene chloride (200 ml) was added thionyl chloride (18.34 ml, 0.25 mol) with stirring at -20C to -30C over a period of 10 min. The stirring was continued at I OC until the reaction mixture became almost transparent. Then a solution of 2-aminobenzophenone (25 g, 0.125 mol) in methylene chloride (100 ml) was added to the reaction mixture at -30C with stirring. The stirring was continued at ambient temperature for another 10 h and a solution of sodium carbonate (40 g) in water (150 ml) was added to the reaction mixture with stirring at OC. The organic layer was separated, the aqueous layer extracted several times with methylene chloride and the organic solutions were combined and evaporated. The product (benzyl proline-aminobenzophenone adduct) was crystallized from ethanol and was isolated in 85 % yield. [001631 A solution of KOH (23.1 g, 0.35 mol) in methanol (75 ml) was poured into a stirred mixture of benzyl proline-aminobenzophenone adduct (19.5 g, 0.05 mol) and nickel nitrate hexahydrate (29.1 g, 0.1 mol), L Ala (8.9 g, 0.1 mol) in methanol (175) under nitrogen at 40-50C. The reaction mixture was stirred at 55C 65C for 2 h and then neutralized with acetic acid (20 ml). The reaction volume was diluted with water (500 ml). After 6 h, the separated crystalline solid was filtered and washed with water (2x) to give the pure compound 2 (red solid, 22 g). M+H obs. 512.4, M+H calc.512.1. 44 WO 2008/104000 Compound 3 PCT/US2008/054922 N' 0 N N

N

3 / [00164] To compound 2 (5.122g, 10.0 mmol) was added dimethyl formamide (45 mL), which was degassed via a freeze-thaw cycle. The solution was cooled to 4C with an ice bath and powdered KOH (6.361g, 100 mmol) was added in one batch. The cold bath was removed and compound I (3.375g, 15 mmol) dissolved in dimethylformamide (4.0 mL) was added via syringe. The reaction was stirred at ambient temperature for 40 min. The reaction was then quenched by adding it slowly to a cold solution of 5 % aqueous acetic acid (200 mL). The crude product was collected by filtration and washed three times with cold water. The product was purified by flash chromatography using a Biotage silica column and hexane ethyl acetate as eluent. Compound 3 was obtained as a red solid, (51% yield), M+H calc.609.2, M+H obs.609.37. The purity was determined as 98 % by UV 254 rim. Compound 4 [00165] To a solution of 5-chloro pentyne (5g, 47.8 mmol) in acetone (80 mL) was added sodium iodide (14.321g, 95.54 mmol). The reaction was heated at reflux overnight. Afterwards, the reaction was diluted with water and the product was extracted with ether (three times). The combined organic extracts were washed with sodium bicarbonate and brine. The organic extracts were dried over magnesium sulfate and concentrated in vacuo. The product 4 was purified by passing it through a plug of neutral alumina. The yield was 92%. Compound 5 O r00 N N 0 [00166] To compound 2 (2.56 Ig, 5.0 mmol) was added dimethyl formamide (23 mL), which was degassed via a freeze-thaw cycle. The solution was cooled to 4C with an ice bath and powdered KOH (3.18 1g, 50 mmol) was added in one batch. The cold bath was removed and compound 4 (1.94g, 10 mmol)) dissolved in 45 WO 2008/104000 IPCT/US2008/054922 dimethyltormamide (2.0 mL) was added via syringe. The reaction was stirred at ammlent temperature for 40 min. The reaction was then quenched by adding it slowly to a cold solution of 5 % aqueous acetic acid (100 mL). The crude product was collected by filtration and washed three times with cold water. The product was purified by flash chromatography using biotage silica column and hexane ethyl acetate as eluent. Compound 5 is a red solid, 1.4g yield 48%. M+H calc.578.19 , M+H obs.578.69. The purity was determined as 97% by UV 254 run. Compound 6 0

HO

FmocHN '" "' N 3 1001671 To a solution (12 ml) of 1/1 3N HCI/MeOH at 70C was added a solution of compound 3 (1 g, 1.65 mmol) in MeOH (3 ml) dropwise. The starting material disappeared within 5-10 min. The reaction mixture was then concentrated in vacuo and residual solvent removed on a high vacuum pump. The crude residue was diluted with 10 % aqueous Na2CO3 (16 ml) cooled to 0 C with an ice bath. Fmoc-OSu (0.84 g, 2.5 mmol) dissolved in dioxane (16 ml) was added and the reaction was allowed to warm up to ambient temperature with stirring overnight. Afterwards, the reaction was diluted with ethyl acetate and I N HCL. The organic layer was washed with 1 N HCl (3 times). The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The pure product was isolated after a flash chromatography purification with a Biotage silica column and ethyl acetate/hexane and methylene chloride/methanol as eluents to give a viscous oil in 36 % overall yield for both steps. M+Na obs 431.89, M+H calc (409.18). Purity was determined as 98% UV 254 nm. Compound 7 0

HO

FmocHN [00168] To a solution (18 ML) of 1/1 3N HCl/MeOH at 70C was added a solution of compound 5 (1.4 G, 2.4 mmol) in MeOH (4 ml) dropwise. The starting material disappeared within 5-10 min. The reaction mixture was then concentrated in vacuo and residual solvent removed on a high vacuum pump. The crude residue was diluted with 10 % aqueous Na2CO3 (24 ml) cooled to 0 C with an ice bath. Fmoc-OSu (0.98g, 2.9 mmol) dissolved in dioxane (24 ml) was added and the reaction was allowed to warm up to ambient temperature with stirring overnight. Afterwards, the reaction was diluted with ethyl acetate and I N HCL. The organic layer was washed with 1 N HCI (3 times). The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The pure product was isolated (30% yield for both steps) after flash chromatography purification with a Biotage silica column and ethyl acetate/hexane and methylene chloride/methanol as eluents. The product was obtained as a viscous oil, that solidifies upon standing. M+H calc. 378.16, M+Na obs 400.85. 46 WO 2008/104000 PCT/US2008/054922 [001691 A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. 47

Claims (57)

1. A pharmaceutically acceptable salt of a peptidomimetic macrocycle, comprising an alpha-helix or beta sheet, of Formula (I): 0 0 R7 R 8 [D] 7 N [A]r[B]y-[C] N {E]. R1 R2 L (Formula (I)), wherein: each A, C, D, and E is independently a natural or non-natural amino acid; R 3 H each B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L 3 CO-], [-NH-L 3 -SO 2 -], or [-NH-L 3 -]; R 1 and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group except -H being optionally substituted with halo-; R 3 is -H, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 ; L is a macrocycle-forming linker of the formula L, L2 NH LI L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -]n, each being optionally substituted with R 5 ; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, C0 2 , or CONR 3 ; each R 5 is independently halogen, alkyl, -OR 6 ,. -N(R) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R6, a fluorescent moiety, a radioisotope or a therapeutic agent; 48 each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with a D residue; Rs is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5.

2. The pharmaceutically acceptable salt of claim 1, comprising the alpha-helix.

3. The pharmaceutically acceptable salt of claim 2, wherein the alpha-helix comprises from I to 5 turns.

4. The pharmaceutically acceptable salt of claim 2, wherein the alpha-helix is more stable than an alpha-helix of a corresponding non-macrocyclic polypeptide.

5. The pharmaceutically acceptable salt of claim 2, wherein the macrocycle-forming linker spans from 1 turn to 5 turns of the alpha helix.

6. The pharmaceutically acceptable salt of claim 2, wherein the macrocycle-forming linker spans approximately 1, 2, 3, 4 or 5 turns of the alpha-helix.

7. The pharmaceutically acceptable salt of claim 2, wherein the macrocycle-forming linker is about 5 A to about 9 A per turn of the alpha-helix.

8. The pharmaceutically acceptable salt of claim 2, wherein the macrocycle-forming linker spans approximately 1 turn of the alpha-helix.

9. The pharmaceutically acceptable salt of claim 1, comprising the beta sheet. 49

10. The pharmaceutically acceptable salt of claim 1, wherein R 1 and R 2 are -H.

11. The pharmaceutically acceptable salt of claim 1, wherein R 1 and R 2 are alkyl.

12. A composition, comprising a peptidomimetic macrocycle, that comprises an alpha-helix or beta sheet, of Formula (I), or its pharmaceutically acceptable salt: 0 0 R 7 R 8 [D]v N[E] R1 R2 L (Formula (I)), wherein: each A, C, D, and E is independently a natural or non-natural amino acid; R 3 $~N H each B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L CO-], [-NH-L 3 -S0 2 -], or [-NH-L 3 -]; R 1 and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group except -H being optionally substituted with halo-; R 3 is -H, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each group except -H group being optionally substituted with R 5 ; L is a macrocycle-forming linker of the formula NH L 1 , L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -]., each being optionally substituted with R 5 ; each R 4 is alkylene, alkenylene. alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, CO 2 , or CONR 3 ; 50 each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR&, -SOR 6 , -S0 2 16, -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with a D residue; R 8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5.

13. The composition of claim 12, further comprising an excipient.

14. The composition of claim 12, comprising the peptidomimetic macrocycle.

15. The composition of claim 12, comprising the pharmaceutically acceptable salt of the peptidomimetic macrocycle.

16. A method of treating a disease in a subject, comprising, administering a peptidomimetic macrocycle, its pharmaceutically acceptable salt, or a composition containing one or more of these, to the subject in an amount sufficient to treat the disease, wherein the peptidomimetic macrocycle is of Formula (1): 0 0 R7 R 8 N[xB - N R1 R 2 L (Formula (I)), wherein: 51 each A, C, D, and E is independently a natural or non-natural amino acid; R3 $N~N HY each B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L 3 CO-], [-NH-L 3 -SO 2 -], or [-NH-L 3 -]; R 1 and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group except -H being optionally substituted with halo-; R 3 is -H, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each group except -H group being optionally substituted with Rs; L is a macrocycle-forming linker of the formula N -N L 1 , L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -],, each being optionally substituted with R 5 ; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, CO 2 , or CONR 3 ; each R 5 is independently halogen, alkyl, -OR 6 , -N(R) 2 , -SR 6 , -SOR 6 , -SO2R6, -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with a D residue; R8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5. 52

17. The method of claim 16, wherein the subject is a human.

18. The method of claim 16, wherein the subject is a human in need thereof.

19. The method of claim 16, wherein the administering is orally administering.

20. The method of claim 16, wherein the peptidomimetic macrocycle and its salt comprise an alpha helix.

21. The method of claim 16, wherein the peptidomimetic macrocycle and its salt comprise a beta sheet.

22. The method of claim 16, wherein the peptidomimetic macrocycle is administered.

23. The method of claim 16, wherein the pharmaceutically acceptable salt of the peptidomimetic macrocycle is administered.

24. The method of claim 16, wherein the composition comprising the peptidomimetic macrocycle is administered.

25. The method of claim 16, wherein the composition comprising the pharmaceutically acceptable salt of the peptidomimetic macrocycle is administered.

26. The method of claim 16, wherein R 1 and R 2 are -H.

27. The method of claim 16, wherein R 1 and R 2 are alkyl.

28. The method of claim 16, wherein R 1 and R 2 are methyl.

29. The method of claim 16, wherein the disease is cancer.

30. The method of claim 29, wherein the cancer is selected from the group consisting of fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, 53 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, Kaposi sarcoma, breast cancer, non invasive carcinoma of the breast, gyecomastia, carcinoma, and primary carcinoma of the liver.

31. A method of making a composition, comprising forming the composition with a peptidomimetic macrocycle, or its pharmaceutically acceptable salt; wherein the peptidomimetic macrocycle is of Formula (I): 0 0 R7 R 8 [D], A,[vy[L-- [E]w R1 R2 L (Formula (I)), wherein: each A, C, D, and E is independently a natural or non-natural amino acid; R 3 N N H each B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L 3 CO-], [-NH-L 3 -SO 2 -], or [-NH-L 3 -]; R 1 and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is optionally substituted with halo-; R 3 is -H, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each non -H group being optionally substituted with R 5 ; L is a macrocycle-forming linker of the formula Li L 2 /_ NH N-N 54 LI L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -],, each being optionally substituted with R 5 ; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, CO 2 , or CONR 3 ; each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -SO 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent: each R is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with a D residue; R 8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each group except -H being optionally substituted with R 5 , or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5.

32. The method of claim 31, comprising making the composition with the peptidomimetic macrocycle.

33. The method of claim 31, comprising making the composition with the pharmaceutically acceptable salt of the peptidomimetic macrocycle. 55

34. Use of a peptidomimetic macrocycle or its pharmaceutically acceptable salt for the manufacture of a medicament for the treatment of a disease, wherein the peptidomimetic macrocycle is of Formula (I): 0 0 R7 R 8 N -N [D] N [AL-[B]y[C N [E]. R 1 R2 L (Formula (I)), wherein: each A, C, D, and E is independently a natural or non-natural amino acid; R3 HY each B is a natural or non-natural amino acid, amino acid analog, 0 , [-NH-L 3 CO-], [-NH-L 3 -SO2-], or [-NH-L 3 -]; R 1 and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is optionally substituted with halo-; R 3 is -H, alkyl, alkenyl. alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each non -H group being optionally substituted with Rs; L is a macrocycle-forming linker of the formula L1 L2 NH N-N; LI, L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -],, each being optionally substituted with R 5 ; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is 0, S, SO, SO 2 , CO, CO 2 , or CONR 3 ; each R 5 is independently halogen, alkyl, -OR 6 , -N(R6) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -CO 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; 56 R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each non -H group being optionally substituted with R 5 , or part of a cyclic structure with a D residue; R8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each non-H group being optionally substituted with R 5 , or part of a cyclic structure with an E residue; v is an integer from 1-1000; w is an integer from 1-1000; x is an integer from 0-10; y is an integer from 0-10; z is an integer from 0-10; and n is an integer from 1-5.

35. The use of claim 34, wherein the peptidomimetic macrocycle is used.

36. The use of claim 34, wherein the pharmaceutically acceptable salt of the peptidomimetic macrocycle is used.

37. The use of claim 34, wherein the disease is cancer.

38. The use of claim 37, wherein the cancer is selected from the group consisting of 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, Kaposi sarcoma, breast cancer, non invasive carcinoma of the breast, gyecomastia, carcinoma, and primary carcinoma of the liver. 57

39. A compound of Formula Ila or Ilb: R12 N3 Tg 9g T Qh R , h R 2 R7 N C0 2 R 11 8 N CO2R R 1 Rio Ila Ilb, wherein R 1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which is unsubstituted or substituted with halo-; each Q and T is independently selected from the group consisting of alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, and [-R 4 -K-R 4 -],, each of which is unsubstituted or substituted with R 5 ; K is 0, S, SO, S02, CO, C0 2 , or CONR 3 ; R 3 is -H, alkyl optionally substituted with R 5 , alkenyl optionally substituted with R 5 , alkynyl optionally substituted with R 5 , arylalkyl optionally substituted with R 5 , heteroalkyl optionally substituted with R 5 , cycloalkyl optionally substituted with R 5 , heterocycloalkyl optionally substituted with R 5 , cycloalkylalkyl optionally substituted with R 5 , cycloaryl optionally substituted with R 5 , or heterocycloaryl optionally substituted with R 5 ; R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R 7 and Rs are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl; Rio and R, 1 are independently -H or any protecting group suitable for peptide synthesis; 58 R 12 is -H or alkyl; g and h are each independently an integer from I to 5; and n is an integer from I to 5.

40. The compound of claim 39, wherein in the compound of Formula Ila R 1 is alkyl unsubstituted or substituted with halo-, or in the compound of Formula Ilb R 2 is alkyl unsubstituted or substituted with halo-.

41. The compound of claim 39, that is a compound of Formula Ila.

42. The compound of claim 39, wherein in the compound of Formula Ia R is unsubstituted alkyl, or in the compound of Formula Ilb R 2 is unsubstituted alkyl.

43. The compound of claim 39, that is a compound of Formula Ilb.

44. The compound of claim 39, wherein in the compound of Formula Ia R 1 is methyl, or in the compound of Formula Ilb R 2 is methyl.

45. The compound of claim 39, wherein in the compound of Formula Ila R 12 is hydrogen.

46. The compound of claim 39, wherein in the compound of Formula IHa or Ilb, at least one of R 10 and R 11 is a protecting group suitable for peptide synthesis. 59

47. A kit comprising a) at least one compound selected from the group consisting of compounds of Formulas Ila and Ilb: R 1 2 N3 Tg T9 Q R Ih R 2 R7 N C0 2 R 1 1 R8NN C0 2 R 11 R10 R, 0 Ila Ib, wherein R 1 is alkyl unsubstituted or substituted with halo-, alkenyl unsubstituted or substituted with halo-, alkynyl unsubstituted or substituted with halo-, arylalkyl unsubstituted or substituted with halo-, cycloalkylalkyl unsubstituted or substituted with halo-, heteroalkyl unsubstituted or substituted with halo-, or heterocycloalkyl unsubstituted or substituted with halo-; R 2 is -H, alkyl unsubstituted or substituted with halo-, alkenyl unsubstituted or substituted with halo-, alkynyl unsubstituted or substituted with halo-, arylalkyl unsubstituted or substituted with halo-, cycloalkylalkyl unsubstituted or substituted with halo-, heteroalkyl unsubstituted or substituted with halo-, or heterocycloalkyl unsubstituted or substituted with halo; each Q and T is independently selected from the group consisting of alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, and [-R4-K-R 4 -], each of which is unsubstituted or substituted with R 5 ; K is 0, S, SO, SO 2 , CO, C0 2 , or CONR 3 ; R 3 is hydrogen, alkyl optionally substituted with R 5 , alkenyl optionally substituted with R 5 , alkynyl optionally substituted with R 5 , arylalkyl optionally substituted with R 5 , heteroalkyl optionally substituted with R 5 , cycloalkyl optionally substituted with R 5 , heterocycloalkyl optionally substituted with R 5 , cycloalkylalkyl optionally substituted with R 5 , cycloaryl optionally substituted with R 5 , or heterocycloaryl optionally substituted with R 5 ; 60 R4 is alkylene, alkenylene, alkynylene, heteroalkylene. cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each R5 is independently halogen, alkyl, -O1(, -N(R6) 2 , -SR 6 , -SOR 6 , -S0 2 R6, -C02R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R 7 and R8 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl; RIO and R, are independently -H or any protecting group suitable for peptide synthesis; R12 is -H or alkyl; g and h are each independently an integer from 0 to 5; n is an integer from I to 5; and b) a macrocyclization reagent.

48. The kit of claim 47, wherein in the compound of Formula Ila R 1 is alkyl unsubstituted or substituted with halo-, or in the compound of Formula Ilb R2 is alkyl unsubstituted or substituted with halo-.

49. The kit of claim 47, wherein the kit comprises a compound of Formula Ilb and the macrocyclization reagent.

50. The kit of claim 47, wherein in the compound of Formula Ila R1 is unsubstituted alkyl, or in the compound of Formula Ilb R 2 is unsubstituted alkyl.

51. The kit of claim 47, wherein in the compound of Formula Ila R12 is -H.

52. The kit of claim 47, wherein in the compound of Formula Ila R 1 is methyl, or in the compound of Formula Ilb R 2 is methyl.

53. The kit of claim 47, wherein the kit comprises a compound of Formula Ila and the macrocyclization reagent.

54. The kit of claim 47, wherein in the compound of Formula Ila or Ib, at least one of RIO and R, is a protecting group suitable for peptide synthesis. 61

55. The kit of claim 47, comprising at least one compound of Formula Ila and at least one compound of Formula Ilb.

56. The kit of claim 47, wherein the macrocyclization reagent is a Cu reagent and in the compound of Formula Ila, R 12 is -H.

57. The kit of claim 47, wherein the macrocyclization reagent is a Ru reagent. 62