Bicyclic mimics-rigid, cell permeable, serum stable helical mimics

Abstract

Embodiments of the disclosure include compositions comprising peptides with an N-terminal capping motif or with the combination of an N-terminal capping motif and a C-terminal capping motif, and methods for synthesizing the peptides. The capping motifs can impart helicity to peptides that otherwise do not exhibit helicity. The helix capping motifs are conformationally rigid, improve peptide serum stability, and increase cell permeability of peptides.

Description

BICYCLIC MIMICS-RIGID, CELL PERMEABLE, SERUM STABLE HELICAL MIMICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the U.S. provisional application number 63 / 519,491 filed August 14, 2023, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted via Patent Center in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on August 12, 2024 is named TAMU.P0004WO_SequenceListing.xml and is about 288,335 bytes in size.BACKGROUND

[0003] This invention was made with government support under R01EY029695 awarded by the National Institutes of Health and CHE 1608009 awarded by the National Science Foundation. The government has certain rights in the invention.I. Technical Field

[0004] This disclosure relates at least to the fields of synthetic organic chemistry and peptide chemistry.II. Background

[0005] The simplest level of protein structure is the sequence of amino acids in a polypeptide chain, and the amino acid sequence is commonly referred to as the primary structure. While primary structure describes the sequence of amino acids, polypeptides also have higher order structural patterns that affect the overall structure. Secondary structure refers to the local spatialconformation of the polypeptide backbone, excluding the side chains. The most common secondary structural elements are alpha helices, beta sheets, beta turns, and omega loops.

[0006] The alpha helix (a-helix) is a right hand-helix conformation in which hydrogen atoms within a N~H group within the peptide chain (at position i) bond to a peptide chain carbonyl group oxygen atom located four residues away (at position i + 4) along the protein sequence. The side chains of residues within the a-helix are directed outwards and away from the helical axis, and the occurrence of polar or charged side chains in the helix can facilitate additional interactions with other side chains in the helix or with other elements outside of the helical structure, imparting stability to the peptide.

[0007] Two techniques for mimicking a-helices include stapling one or two peripheral fragments to produce monocyclic rings and capping N- or C-termini with synthetic capping structures. Most stapled peptides feature N- and C-terminal exocyclic peptide regions that are vulnerable to conformational fraying, and proteolytic trimming by exopeptidases. Capping motifs kink terminal backbones out of helical torsions to terminate helices and satisfy overhanging H- bond donors or acceptors. It is common for protein ligands to present interface a-helices in proteinprotein interactions (PPIs). For example, multiple a-helices composed primarily of hydrophobic amino acids can give rise to transmembrane bundles through protein-protein and protein-lipid interactions. Consequently, interface a-helix mimicry has emerged as a new direction in treating diseases and strategy for the development of new drugs.

[0008] Capping moieties act as protective shields and can protect proteins from proteolytic degradation at N- and C-termini. This increases peptide half-life in biological systems and can be used to improve pharmacokinetic parameters by reducing degradation rate and increasing increase half-lives of therapeutic peptides, a-helix structures have been associated with cellular uptake, and inducing helix structures through the use of capping moieties can be used to increase uptake of therapeutic peptides.

[0009] Consequently, there is a need for new peptide capping structures that can improve pharmacokinetic properties of existing peptide-based therapeutics.SUMMARY

[0010] Some embodiments of the disclosure are directed to a dual capped peptide comprising a synthetic N-cap and a synthetic C-cap and having a structure of Formula I:any natural or unnatural amino acid and q is an integer from 1 to 40. In some embodiments, [Xaa]qis peptide comprising at least one helical domain. In some embodiments, the compound of Formula I, does not comprise a linkage between the synthetic N-cap and the synthetic C-cap other than [Xaa]q. In some embodiments, the synthetic N cap and / or the synthetic C-cap are bicyclic, tricyclic, or have a higher order cyclic structure. In some aspects, the synthetic N-cap and / or the synthetic C cap are bicyclic or tricyclic. In some aspects, the synthetic N-cap and / or the synthetic C cap are bicyclic. In some aspects, the synthetic N-cap and / or the synthetic C cap are tricyclic.

[0011] In some embodiments, the synthetic N-cap is represented by formula II:Wherein each U is independently S, O, or CH2, each V is independently H or alkyl, W isX1, X2, and X3are each independently S, O, Se, or NV, Y1, Y2, and Y3are each independently CH2, (CH2 or CMe2, A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid, AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond, and n is 1, 2, or 3. In some embodiments, AA1is D- or L-aspartic acid. In some embodiments, Y1, Y2, and Y3are CH2. In some embodiments, X1, X2, and X3are S. In specific embodiments, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In some embodiments, W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. In some embodiments, A1, A2, and A3are each H. In further embodiments, Z is H, alkyl, or an amine protecting group. In some embodiments, the N-cap further comprises at least one2H,3H,nC,13N, or15O isotope.

[0012] In some embodiments, the C-cap is represented by formula 111:wherein B is O, S, NH, NR, or a bond, each U is independently S, O, or CH2, each V is independently H or alkyl, W isX4, X5, and X6are each independently S, O, Se, or NV, Y4, Y3, and Y6are each independently CH2, (CH2)2, or CMe2, A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one nonnatural amino acid, AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond, and n is 1, 2, or 3. In some embodiments, Y4, Y5, and Y6are CH2. In some embodiments, X4, X5, and X6are S. In further embodiments, X4, X3, and X6are S and Y4, Y5, and Y6are CH2. In some embodiments, AA6is glycine. In some embodiments, W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. For instance, in some embodiments, A1, A2, and A3are each H. In further embodiments, R is H, alkyl, aryl, or a carboxy protecting group. In some embodiments, the C-cap further comprises at least one2H,3H, "C,13N, or15O isotope. In some embodiments, the peptide is represented by formula I-A:wherein B is O, S, NH, NR, or a bond, each U is independently S, O, or CH2,, each V is independently H or alkyl; each W is independentlyX1, X2, X3, X4, X5, and X6are each independently S, O, Se, or NV, Y1, Y2, Y3, Y4, Y5, and Y6are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid, Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid, AA1, AA2, AA3, AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond, n is 1, 2, or 3, Xaa is any natural amino acid or any unnatural amino acid, and q is an integer from 1 to 40. In someembodiments, Y1, Y2, and Y3are CH2. In some embodiments, Y4, Y5, and Y6are CH2. In further embodiments, Y1, Y2, Y3, Y4, Y5, and Y6are CH2. In some embodiments, X1, X2, and X3are S. In some embodiments, X4, X5, and X6are S. In further embodiments, X1, X2, X3, X4, X5, and X6are S. In some embodiments, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In some embodiments, X4, X5, and X6are S and Y4, Y5, and Y6are CH2. In further embodiments, X1, X2, X3, X4, X5, and X6are S and Y1, Y2, Y3, Y4, Y5, and Y6are CH2. In some embodiments, each W isand A1, A2, and A3are each independently hydrogen, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. For instance, in some embodiments, A1, A2, and A3are each hydrogen. In further aspects, R is H, alkyl, aryl, or a carboxy protecting group and Z is H, alkyl, or an amine protecting group. In some embodiments, a dual capped peptide that includes a synthetic N-cap and a synthetic C-cap does not comprise a 53-atom macrocycle (e.g., as used in Sulanemadlin (ALRN-6924) as described in Guerlavis et al., J Med Chem. 2023 Jul 27;66(14):9401-9417, which is incorporated herein by reference in its entirety). That is, the dual capped peptide does not comprise Sulanemadlin (ALRN-6924).

[0013] Some embodiments of the disclosure are directed to an N-capped peptide of formulaIV:wherein each U is independently S, O, or CH2, each V is independently H or alkyl, W isX1, X2, and X3are each independently S, O, Se, or NV, Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2, A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid, AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond, n is 1, 2, or 3, Xaa is any natural amino acid or any unnatural amino acid; and q is an integer from 1 to 40. In some embodiments, AA1is D- or L-aspartic acid. In some embodiments, Y1, Y2, and Y3are CH2. In some embodiments, X1, X2, and X3are S. In further embodiments, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In some embodiments, W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. In some embodiments, A1, A2, and A3are each H. In some embodiments, Z is H, alkyl, or an amine protecting group. In some embodiments, the peptide further comprises at least one2H,3H,nC,13N, or15O isotope.

[0014] Some embodiments of the present disclosure are directed to a capping moiety for capping the N-terminal region of a peptide, comprising a compound of formula II as provided above.

[0015] Some embodiments of the disclosure are directed to a method of imparting helicity to at least a portion of an N-terminal region of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide. Some embodiments of the disclosure are directed to a methodof reducing systemic degradation of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide. Some embodiments of the disclosure are directed to a method of increasing cellular uptake of a peptide, comprising attaching a compound of formula II to an N- terminus of the peptide.

[0016] Some embodiments of the disclosure are directed to a method of imparting helicity to at least a portion of an N-terminal region of a peptide and to at least a portion of a C-terminal region of the peptide, comprising attaching a compound of formula II to an N-terminus of the peptide and attaching a compound of formula III, as provided above, to the C-terminus of the peptide.

[0017] Some aspect of the disclosure are directed to a method of reducing systemic degradation of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide and attaching a compound of formula III to a C-terminus of the peptide. Some aspect of the disclosure are directed to a method of increasing cellular uptake of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide and attaching a compound of formula III to a C-terminus of the peptide.

[0018] Some aspects of the disclosure are directed to a method of synthesizing an N-capped peptide, the method comprising reacting a peptide of Formula IV-A:wherein each U is independently S,O, or CH2; each V is independently H or alkyl; K1, K2, and K3are each independently SH, OH, SeH, NH2, or NHV, Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; with a compound selectedfrom the group consisting of;wherein: n is 1, 2, or 3; and L1, L2, and L3are each independently selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate; and A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl.

[0019] Some aspects of the disclosure are also directed to methods of synthesizing a dualcapped peptide of Formula I. Accordingly, in a particular embodiment, a method for synthesizing a peptide of Formula I-AI is provided. In an aspect, the method comprises:(a) reacting an immobilized peptide of Formula III-A:wherein each U is independently S,O, or CH2; each V is independently H or alkyl, K4, K?, and K6are each independently SH, OH, SeH, NH2, or NHV; Y4, Y5, and Y6are each independently CH2, (CH2)2, or CMe2; AA4, AA5, and AA6are each independently any natural amino acid, unnatural amino acid, or a bond; with a compound selected from the group consisting of:and;wherein: n is 1, 2, or 3; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; and L1, L2, and L3are each a leaving group (e.g., selected from the group consisting of F, Cl, Br, I, mesylate, triflate, and tosylate, and optionally further selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate) to form an immobilized cyclized peptide of Formula III-B:independently S, O, Se, or NV; and W is W is(b) ligating the immobilized cyclized peptide of Formula III-B to a second peptide comprising Formula IV-A:wherein U and V are as previously defined; K1, K2, and K3are each independently SH, OH, SeH, NH2, or NHV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; AA1, AA2and AA3are each independently any naturalamino acid, any unnatural amino acid, or a bond; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; to form an immobilized peptide of Formula I-A-a:wherein U, Z, V, W, AA1, AA2, AA3, AA4, AA5, AA6, K1, K2, K3, X4, X5, X6, Y1, Y2, Y3, Y4, Y3, and Y6, Xaa , and q are as previously defined;(c) cleaving the peptide of Formula (I-A-a) to form a non-immobilized peptide of Formula I-A-b:wherein B is O, S, NH, NR, or a bond; R is alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; and Z, U, V, W, K1, K2, K3, X4, X3, X6, Y1, Y2, Y3, Y4, Y3, Y6, AA1, AA2, AA3, AA4, AA5, AA6, Xaa, n and q are as previously defined; (d) reacting the peptide of Formula IX-B with a compound selected from the group consisting of:3; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; and L1, L2, and L3are each independently a leaving group (e.g., selected from the group consisting of F, Cl, Br,I, mesylate, triflate, and tosylate, and optionally further selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate), to form a dual capped peptide of Formula I-A:

[0020] Some aspects of the present disclosure are also directed to compounds comprising the structure of Formula V:wherein each U is independently S, O, orCH2; each V is independently H or alkyl;; X , X , and X are each independently S, O, Se, or NV; Y , Y , and Y are each independently CH2, (CH2 or CMe2 ; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; B is O, S, NH, NR, or a bond; R is hydrogen, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3. In some aspects, a compound of Formula V is provided wherein R is H, alkyl, aryl, or a carboxy protecting group.

[0021] Also provided herein are methods of imparting helicity to at least a portion of an N- terminal region of a peptide and to at least a portion of a C-terminal region of the peptide, the method comprising attaching a synthetic N-terminal cap to the N-terminal region of the peptide and a synthetic C-terminal cap to the C-terminal region of the peptide. In some aspects, one or more of the synthetic N-terminal cap and the synthetic C-terminal cap is bicyclic, tricyclic, or has a higher order cyclic structure. For instance, one or more of the synthetic N-terminal cap and the synthetic C-terminal cap may be bicyclic. In another aspect, one or more of the synthetic N-cap and the synthetic C-cap may be tricyclic.

[0022] Also provided herein are methods of reducing systemic degradation of a peptide, the methods comprising attaching a synthetic N-terminal cap to an N-terminal region of the peptide and a synthetic C-terminal cap to a C-terminal region of the peptide. In some aspects, one or more of the synthetic N-terminal cap and the synthetic C-terminal cap is bicyclic, tricyclic, or has a higher order cyclic structure. For instance, one or more of the synthetic N-terminal cap and the synthetic C-terminal cap may be bicyclic. In another aspect, one or more of the synthetic N-cap and the synthetic C-cap may be tricyclic.

[0023] Also provided herein are methods of increasing cellular uptake of a peptide, the method comprising attaching a synthetic N-terminal cap to an N-terminal region of the peptide and asynthetic C-terminal cap to a C-terminal region of the peptide. In some aspects, one or more of the synthetic N-terminal cap and the synthetic C-terminal cap is bicyclic, tricyclic, or has a higher order cyclic structure. For instance, one or more of the synthetic N-terminal cap and the synthetic C-terminal cap may be bicyclic. In another aspect, one or more of the synthetic N-cap and the synthetic C-cap may be tricyclic.

[0024] Also provided are compounds comprising, consisting essentially of or consisting of a synthetic N-cap covalently linked to a synthetic C-cap. In an aspect, one or more of the synthetic N-cap and the synthetic C-cap may be bicyclic, tricyclic, or has a higher order cyclic structure. In an aspect, one or more of the synthetic N-cap and the synthetic C-cap may be bicyclic. In an aspect, one or more of the synthetic N-cap and the synthetic C-cap may be tricyclic. In an aspect, compounds comprising, consisting essentially of, or consisting of a synthetic N-cap covalently linked to a synthetic C-cap may comprise a structure of Formula V:wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is independently H or alkyl; each W is independentlyor; X1, X2, X3, X4, X5, and X6are each independently S, O, Se, or NV; Y1, Y2,Y3, Y4, Y3, and Y6are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, AA3, AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond; n is 1, 2, or 3; and Q is a bond or alkyl.

[0025] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the disclosure.

[0026] Other objects, features and advantages of the subject matter of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be betterunderstood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0028] FIGS. 1A-1C: Synthetic N-Caps And Natural ASX Motifs. FIG. 1 A Hydrogen bond surrogate (PDB 4MZL) where an alkene mimics mNcap,mN4. FIG. IB Peptidic strap N-cap mimic (5GS4) where an D-iso-Asp at Neap, is amide-linked to L-Dap at N3. FIG. 1C An illustrative, natural ASX motif (1A2X) showing the two key H-bonds.

[0029] FIGS. 2A-2L: Discovery and Analyses of Hydrophobic Triangles. FIG. 2A ASX / ST Search flow. FIG. 2B Typical capping box (mNcap,sN3 H-bond) incorporating an Asp Neap and Glu N3. FIG. 2C Hydrophobic patches formed by hydrophobic side chains at N’ and N4. FIG. 2D ASX / ST Search revealed hydrophobic patches are frequently augmented by hydrophobic side chain fragments at N3 to form hydrophobic triangles. FIG. 2E Numbers, percentages and relationships of hydrophobic patches and triangles in the key data set.. FIG. 2F Hydrophobic triangles favor Leu, Leu at N’,N4. FIG. 2G Hydrophobic side chain fragments at N3 can be provided by conventionally hydrophilic residues. FIG. 2H N’ - N3 and N’ - N4 distances in the absence of a capping box. FIG. 21 N’ - N3 and N’ - N4 distances with a capping box. FIG. 2J Illustrative example of the dual role of N3 Glu, ie facilitating hydrophobic triangles and capping boxes. FIG. 2K Potential cross-turn hydrophobic interactions between side chains of (N’,N4), and / or (N’,N3). A ID array, [(N’,N4), (N’,N3)], was used to describe hydrophobic contacts. FIG. 2L Illustrative hydrophobic triangle at helical N-cap from PDB 1Q5V.

[0030] FIGS. 3 A-3M: Model Peptides. FIG. 3 A Systematic incorporation of ASX motif. FIG. 3B Systematic incorporation of a patch. FIG. 3C Systemic incorporation of a triangle. FIG. 3D Helicity trend on sequentially introducing stabilizing factors. FIG. 3E Ala scan over the four key residues shown. FIG. 3F Helicity of a 12-mer on inclusion of an ASX motif and a triangle. FIG. 3G shows representative CD spectra of AALA, ADAA, LDAL, and LDLL peptides (e.g., see FIG. 3D). FIG. 3H shows representative CD spectra of LALL, ADLL, LDLA, LDAL and LDLL peptides (e.g., see FIG. 3E). FIG. 31 shows representative CD spectra of 12, 17 - and 22-mers of AAKA and LDLL. FIG. 3J- 3K show representative CD spectra (FIG. 3J) and % helicity (FIG. 3K) of AAKA, ADAA, LDEL, LDQL, LDTL, LDSL LDAL, LDAL, LDLL, and LDFL peptides.FIG. 3L-3M show representative CD spectra (FIG. 3L) and % helicity (FIG. 3M) of LDLL, IDLL, LDLI, VDLL, LDLV, FDLL, and LDLF peptides.

[0031] FIGS. 4A-4F: Bicyclic ASX Motif Mimics (BAMMs). FIG. 4A Conceptual relationship between an ASX motif hydrophobic triangle and a BAMM. FIG. 4B Quantitated helical residence times in MD for a 13-mer linear control VFW and BAMMs with all possible Cys-stereochemical combinations. FIG. 4C Structures of control peptides without (linear) or with only one (monocyclic) ring constraining groups compared with the bicyclo 12-mer. FIG. 4D CD data for two linear and two monocyclic controls and the bicyclic 12-mer in 10% trifluoroethanol (TFE) / phosphate buffered saline (PBS). FIG. 4E Relative %helicity for this peptide series show the same trends in 10% TFE / PBS and PBS. FIG. 4F VT-CD for the featured peptides in 10% TFE / PBS.

[0032] FIGS. 5A-5I: Solution Structures Of LDLL And Bicyclo 12-mers From Conformational Sampling With NMR Constraints. FIG. 5A Comprehensive ensemble of structures within 3 kcal’mol-1 of the minimum energy conformer located for the LDLL 12-mer. FIG. 5B Stick diagram of representative conformers of LDLL 12-mer highlighting three Leu side chains that comprise the hydrophobic triangle. Dashed lines represent hydrophobic interactions. FIG. 5C Space-fdling diagram of representative conformers of LDLL 12-mer highlighting three Leu side chains that comprise the hydrophobic triangle. FIGS. 5D-5E Corresponding diagrams for the bicyclo 12-mer where the TMB fragment mimics the hydrophobic triangle. Dashed lines highlighting H-bonds in ASX motifs. FIG. 5F Diagram demonstrating N’ Cys CaH and Neap Asp NH of the bicyclo 12-mer are both under the TMB aromatic ring, with the Ca being closest. Ramachandran plots of the low energy conformers for linear LDLL (FIG. 5G), and bicyclo (FIG. 5H) 12-mer. FIG. 51 Graph demonstrating protection factors for the bicyclo 12-mer around the capping region are significantly higher than for the linear LDLL 12-mer consistent with reduced H / D exchange rates caused by the rigidity of the bicyclo N-cap.

[0033] FIGS. 6A-6L: BAMM Mimics Of Natural Interface Helices. FIGS 6A-6C Diagrams depicting which interface helices can interact with their receptors (FIG 6A PDB 1L8C; FIG. 6B 6M0J; FIG. 6C 4A0M). FIG. 6D CD spectra of interface helix from BH3 in BH3«Bcl-Xl. FIG. 6E CD spectra of interface helix HIF-la in HfF-la’p300. FIG. 6F CD spectra of interface helix fromCDK2 in CDK2»cyclin E. FIG. 6G CD spectra of interface helix from myoA in myoA’MTIP. FIG. 6H CD spectra of interface helix from ACE2 in ACE2*RBD of spike protein. FIG. 61 Half-lives of 6D-6H in 25% human serum measured at 37 °C. FIG. 6J CDK2*cyclin E from PDB 1W98 showing a tryptophan in cyclin E directly impacting the c-helix of CDK2. FIG. 6K Relative fluorescence intensity as a function of molar ratios of 3:cyclin (left panel) and change in fluorescence over max as a function of concentration of peptides 3 (right panel). FIG. 6L Uptake of peptides 3 into breast cancer cells after incubation at 37 °C: triple negative (MDA-MB-231), and ER+ (MCF-7).

[0034] FIG. 7: Table depicting linear peptides and respective percent helicity in PBS buffer.

[0035] FIG. 8: Table depicting percent helicity and ellipticity ratios 222 / 208 nm of controls and bicyclo 12-mers in PBS and in 10% TFE / PBS.

[0036] FIG. 9: Table depicting percent helicity, ellipticity ratio 222 / 208 nm and half-life in 25% human serum of wild-type and modified biological sequences. Bold residues in linear sequences for residues which may be mutated to form BAMM A'-cap; C* for Cys(Me).

[0037] FIG. 10: Table depicting sequences and binding affinities of fluorescent labeled peptides examined in fluorescence polarization assay.

[0038] FIG. 11 : Diagram depicting synthesis route of linear peptides.

[0039] FIG. 12: Diagram depicting synthesis route of bicyclic capped peptides.

[0040] FIGS. 13A-13E: Degradation progression of series 1 peptide IB. FIG. 13A Speculated degradation process of IB. FIGS. 13B-13E RP-HPLC traces of IB, intermediates, and A1B at 0 h (FIG.13B), 6 h (FIG. 13C), 24 h (FIG. 13D), and 72 h (FIG. 13E).

[0041] FIGS. 14A-14B: Degradation kinetic curves of series 1 peptides. FIG. 14A Kinetic curves of series 1 peptides over 8 hours. FIG. 14B A1B formed as IB was exposed to 25% serum solution.

[0042] FIGS. 15A-15D: Degradation progression of series 2 peptide 2B. FIG. 15A Speculated degradation process of 2B. FIGS. 15B-15D RP-HPLC traces of 2B, intermediates, and A2B at 4 h (FIG.15B), 8 h (FIG. 15C), and 72 h (FIG. 15D).

[0043] FIGS. 16A-16B: Degradation kinetic curves of series 2 peptides. FIG. 16A Kinetic curves of series 2 peptides over 8 hours. FIG. 16B A2B formed as 2B was exposed to 25% serum solution.

[0044] FIGS. 17A-17E: Degradation progression of series 3 peptide 3B. FIG. 17A Speculated degradation process of 3B. FIGS. 17B-17E RP-HPLC traces of 3B, intermediates, and A2B at 0 h (FIG.17B), 8 h (FIG. 17C), 24 h (FIG. 17D), and 72 h (FIG. 17E).

[0045] FIGS. 18A-18B: Degradation kinetic curves of series 3 peptides. FIG. 18A Kinetic curves of series 3 peptides over 8 hours. FIG. 18B A3B formed as 3B was exposed to 25% serum solution.

[0046] FIGS. 19A-19C: Degradation progression and kinetic curves of series 4 peptide 4B. FIG. 19A Speculated degradation process of 4B. FIG. 19B RP-HPLC trace of 4B, intermediates, and A4B at 24 h. FIG. 19C Kinetic curves of series 4 peptides over 8 hours.

[0047] FIGS. 20A-20C: Degradation progression and kinetic curves of series 5 peptide 5B. FIG. 20A Speculated degradation process of 5B. FIG. 20B RP-HPLC trace of 5B, intermediates, and A5B at 24 h. FIG. 20C Kinetic curves of series 5 peptides over 8 hours.

[0048] FIG. 21 : Table that includes sequences of N-capped and dual-capped (N-capped and C-capped) peptides examined in cellular uptake experiments.

[0049] FIGS. 22A-22C: Quantification of cellular uptake in different cell lines. FIG. 22A Quantification of peptides at 1 pM concentration in HepG2 cells. FIG. 22B Quantification of peptides at 1 pM concentration in MCF-7 cells. FIG. 22C Quantification of peptides at 1 pM concentration in MDA-MB-231 cells.

[0050] FIGS. 23A-23C: Quantification of cellular uptake in different cell lines. FIG. 23A Quantification of peptides at 10 pM concentration in HepG2 cells. FIG. 23B Quantification ofpeptides at 10 pM concentration in MCF-7 cells. FIG. 23C Quantification of peptides at 10 pM concentration in MDA-MB-231 cells.

[0051] FIGS. 24A-24E: Confocal imaging of MCF-7 cells treated with test compounds at 4 hours. FIG. 24A Control. FIG. 24B Linear peptide. FIG. 24C N-capped peptide. FIG. 24D C- capped peptide. FIG. 24E Dual-capped peptide.

[0052] FIGS. 25A-25E: Confocal imaging of MCF-7 cells treated with test compounds at 25 hours. FIG. 25 A Control. FIG. 25B Linear peptide. FIG. 25C N-capped peptide. FIG. 25D C- capped peptide. FIG. 25E Dual-capped peptide.

[0053] FIG. 26: Synthetic approach for synthesis of a dual -capped peptide.

[0054] FIGS. 27A-27B: FIG. 27A Table that includes sequences of N-capped and dual -capped (N-capped and C-capped) peptides examined in CD and degradation experiments. FIG. 27B CD spectra of peptides in PBS buffer at 25 °C.

[0055] FIGS. 28A-28B: CD and Variable temperature circular dichroism (VTCD) studies. FIG. 28 A VTCD results with dual-capped peptide tested at 5 pM and other peptides at 10 pM. FIG. 28B Concentration-dependent CD of dual-capped peptide.

[0056] FIGS. 29A-29B: Degree of helical induction. FIG. 29A Concentrations of stock solutions were determined by CD spectra. FIG 29 Dual-capped peptides formed coiled coils at increased concentrations.

[0057] FIGS. 30A-30B: Examination of serum stability over time. FIG. 30A Graph depicting serum stabilities of peptides over 8 hours. FIG. 30B Graph depicting serum stabilities of peptides over 96 hours.

[0058] FIGS. 31 A-3 ID: Binding affinities for fluorescent peptides. FIG. 31 A Linear peptide. FIG. 3 IB C-capped peptide. FIG. 31C N-capped peptide. FIG. 3 ID Dual-capped peptide.

[0059] FIGS. 32A-32D: FIG. 32A-32B show differences between N- and C-terminal hydrophobic triangles, respectively. FIG. 32C-32D show diagrams of linear and BAMM N- capped, and BSM C-capped systems (FIG. 32C) and results from VT-CD experiments showing222 nm ellipticity for BAMM N-capped, BSM C-capped systems (blue and red, respectively) relative to linear controls (black) from 5 to 90 °C.

[0060] FIG. 33: Representative schematic of a helical interface mimic prepared with dual bicyclic caps.

[0061] FIGS. 34A-34D: Typical: (FIG. 34A) hydrocarbon staple (PDB 4MZK); (FIG. 34B) synthetic N-caps (PDB 4MZL and 5GS4); (FIG. 34C) natural caps with hydrophobic triangles (upper row) and corresponding synthetic bicyclic caps (bottom row); and (FIG. 34D) a dual cap comprising a BAMM and BSM.

[0062] FIG. 35A-35C shows schematics (FIG. 35A) and MD traces of linear, N-terminal, C- terminal capped and dual-capped peptides (FIG. 35B) and staple-a, dual-a, HBS-a, and strap-a peptides (FIG. 35C).

[0063] FIGS. 35D shows a flow chart with different reaction protocols to synthesize dualcapped peptides.

[0064] FIG. 35E shows crude purity of dual by anal-HPLC: construction of the BSM first (red trace, I— II in part c) and then the BAMM fragments (blue, III, and purple, IV).

[0065] FIG. 35F shows a schematic of a synthesis protocol to prepare dual-capped peptides.

[0066] FIGS. 36A-36G: FIG. 36A shows a C-helix binding cyclin E (1W98). Cyclin E hot spots are in magenta, and tryptophan (W, cyan) is used for FQ. FIG. 36B shows sequences of the test compounds. FIG. 36C depicts UV absorbance spectra (260-320 nm) of tested peptides normalized by absorbance intensities at 275 nm. FIG. 36D shows calibration of the UV absorbance coefficient at 275 nm of trithiol-alkylated 1,3, 5 -trimethylbenzene (TMB) by Beer-Lambert Law. FIG. 36E shows CD spectra for linear, BAMM, BSM, and dual in PBS pH 7.4, 25 °C. FIG. 36F shows CD spectra for dual as well as staples 1 and 2 under the same conditions. FIG. 36G shows experimental percent helicity of the tested peptides.

[0067] FIGS. 37A-37D: FIG. 37A shows CSIs of dual C-helix, showing transitions between the N-cap (blue) to helix (green) to C-cap (red). FIG. 37B shows 31 low-energy conformers of dual. FIG. 37C shows ramachandran plot of backbone dihedrals in dual low-energy conformers(favored and allowed (p,v ’nprotein conformations (excluding Pro and Gly) shown in deep and light blue backgrounds). FIG. 37D shows lowest-energy conformer with BAMM N-cap (blue dotted boxes) and BSM C-cap (red) in the dual peptide.

[0068] FIGS. 38A-38G: FIG. 38A shows FQ binding data for six unlabeled peptides / peptidomimetics with cyclin E. FIGS. 38B-38C shows FP binding of dual-f and staple-1- f by cyclins E and Al. FIG. 38D shows mimic degradation profdes in 25% human serum at 37 °C. FIG. 38E shows major degradation products of BAMM and BSM-capped peptides after 48 h. FIG. 38F-38G shows the degradation process of dual-capped C-helix in 48 h (FIG. 38F) and corresponding analytical HPLC traces at 0, 8, 24, and 48 h (FIG. 38G).

[0069] FIGS. 39A-39F: FIG. 39A-39C show cell uptake into three cell lines (MDA-MB-231 , triple negative breast cancer (FIG. 39A); MCF-7, ER+ breast cancer (FIG. 39B); HepG2, liver cancer (FIG. 39C)) after incubation of 1 pM test compounds at 37 °C, as a function of time. FIG. 39D shows fluorescence spectra as a function of concentrations indicate a difference in quantum yields corresponding to variable fluorescein environments. FIG. 39E shows an illustrative western blot of cyclin E shows relative levels of expression in those three cell lines. FIG. 39F shows a comparison of levels of cyclin E expression with uptake of the dual peptide for the three cell lines.

[0070] FIGS. 40A-40B: FIG. 40A shows comparisons between linear, BAMM or BSM monocapped, BAMM / BSM dual-capped, and hydrocarbon-stapled peptides in terms of cost and biophysical properties. FIG. 40B show diagrams of helices in protein ligands in the context of a receptor - and show how they can be totally enclosed by the receptor, exposed on one flank, or extended beyond the receptor. BAMM N-caps, for instance, can be used in all three situations, as described in the text. Green for native helices; blue for added or mutated residues or fragments to install BAMM N-caps.

[0071] FIGS. 41A-41D shows CD curves of dual C-helix (FIG. 41 A), staple-1 (FIG. 41B), and staple-2 (FIG. 41C) at concentrations in the range of 5 to 30 pM in PBS buffer. FIG. 41D shows variations of molar 9

[0222] against various concentrations.

[0072] FIGS. 42A-42F shows smoothed CD curves of tested peptides in TFE / PBS solutions. FIG. 42A is Linear C-helix in PBS, 10%, 50% and 60% TFE / PBS; FIG. 42B is BAMM C-helixin PBS, 40% and 50% TFE / PBS; FIG. 42C is BSM C-helix in PBS, 40% and 50 % TFE / PBS; FIG. 42D is dual C-helix in PBS and 10% - 50 % TFE / PBS; FIG. 42E is staple-1 in PBS, 40% and 50 % TFE / PBS; FIG. 42F is staple-2 in PBS, 40% and 50 % TFE / PBS.

[0073] FIGS. 43A-43E show: (FIG. 43A): analytical HPLC traces of BAMM degradation mixture at 48 hour time point, and the MS spectra of the major degradation product obtained from LCMS, (FIG. 43B): analytical HPLC trace of BSM degradation mixture at 48 hour time point, and the MS spectra of the major degradation product obtained from LCMS (FIG. 43C): Analytical HPLC trace of dual degradation mixture at 48 hour time point, and the MS spectra of the major remnants obtained from LCMS; (FIG. 43D) Analytical HPLC trace of staple- 1 degradation mixture at 48 hour time point, and the MS spectra of the major remnants obtained from LCMS; and (FIG. 43E): analytical HPLC trace of staple-2 degradation mixture at 48 hour time point, and the MS spectra of the major remnants obtained from LCMS.

[0074] FIGS. 44A-44B shows smoothed CD curves of dual C-helix in 0 - 50% TFE / PBS solution (FIG. 44A) and variations of molar ellipticity 0

[0223] with increase of %TFE (FIG. 44B).

[0075] FIGS. 45A-45C shows fingerprint regions (6.5 - 9.0 ppm) of ID H-H spectra for C- helix dual in aqueous solutions with 20% TFE-di (FIG. 45 A), 30% TFE-ds (FIG. 45B) or 35% TFE-d3(FIG. 45C).

[0076] FIGS. 46A-46B shows a side views of benzene ring and the six affected Hs illustrating that they are partially overlapped in the BAMM N-cap (FIG. 46A) and BSM C-cap region (FIG. 46B).

[0077] FIGS. 47A-47D shows raw data of fluorescence polarization assays for linear-f (FIG. 47A), BAMM-f (FIG. 47B), BSM-f (FIG. 47C) or staple-2-f (FIG. 47D).

[0078] FIG. 48 is a representative schematic of a protocol for synthesizing linear peptides.

[0079] FIG. 49 is a representative schematic of a protocol for synthesizing bicyclic C- capped peptides.

[0080] FIG. 50 is a representative schematic of a protocol for synthesizing bicyclic A-capped peptides.

[0081] FIG. 51 is a representative schematic of a protocol for synthesizing hydrocarbon stapled peptides.

[0082] FIG. 52 is a representative schematic of a protocol for synthesizing dual capped peptides. Residues E, Y, D, S, R were in the form of E(t-Bu), Y(t-Bu), D(t-Bu), S(t-Bu), R(Pbf) before cleavage in 95% TFA solution.

[0083] FIG. 53 is a representative HPLC trace showing a reaction mixture formed from a one- pot synthesis scheme, where both BAMM and BSM caps were prepared in solution.

[0084] FIG. 54A-54C shows representative HPLC traces of starting linear peptides and reaction mixtures after cyclization after protocols using BSM-C-Cap on a solid phase (FIG. 54A) or BAMM-N-Cap on a solid phase (FIG. 54B) as well as a graphic depicting differences between resin-linked C- and N-terminal Cys groups (FIG. 54C).

[0085] FIGS. 55A-55B show crude analytical HPLC traces of starting peptides and reaction mixtures obtained following formation of BSM-C-cap on a solid phase (FIG. 55A) and BAMM N-Cap in a solution phase (FIG. 55B) before synthesis of dual C-helix (dual-capped peptides).DETAILED DESCRIPTIONExamples of Definitions

[0086] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0087] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0088] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0089] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0090] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0091] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0092] A synthetic N-cap is a non-natural, man-made capping moiety that terminates the helical character of the N-terminal region of a peptide. The term N-cap refers to a capping moiety that is located at the N-terminus of a peptide. In some embodiments, the N-cap includes structural features that are chemically bonded to the N-terminal amino acid, N-terminal amino acid +1, N- terminal amino acid +2, N-terminal amino acid +3, N-terminal amino acid +4, N-terminal amino acid +5, N-terminal amino acid +6, and / or N-terminal amino acid +7. The phrase “N-terminal amino acid +1” refers to the second amino acid, when counting from the N-terminal end. The phrase Bicyclic ASX Motif Mimic or “BAMM” refers to an N-cap. Unless otherwise noted, the terms N-cap, N-cap moiety, and N-capping moiety are used interchangeably herein. A natural N- cap is a capping moiety that is synthesized by natural means and caps the N-terminal region of a peptide. In an aspect, a synthetic N-cap can mimic the structure of a natural N-cap, through use of certain structural domains. For instance, the synthetic N-cap may mimic the structure of an ASX motif, as described further in the Examples below.

[0093] A synthetic C-cap is a non-natural, man-made capping moiety that terminates the helical character of the C-terminal region of a peptide. The term C-cap refers to a capping moiety that is located at the C-terminus of a peptide. In some embodiments, the C-cap includes structural features that are chemically bonded to the C-terminal amino acid, C-terminal amino acid +1, C- terminal amino acid +2, C-terminal amino acid +3, C-terminal amino acid +4, C-terminal amino acid +5, C-terminal amino acid +6, and / or C-terminal amino acid +7. The phrase “C-terminal amino acid +1” refers to the second amino acid, when counting from the C-terminal end. The phrase Bicyclic Schellman Loop Mimic or “BSM” refers to a C-cap. Unless otherwise noted, the terms C-cap, C-cap moiety, and C-capping moiety are used interchangeably herein. A natural C- cap is a capping moiety that is synthesized by natural means and caps the C-terminal region of a peptide. In an aspect, a synthetic C-cap can mimic the structure of a natural C-cap, through use of certain structural domains. For instance, the synthetic C-cap may mimic the structure of a natural Schellman Loop, as described further in the Examples below.

[0094] The term dual-capped peptide refers to a peptide that includes a first capping moiety at its N-terminus and a second capping moiety at its C-terminus. Preferably, the dual capped peptide does not comprise any linkage between the synthetic N cap and the synthetic C cap other than the peptide that connects them. That is, the synthetic N cap and the synthetic C cap are not covalently linked by any other moiety (e.g., a staple moiety or a macrocyclic compound). The term “staple” refers to a moiety in which a first amino acid side chain is covalently linked to a second amino acid side chain, where the first and second amino acids are in different loops within a helix. One example of a staple is a one-loop staple between amino acids at positions i and i+4. Another example of a staple is a two-loop staple between amino acids at positions i and i+7.

[0095] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a linear (i.e. unbranched) or branched carbon chain, which may be fully saturated, mono- or polyunsaturated. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Saturated alkyl groups include those having one or more carbon-carbon double bonds (alkenyl, also olefinic) and those having one or more carbon-carbon triple bonds (alkynyl). The groups, -CHi (Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr), -CH(CH3)2(zso-Pr), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 ( / .w-butyl), -C(CH3)3 (terZ-butyl), -CH2C(CH3)3 (N -pentyl), are all non-limiting examples of alkyl groups. The alkyl groups employed herein can be substituted or unsubstituted.

[0096] The term "aryl" means a polyunsaturated, aromatic, hydrocarbon substituent. Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3 rings that are fused together or linked covalently). The term "heteroaryl" refers to an aryl group that contains one to four heteroatoms selected from N, O, and S. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The aryl groups employed herein can be substituted or unsubstituted.

[0097] The term “acyl” refers to the group — C(O)R, in which R is alkyl, aryl, aralkyl or heteroaryl, as those terms are defined above. The groups, — C(O)CHs (acetyl, Ac), — C(O)CH2CH3, — C(O)CH2CH2CH3, — C(O)CH(CH3)2, — C(O)CH(CH2)2, — C(O)C6H5, — C(O)CeH4CH3, — C(O)CH2CeH5, — C(O)(imidazolyl) are non-limiting examples of acyl groups.

[0098] The term “bicyclic” refers to a compound with two conjoined rings. The two conjoined rings can be conjoined in a fused, bridged, or spiro arrangement. A fused bicyclic compound includes two rings that share two atoms. A bridged bicyclic compound includes two rings that share three or more atoms and a bridge that contains at least one atom, where the bridge atom is covalently bound to the shared ring atoms. A spiro bicyclic compound includes two rings that are joined at a common atom. The term “tricyclic” refers to a compound with three conjoined rings. The tricyclic compound may, optionally, contain fused, bridged or spiro moieties as applied to bicyclic compounds above. Higher order cyclic structures refer to compounds with more than three fused rings, including one or more fused, bridged, or spiro moieties as described herein.

[0099] Various groups are described herein as substituted or unsubstituted (i.e., optionally substituted). Optionally substituted groups may include one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and other substituting groups known in the art. In certain aspects the optional substituents may be further substituted with one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, or unsubstituted heteroaryl. Exemplary optional substituents include, but are not limited to: -OH, oxo (=0), -Cl, -F, Br, Ci-aalkyl, phenyl, benzyl, -NH2, -NH(Ci-4alkyl), -N(Ci- 4alkyl)2, -NO2, -S(Ci-4alkyl), -SO2(Ci-4alkyl), -CO2(Ci-4alkyl), and -O(Ci-4alkyl).

[0100] Examples of amine protecting groups include tert-butyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (FMOC), benzyloxycarbonyl (Cbz), a,a-dimethyl-3,5- dimethoxybenzyloxycarbonyl (Dbz), acetyl, trifluoroacetamide, phthalmide, benzyl, trityl, benzylideneamine, tosyl, 2-(4-biphenyl)isopropoxycarbonyl (Bpoc), 2-nitrophenylsulfenyl (Nps), ethanesulfonylethoxycarbonyl (Esc), benzyloxycarbonyl, Allyloxycarbonyl, 2,4- dinitrobenzenesulfonyl, benzothiazole-2-sulfonyl, 2-nitrophenylsulfanyl, acyl, Dithiasuccinoyl, p- nitrobenzyloxycarbonyl, propargyloxycarbonyl, o-nitrobenzyl oxy carbonyl, 2-(2- nitrophenyl)propyloxycarbonyl, 2-(3,4-methylenedioxy-6-nitrophenyl)propyloxy carbonyl, 9-(4- bromophenyl)-9-fluorenyl, and azidomethyloxycarbonyl. Additional amine protecting groups include any of the protecting groups in Greene's Protective Groups in Organic Synthesis by Peter G. M. Wuts and Theodora W. Greene, and Isidro-Llobet et al., Amino Acid-Protecting Groups 2009 Chem. Rev. 109, 2455-2504.

[0101] Examples of carboxy protecting groups include alkyl groups (to form esters), benzyl, phenylacyl (Pac), p-nitrobenzyl, 2-trimethylsilylethyl (TMSE), 2-alkyl-l,3-oxazoline, 2- chlorotrityl, 2,4-Dimethoxybenzyl, 2-phenylisopropyl, 5-Phenyl-3,4-ethylenedioxythenyl derivatives, 9-fluorenylmethyl, 4-(N-[l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino)benzyl (Dmab), carbamoyl methyl (Cam), (2-phenyl-2-trimethylsiylyl)ethyl (PTMSE), 2-(trimethylsilyl)isopropyl (Tmsi), 2,2,2-trichloroethyl (Tee), p-hydroxyphenacyl (pHP), 1,1 -dimethylallyl (Dma), allyl, 2,2,2-trichloroethyl (Tee), p-hydroxyphenacyl (pHP), and 4,5-dimethoxy-2-nitrobenzyl (Dmnb). Additional carboxy protecting groups include any of the protecting groups in Greene's Protective Groups in Organic Synthesis by Peter G. M. Wuts and Theodora W. Greene, and Isidro-Llobet et al., Amino Acid-Protecting Groups 2009 Chem. Rev. 109, 2455-2504.

[0102] A “leaving group” is understood in the art as an atom or a group of atoms that is displaced from a molecule, taking with it bonding electrons. Suitable “leaving groups” include, but are not limited to halogens (F, Cl, Br, I) as well as sulfoxide containing functional groups such as mesylate, triflate and tosylate.

[0103] The compounds disclosed herein may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S- or the R- configuration, as defined by the IUPAC 1974 Recommendations. Compounds may be of the D- or L- form, for example. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic form, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.Embodiments of Compounds

[0104] In certain embodiments, the disclosure provides synthetic caps for peptides. In particular embodiments, the synthetic cap caps the N-terminus of a peptide.

[0105] In further embodiments, the peptides can include a combination of a synthetic N-cap and a synthetic C-cap.

[0106] In some embodiments, methods of synthesizing N-capped peptides are provided herein. In further embodiments, methods of synthesizing dual-capped peptide are provided herein.

[0107] In some embodiments, compounds of the disclosure for use in the methods comprise a peptide that comprises an N-cap. In some aspects, an N-cap moiety can be attached to an existing peptide using native ligation methods.

[0108] In some embodiments, compounds of the disclosure for use in the methods comprise a dual-capped peptide. In various aspects, the dual-capped peptides of the instant disclosure comprise a structure of Formula I:

[0109] In some embodiments, the dual-capped peptide comprises a synthetic N-cap at its N- terminus and a synthetic C-cap at its C-terminus. In some aspects, an N-cap moiety can be attached to an existing peptide using native ligation methods. In some aspects, a C-cap moiety can be attached to an existing peptide using native ligation methods. Preferably, the synthetic N-cap and the synthetic C-cap are not linked in any way except through the peptide that connects them. That is, the dual-capped peptide does not comprise a linkage between the synthetic N-cap and the synthetic C-cap. Advantageously, it has been found that the synthetic N-caps and synthetic C- caps of the instant disclosure can impart helicity to a peptide or portion thereof. Accordingly, in various aspects, the dual capped peptide provided herein comprises at least one helical domain.

[0110] In some embodiments, the synthetic N cap is bicyclic, tricyclic, or has a higher order cyclic structure. For instance, in some aspects the synthetic N cap is bicyclic. In some instances the synthetic N cap is tricyclic.

[0111] In some embodiments, the synthetic C cap is bicyclic, tricyclic, or has a higher order cyclic structure. For instance, in some aspects the synthetic C cap is bicyclic. In some instances the synthetic C cap is tricyclic.Embodiments of N-Caps

[0112] In particular embodiments, the disclosure encompasses an N-cap represented by Formula II:Wherein each U is independently S, O, or CH2; each V is independently H or alkyl; W is; X1, X2, and X3are each independently S, O, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1 , 2, or 3.

[0113] In some embodiments, AA1is D- or L-aspartic acid. In some embodiments, Y1, Y2, and Y3are CH2. In some embodiments, X1, X2, and X3are S. In some embodiments, X1, X2, and X3are S and Y1, Y2, and Y3are CH2 In some embodiments, Z is H, alkyl, or an amine protecting group.

[0114] In some embodiments, W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. For instance, in some aspects, A1, A2, and A3are each H.

[0115] In some embodiments, Z is H, alkyl, an amine protecting group. Suitable amine protecting groups are known in the art and may include, for instance, include tert-butyloxy carbonyl (Boc), 9-fluorenylmethoxycarbonyl (FMOC), benzyloxycarbonyl (Cbz), a,a-dimethyl-3,5- dimethoxybenzyloxycarbonyl (Dbz), acetyl, trifluoroacetamide, phthalmide, benzyl, trityl, benzylideneamine, tosyl, 2-(4-biphenyl)isopropoxycarbonyl (Bpoc), 2-nitrophenylsulfenyl (Nps), ethanesulfonylethoxycarbonyl (Esc), benzyloxycarbonyl, Allyloxycarbonyl, 2,4- dinitrobenzenesulfonyl, benzothiazole-2-sulfonyl, 2-nitrophenyl sulfanyl, acyl, Dithiasuccinoyl, p- nitrobenzyloxycarbonyl, propargyloxycarbonyl, o-nitrobenzyloxycarbonyl, 2-(2- nitrophenyl)propyloxycarbonyl, 2-(3,4-methylenedioxy-6-nitrophenyl)propyloxycarbonyl, 9-(4- bromophenyl)-9-fluorenyl, and azidomethyloxycarbonyl. Additional amine protecting groups include any of the protecting groups in Greene's Protective Groups in Organic Synthesis by Peter G. M. Wuts and Theodora W. Greene, and Isidro-Llobet et al., Amino Acid-Protecting Groups 2009 Chem. Rev. 109, 2455-2504.

[0116] In some embodiments, the N-cap further comprises at least one2H,3H,nC,13N, or13O isotope.

[0117] In some embodiments, the N-cap comprises an amino acid sequence of C*DXXC*C* where each X is any natural or unnatural amino acid and each C* is L-cysteine alkylated with 1,3,5 tri methylbenzene (TMB) (SEQ ID NO: 1). In some instances, each X is independently A, S, Q, or V. For instance in some aspects, the N-cap comprises an amino acid sequence of C*DASC*C* (SEQ ID NO: 2), C*DAAC*C (SEQ ID NO: 3), C*DQAC*C (SEQ ID NO: 4), or C*DQVC*C(SEQ ID NO: 5). For instance, the N-cap can comprise an amino acid sequence of C*DASC*C* (SEQ ID NO: 2).Embodiments of C-Caps

[0118] In particular embodiments, the disclosure encompasses a C-cap represented byFormula III:Wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V isX4, X5, and X6are each independently S, O, Se, or NV; Y4, Y5, and Y6are each independently CH2, (CEE or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one nonnatural amino acid; AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

[0119] In certain aspects, Y4, Y5, and Y6are CH2. In some aspects, X4, X3, and X6are S. In some aspects, X4, X5, and X6are S and Y4, Y5, and Y6are CH2. In still other aspects, AA6is glycine.

[0120] In some embodiments, W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. For instance, in some aspects, A1, A2, and A3are each H.

[0121] In various aspects, R is H, alkyl, aryl, or a carboxy protecting group. Suitable carboxy protecting groups are known in the art and can include, but are not limited to, alkyl groups (to form esters), benzyl, phenylacyl (Pac), p-nitrobenzyl, 2-trimethylsilylethyl (TMSE), 2-alkyl-l,3- oxazoline, 2-chlorotrityl, 2,4-Dimethoxybenzyl, 2-phenylisopropyl, 5-Phenyl-3,4- ethylenedi oxythenyl derivatives, 9-fluorenylmethyl, 4-(N-[l-(4,4-dimethyl-2,6- dioxocyclohexylidene)-3-methylbutyl]-amino)benzyl (Dmab), carbamoylmethyl (Cam), (2- phenyl-2-trimethylsiylyl)ethyl (PTMSE), 2-(trimethyl silyl )isopropyl (Tmsi), 2,2,2-trichloroethyl (Tee), p-hydroxyphenacyl (pHP), 1,1 -dimethylallyl (Dma), allyl, 2,2,2-trichloroethyl (Tee), p- hydroxyphenacyl (pHP), and 4,5-dimethoxy-2-nitrobenzyl (Dmnb). Additional carboxy protecting groups include any of the protecting groups in Greene's Protective Groups in Organic Synthesis by Peter G. M. Wuts and Theodora W. Greene, and Isidro-Llobet et al., Amino Acid-Protecting Groups 2009 Chem. Rev. 109, 2455-2504.

[0122] In various aspects, the C-cap and / or compound comprising the C-cap further comprises at least one2H,3H,nC,13N, or15O isotope.

[0123] In various aspects, the C-cap may comprise an amino acid sequence of C*XXC*GC*, where each X is any natural or unnatural amino acid and each C* is L-cysteine alkylated with 1,3,5 trimethylbenzene (TMB) (SEQ ID NO: 6). In some instances, each X is independently E, Y, K, or I. For instance, in some aspects, the C-cap may comprise an amino acid sequence of C*EYC*GC* (SEQ ID NO: 7), C*YKC*GC* (SEQ ID NO: 8), or C*IYC*GC* (SEQ ID NO: 9). For instance, the C-cap may comprise an amino acid sequence of C*EYC*GC* (SEQ ID NO: 7).Embodiments of Capped Peptides

[0124] In particular embodiments, the disclosure encompasses peptides comprising a synthetic N-cap and a synthetic C-cap. In an aspect, these dual capped peptides comprise a structure of Formula I:any natural or unnatural amino acid and q is an integer from 1 to 40.

[0125] In various aspects, peptides comprising the synthetic N-cap and synthetic C-caps do not comprise any linkages between the N-cap and the C-cap other than the peptide that connects them. That is, the N-cap is understood to not be covalently bound to the C-cap (e.g., via a staple or macrocycle moiety), except through the intervening peptide. In further aspects, the dual capped peptide does not comprise a staple moiety. In further aspects, the dual capped peptide does not comprise a 53 atom macrocycle such as used in Sulanemadlin (ALRN-6924) and described in Guerlavais V, et al., Discovery of Sulanemadlin (ALRN-6924), the First Cell-Permeating, Stabilized a-Helical Peptide in Clinical Development. J Med Chem. 2023 Jul 27;66(14):9401- 9417. That is, in preferred embodiments, the dual-capped peptides provided herein do not comprise Sulanemadlin (ALRN-6924).

[0126] In various aspects, the N-cap is represented by formula II, above. In various aspects the C-cap is represented by Formula III, above. Accordingly, in aspect, a peptide provided herein is represented by Formula LA:LA. wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is independently H or alkyl; each W is independentlyorare each independently S, 0, Se, or NV; Y , Y , and Y are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independentlyany natural amino acid, any unnatural amino acid, or a bond; n is 1 , 2, or 3; Xaa is any natural amino acid or any unnatural amino acid; and q is an integer from 1 to 40.

[0127] In various aspects, Y1, Y2, and Y3are CEE. In various aspects, Y4, Y5, and Y6are CH2. In various aspects, Y1, Y2, Y3, Y4, Y5, and Y6are CH2.

[0128] In various aspects, X1, X2, and X3are S. In various aspects, X4, X3, and X6are S. In various aspects, X1, X2, X3, X4, X5, and X6are S.

[0129] In various aspects, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In various aspects, X4, X5, and X6are S and Y4, Y5, and Y6are CH2. In various aspects, X1, X2, X3, X4, X5, and X6are S and Y1, Y2, Y3, Y4, Y5, and Y6are CH2.

[0130] In some embodiments, at least one W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. In some embodiments, each W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. In some of these embodiments, A1, A2, and A3are each H.

[0131] In various aspects, R is H, alkyl, aryl, or a carboxy protecting group and Z is H, alkyl, or an amine protecting group. Suitable carboxy protecting groups and amine protecting groups are known in the art and described above.

[0132] In an aspect, the dual-capped peptide provided herein may comprise an amino acid sequence of C*XXC*GC* (SEQ ID NO: 6) at the C-terminus, wherein each C* is L-Cys alkylated with TMB and each X is a natural or unnatural amino acid. In an aspect, the dual-capped peptidecomprises the amino acid sequence of C*DXXC*C* (SEQ ID NO: 1) at the N-terminus, where each C* is L-Cys alkylated with TMB and each X is a natural or unnatural amino acid. In some embodiments, the dual-capped peptide provided herein comprises an amino acid sequence selected from any one of SEQ ID Nos: 2-5 at the N-terminus. In some embodiments, the dual-capped peptide provided herein comprises an amino acid sequence selected from any one of SEQ ID Nos: 7-9. In some embodiments, the dual-capped peptide provided herein comprises SEQ ID NO: 2 at the N-terminus and SEQ ID NO: 7 at the C-terminus.

[0133] In particular embodiments, the disclosure encompasses an N-capped peptide ofFormula IV:Wherein each U is independently S, O, or CH2; each V is independently H or alkyl; W is; X1, X2, and X3are each independently S, O, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CFE or Cme2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

[0134] In an aspect, AA1is D- or L-aspartic acid.

[0135] In an aspect, Y1, Y2, and Y3are CH2. In an aspect, X1, X2, and X3are S. In an aspect, X1, X2, and X3are S and Y1, Y2, and Y3are CH2.

[0136] In some embodiments, W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. For instance, in some embodiments, A1, A2, and A3are each H.

[0137] In some embodiments, Z is H, alkyl, or an amine protecting group. Suitable amine protecting groups are known in the art and described above.

[0138] In an aspect, the dual-capped peptide and / or N-capped peptide provided herein further comprises at least one2H,3H,nC,13N, or15O isotope.Embodiments of Capping Moieties

[0139] In particular embodiments, the disclosure encompasses a capping moiety for capping the N-terminal region of a peptide comprising a compound of Formula II as provided above. In further embodiments, the disclosure encompasses a capping moiety for capping the C-terminal region of a peptide comprising a compound of Formula III, as provided above. A compound of formula II or formula III can be attached to an existing peptide using native ligation methods, for example.

[0140] In an aspect, an isolated N-cap of Formula V is provided herein:wherein: each U is independently S, O, orCH2; each V is independently H or alkyl;; X1, X2, and X3are each independently S, O, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or Cme2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; B is O, S, NH, NR, or a bond; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

[0141] In some embodiments, R is H, alkyl, aryl, or a carboxy protecting group and Z is H, alkyl, or an amine protecting group. Suitable amine protecting groups are known in the art and described further herein above.Embodiments of Methods of Synthesizing Capped Peptides

[0142] In various embodiments, the disclosure encompasses methods of synthesizing capped peptides (e.g., N-capped or dual-capped peptides provided herein). For instance, the disclosure encompasses methods of synthesizing an N-capped peptide of Formula IV.

[0143] In an aspect, a method of synthesizing an N-capped peptide of Formula IV may comprise reacting a peptide of Formula IV-A:wherein each U is independently S,O, or CH2; each V is independently H or alkyl; K1, K2, and K3are each independently SH, OH, I, NH2, or NHV, Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe?; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; with a compound selected from the group consisting ofand;wherein: n is 1, 2, or 3; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; and L1, L2, and L3are each independently a leaving group (e.g., selected from the group consisting of F, Cl, Br, I, mesylate, triflate, and tosylate, and optionally further selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate). In some embodiments, the compound of Formula IV-A is reacted with a compound of, where L1, L2, and L3are as defined above and A1, A2, and A3are eachindependently hydrogen, deuterium, halogen, or a C1-C4 unsubstituted alkyl.. In some embodiments, the compound of Formula IV-A is reacted with l,3,5-tris(bromomethyl)benzene (TBMB). In some aspects the peptide of Formula IV-A comprises the amino acid sequence of CDXXCC (SEQ ID NO: 10). In some aspects, the peptide of Formula IV-A comprises the amino acid sequence of CDASCC (SEQ ID NO: 11), CDAASCC (SEQ ID NO: 12). CDQACC (SEQ ID NO: 13), or CDQVCC (SEQ ID NO: 14). For instance, in some aspects, the peptide of Formula IV-A comprises the amino acid sequence of CDASCC (SEQ ID NO: 11).

[0144] In an aspect, a method of synthesizing a dual-capped peptide of Formula III is provided. In various aspects, a method for synthesizing a dual-capped peptide may comprise a two step process, where a C-cap moiety is synthesized on a solid substrate before generating an N-capped moiety in a solution phase. It was surprisingly found that, while it is technically possible to prepare a dual capped peptide by generating an N-cap moiety on a solid substrate and a C-cap moiety in solution, that highest purity was obtained when C-cap moiety is synthesized on the solid substrate and the N-capped moiety is synthesized in solution.(66) Accordingly, in a particular embodiment, a method for synthesizing a peptide of Formula III is provided. In an aspect, the method comprisesa) reacting an immobilized peptide of Formula III-A:wherein each U is independentlyS, O, or CH2; each V is independently H or alkyl, K4, K5, and K6are each independently SH, OISeH, NH2, or NHV; Y4, Y5, and Y6are each independently CH2, (CH2)2, or CMe2; AA4, AA5, and AA6are each independently any natural amino acid, unnatural amino acid, or a bond; with a, and; wherein: n is 1, 2, or 3; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; and L1, L2, and L3are each independently a leaving group (e.g., selected from the group consisting of F, Cl, Br, I, mesylate, triflate, and tosylate, and optionally further selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate) to form an immobilized cyclized peptide of Formula III-B:are as previously defined; (b) ligating the immobilized cyclized peptide of Formula III-B to a second peptide comprising Formula IV-A:wherein U and V are as previously defined; K1, K2, and K3are each independently SHIH, SeH, NH2, or NHV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; AA1, AA2and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; to form an immobilized peptide of Formula I-A-a:wherein U, Z, V, W, AA1, AA2, AA3, AA4, AA5, AA6, K1, K2, K3, X4, X5, X6, Y1, Y2, Y3, Y4,Y3, Y6, Xaa , and q are as previously dined;(c) cleaving the peptide of Formula (I-A-a) to form a non-immobilized peptide of Formula I-A-b:wherein B is O, S, NH, NR, or a bond; R is alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; and Z, U, V, K1, K2, K3, X4, X5, X6, Y1, Y2, Y3, Y4, Y5, Y6, AA1, AA2, AA3, AA4, AA5, AA6, Xaa, n and q are as previously defined; (d) reacting the peptide of Formula I-A-b with a compound selected from the group consisting of:A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl, and L1, L2, and L3are each independently selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate , to form a dual capped peptide of Formula I-A:

[0145] In various aspects, the method may comprise reacting the peptide of Formula IV-Aand / or the peptide of Formula I-A-b with a compound of where L1, L2, andL3are as previously defined and A1, A2, and A3are each independently hydrogen, deuterium, halogen, or a C1-C4 unsubstituted alkyl. In an aspect, the method comprises reacting the peptideof Formula IV-A and / or the peptide of Formula I-A-b with l,3,5-tris(bromomethyl)benzene (TBMB).

[0146] In various aspects, Y1, Y2, and Y3are CH2. In various aspects, Y4, Y5, and Y6are CH2. In various aspects, Y1, Y2, Y3, Y4, Y5, and Y6are CH2.

[0147] In various aspects, X1, X2, and X3are S. In various aspects, X4, X3, and X6are S. In various aspects, X1, X2, X3, X4, X5, and X6are S.

[0148] In various aspects, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In various aspects, X4, X5, and X6are S and Y4, Y5, and Y6are CH2. In various aspects, X1, X2, X3, X4, X5, and X6are S and Y1, Y2, Y3, Y4, Y5, and Y6are CH2.

[0149] In an aspect, AA1is D- or L-aspartic acid. In still other aspects, AA6is glycine.

[0150] In an aspect, the immobilized peptide of Formula III-A may comprise CXXCGC* (SEQ ID NO: 15) wherein * represents the attachment point to the solid substrate. For instance, the immobilized peptide of Formula III-A may comprise CEYCGC* (SEQ ID NO: 16), CYKCGC* (SEQ ID NO: 17), or CIYCGC* (SEQ ID NO: 18), wherein each * refers to the attachment point to the solid substrate. In some aspects, the immobilized peptide of Formula III- A comprises CEYCGC* (SEQ ID NO: 16). In an aspect, the peptide of Formula IV-A comprises the amino acid sequence of CDXXCC (SEQ ID NO: 10). For instance, in some aspects, the peptide of Formula IV-A comprises the amino acid sequence of CDASCC (SEQ ID NO: 11), CDAACC (SEQ ID NO: 12), CDQACC (SEQ ID NO: 13), or CDQVCC (SEQ ID NO: 14). For instance, in an aspect, the peptide of Formula IV-A may comprise the amino acid sequence of CDASCC (SEQ ID NO: 11).

[0151] In an aspect, the solid substrate may comprise any substrate known in the art that is used for peptide attachment. In an aspect, the solid substrate may be a bead, a nanoparticle, a microparticle, a microarray, or any other solid substrate known in the art. . Methods of synthesizing peptides (e.g., a peptide of SEQ ID NO: 15 , above) on a solid substrate are known in the art and are described in the Examples below. Likewise, methods of cleaving an immobilized peptide (e.g., Formula I-A-a) from solid substrates are known in the art.Embodiments of Uses of the Compounds

[0152] In particular embodiments, the disclosure encompasses methods of use of the peptides disclosed herein.

[0153] Embodiments of the disclosure include methods of imparting helicity to a peptide by attaching a synthetic N-cap to the peptide.

[0154] Embodiments of the disclosure include methods of imparting helicity to a peptide by attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.

[0155] Embodiments of the disclosure include methods of increasing helicity of a peptide by attaching a synthetic N-cap to the peptide.

[0156] Embodiments of the disclosure include methods of increasing helicity of a peptide by attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.

[0157] Embodiments of the disclosure include methods of locking helicity of a peptide by attaching a synthetic N-cap to the peptide.

[0158] Embodiments of the disclosure include methods of locking helicity of a peptide by attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.

[0159] Embodiments of the disclosure include methods of decreasing cellular degradation of a peptide by attaching a synthetic N-cap to the peptide.

[0160] Embodiments of the disclosure include methods of decreasing cellular degradation of a peptide by attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.

[0161] Embodiments of the disclosure include methods of increasing the half-life of a peptide by attaching a synthetic N-cap to the peptide.

[0162] Embodiments of the disclosure include methods of increasing the half-life of a peptide by attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.

[0163] Embodiments of the disclosure include methods of increasing the cellular uptake of a peptide by attaching a synthetic N-cap to the peptide.

[0164] Embodiments of the disclosure include methods of increasing the cellular uptake of a peptide by attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.

[0165] Embodiments of the disclosure include methods of interrupting protein-protein interactions comprising exposing a protein-protein complex to an N-capped peptide.

[0166] Embodiments of the disclosure include methods of interrupting protein-protein interactions comprising exposing a protein-protein complex to a dual-capped peptide.

[0167] Embodiments of the disclosure include methods of binding a macromolecule, and in particular a protein, comprising exposing the protein to an N-capped peptide.

[0168] Embodiments of the disclosure include methods of interrupting protein-protein interactions comprising exposing a protein-protein complex to an N-capped peptide.

[0169] Embodiments of the disclosure include methods of interrupting protein-protein interactions comprising exposing a protein-protein complex to a dual-capped peptide.

[0170] Embodiments of the disclosure include methods of improving at least one pharmacokinetic property of a peptide comprising attaching a synthetic N-cap to the peptide.

[0171] Embodiments of the disclosure include methods of improving at least one pharmacokinetic property of a peptide comprising attaching a combination of a synthetic N-cap and a synthetic C-cap to the peptide.EXAMPLES

[0172] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention-Example 1 - In Silico ASX Motif Exploration

[0173] Two unnatural A'-capping strategies are prevalent in the contemporary literature: H- bond surrogates (FIG. 1A), and peptidic straps (FIG. IB). Hydrogen bond surrogates feature synthetic fragments to replace an N4H to Ncap(CO) / -bond (“mNcap,mN4” using the / / -bond nomenclature introduced in FIG. 1C). The existence of these caps led the present inventors to investigate structural peptidic motifs that could be used to develop new synthetic caps.

[0174] A searchable database of ASX motifs revealed existence of hydrophobic triangles (FIG. 2A). Discovery of new trends was unanticipated because ASX motifs had been known for more than two decades. Experiments on linear peptides proved hydrophobic triangles had a more significant helix-inducing effect on 17-mers, but less so on 12-mers (FIG. 3). A rigid mimic of hydrophobic triangles was designed in an attempt to overcome the unfolding tendency in short peptides and continuously induce helicity—Example 2 - Bioinformatics of ASX Motifs

[0175] An ASX motif is a commonly occurring feature in proteins and polypeptides that consists of four or five amino acid residues with either aspartate or asparagine as the first residue (residue i). ASX motifs include two internal hydrogen bonds, one is between the side chain oxygen of residue i and the main chain NH of residue i+2 or i+3, and the other is between the main chain oxygen of residue i and the main chain NH of residue i+3 or i+4. Asx motifs occur commonly in proteins and polypeptides.

[0176] The existing searchable database to access crystallographically characterized ASX motifs (PDBeMotif, as described in Golovin, A. & Henri ck, K. BMC Bioinformatics 9, 312 (2008), incorporated herein by reference in its entirety) has limited capacity to probe on structural details (eg atom-atom distances, dihedral angles, and sequence ranges). Consequently, a search routine was devised using the strategy in FIG. 2A which has enhanced utility. Briefly, the whole PDB was downloaded and filtered based on <95% primary sequence similarities (using an algorithm described in Huang, Y et al., Bioinformatics 26, 680-682 (2010) and Li, W. et al.,Bioinformatics 22, 1658-1659 (2006) which are both incorporated herein by reference in their entirety) to leave only unique protein chains. Two criteria were used to search all ASX motifs: an Asp or Asn, and characteristic H-bond pattern for ASX motifs. This ASX data set was processed to isolate ASX motifs at helical N-caps using DSSP (Define Secondary Structure of Proteins;, described in Touw, W. G. et al. Nucleic Acids Res 43 , D364-D368 (2015) and incorporated herein by reference in its entirety). H-Bonds and hydrophobic contacts were detected based on distance and angle requirements. The search process is referred to herein as “ASX / ST Search”.

[0177] Two factors known to reinforce helical ASX motifs are capping boxes which provide an additional mNcap,sN3 H-bond (FIG. 2B), and patches where hydrophobic side chains atN’ and N4 interacts across the first helical turn (FIG. 2C). Interrogating potential hydrophobic contacts between side chains of N’, N3 and N4 in helical ASX motifs revealed a previously undiscovered type of structural motif which can augment hydrophobic patches. Specifically, a script was designed to probe potential side chain hydrophobic contacts between residue pairs (N’, N3) and (N’, N4) in a subset of ASX motifs where N’ and N4 were both hydrophobic amino acids; the subset includes 12,175 motifs (36% of total). Contacts between (N3, N4) were not considered because these residues were naturally adjacent. The script measured nearest carbon distances between side chains of residue pairs, with a 4.5 A distance cut-off; side chains within this were considered for potential hydrophobic contacts. Outputs were recorded in ID arrays, [(N’,N4), (N’,N3)] where ‘ 1 ’ would be assigned if potential interactions could occur. Results from this analysis are shown in Venn diagram FIG. 2E. Of the 12,175 ASX motifs in the key data set, 71% could form at least one hydrophobic contact, and 29% could simultaneously have (N’,N4) and (N’,N3) contacts. The previously underappreciated hydrophobic interaction (N’,N3), occurred alone only rarely, but frequently with the (N’,N4) contact: isolated (N’,N3) contacts and coexisting ones were, 1139 and 3492, respectively. The structural motif with both (N’,N4) and (N’,N3) contacts is referred to herein as a “hydrophobic triangle” because ASX motifs are stabilized by packing three hydrophobic fragments in the N’, N4 and N3 into triangular shapes (FIG. 2D and FIG. 2K, which specifically shows a hydrophobic triangle from PDB 1Q5V where all residues are leucine). All hydrophobic triangles include hydrophobic patches, but not all hydrophobic patches include hydrophobic triangles. FIG. 2F illustrates the most common N’ and N4 combinations in hydrophobic triangles feature Leu at either position, vaguely reminiscent of interdigitated Leu-zippers. Amino acids with shorter side chains are less common, probablybecause these do not contact each other well across helical turns. “Extended” aromatic residues like Phe and Trp do not occur frequently in hydrophobic triangles. These observations may be useful for including hydrophobic triangles into helix-inducing ASX motifs in peptides designed to be helical.

[0178] FIG. 2G shows N3 residue abundancies in hydrophobic triangles. The N3 side chain methylene groups of Glu, Gin and Asp residues can contribute to hydrophobic packing, even though their termini are carboxylates or amides. Nearly three quarters (72.3%) of ASX motifs featuring hydrophobic triangles with Glu and Gin at N3 also form capping boxes. Capping boxes enhance ASX motif stabilities by providing mNcap,sN3 H-bonds, bringing N-terminal loops (• ■ N’-Ncap) and helical fragments (N3-N4- • •) closer and better oriented for hydrophobic packing. FIGS. 2H-2I showN’,N3, and, N’,N4 Ca-Ca distances in typical triangular cores are significantly shorter when there is a capping box than when there is not a capping box present. This allows the N3 methylene groups to pack with nearby hydrophobic side chains. Therefore, capping boxes and hydrophobic triangles can mutually stabilize each other in ASX motifs (FIG. 2J). -Example 3 - Synthesis of Linear Peptides

[0179] A series of linear peptides were prepared to study impacts of the featured structural motifs on helicities. Systematic changes were made at N’, Neap, N3 and N4 in 17-mers (FIGS. 3A-3C and FIG. 7) caused step-wise helicity increases by adding an ASX motif, a patch, then a triangle (FIG. 3D). The most helical peptide was LDLL (LDAALLAAAKAAAAKAW (SEQ ID NO: 23); throughout, four letter codes like AAKA (for AAAAKAAAAKAAAAKAW (SEQ ID NO:20)) this define the N’, Neap, N3, and N4 residues in linear peptides; other parts of the peptide sequence were unchanged in this series). First, an A at Neap was replaced with D to potentially add an ASX motif, then K at N3 was mutated to A to avoid extra H-bond contacts with D, giving “ADAA” (SEQ ID NO: 21). Then abundant residue pairs of hydrophobic amino acids in FIG. 2F were installed at (N’,N4) to form hydrophobic patches; “LDAL” (SEQ ID NO: 22) represents the most abundant pairing, (L,L). Finally, A at N3 was replaced by the set of abundant amino acids (FIG. 2G) to build a hydrophobic triangle giving the most helical system “LDLL” (SEQ ID NO: 23). LDLL, which has an ASX motif and a three-Leu hydrophobic triangle, has 93% relative helicity in PBS buffer, as indicated by a method widely used to estimate peptide helicity.Systematic Leu-to-Ala substitutions at these four key positions resulted in significantly diminished helicities (FIG. 3E), indicating disruption of either an ASX motif or hydrophobic triangle impairs helix inducing effect. A study of 12-mer analogs of LDLL showed the helix stabilizing effect of hydrophobic triangle is less effective in the 12-mer which has weaker helix tendency than 17 mers (FIG. 3F). FIG. 3G-3I shows CD spectra of “AAKA” (SEQ ID NO: 20), “ADAA” (SEQ ID NO: 21), “LDAL” (SEQ ID NO: 22), and “LDLL” (SEQ ID NO: 23) in PBS. CD of the other combinations is in FIGS. 3J-3M, and a summary of sequences and calculated helicities is in FIG. 7.

[0180] Linear peptides were synthesized using standard Fmoc peptide synthesis protocols (FIG. 11) on TentaGel S RAM resin (capacity = 0.22 mmol / g) either on a LibertyBlue peptide synthesizer or manually. Couplings were carried out using 5 equivalents (equiv) of Fmoc-protected amino acid, 10 equiv of Oxyma, 10 equiv of N,N'-Diisopropylcarbodiimide (DIC). Coupling reactions were allowed to proceed for 2 minutes in DMF at 90 °C under microwave, after which Fmoc deprotection was carried out with 20% (vol / vol) piperidine in DMF at 90 °C for 1 minutes. Upon completion of the peptide sequence assembly on resin and deprotection of the final Fmoc group, the N-terminal amine was acetylated by treatment with 25% (vol) acetic anhydride and 3.5% (vol) diisopropylethylamine (DIPEA) in dimethylformamide (DMF). Cleavage and deprotection from the solid support was carried out using trifluoroacetic acid (TFA) / H2O / tri isopropyl si lane (TIPS) (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature (longer time are required if there is Arg on peptides). The resin was then filtered and washed with excess TFA. The isolated TFA solution was then evaporated, and the peptide was precipitated by adding cold diethyl ether. The peptide suspension in ether was centrifuged to pellet followed by decanting the ether. The pellet was then washed twice with cold diethyl ether. Purification of the resulting peptides was achieved by high performance liquid chromatography (HPLC) on a reversed-phase C18 column to yield peptide with purity >95%. Purity and identity were assessed using ESLMS and analytical HPLC on a C18 column-Example 4 - Synthesis of Bicyclic Capped Peptides

[0181] Initial, linear peptides were synthesized using standard Fmoc peptide synthesis protocols (FIG. 12) on TentaGel S RAM resin (capacity = 0.22 mmol / g) either on a LibertyBluepeptide synthesizer or manually. The three cystines were placed at N’, N3 and N4 at the N- terminus. Couplings were carried out using 5 equivalents (equiv) of Fmoc-protected amino acid, 10 equiv of Oxyma, 10 equiv of DIC. Coupling reactions were allowed to proceed for 2 minutes in DMF at 90 °C under microwave, after which Fmoc deprotection was carried out with 20% (vol / vol) piperidine in DMF at 90 °C for 1 minutes. Upon completion of the peptide sequence assembly on resin and deprotection of the final Fmoc group, cleavage and deprotection from the solid support was carried out using TFA / H2O / TIPS (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature (longer time is required for peptides with Arg). The resin was then filtered and washed with excess TFA. The isolated TFA solution was then evaporated, and the peptide was precipitated by adding cold diethyl ether. The peptide suspension in ether was centrifuged to pellet followed by decanting the ether. The pellet was then washed twice with cold diethyl ether and dissolved in 1 : 1 mixture of aqueous buffer (20 mM NH4HCO3) and ceric ammonium nitrate (CAN) to make final concentration around 1 mM. 1.2 equiv of l,3,5-tris(bromomethyl)benzene (TBMB) was added and the solution was stirred for 15 min at room temperature.

[0182] Purification of the resulting peptides was achieved by high performance liquid chromatography (HPLC) on a reversed-phase Cl 8 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a Cl 8 column. Crude bicyclic peptides were semi-pure, demonstrated by only one major peak in HPLC traces during purification (other than solvent). This proved the high accessibility of the new bicyclic caps-Example 5 - Bicyclic ASX Motif Mimics (BAMMs)

[0183] Hydrophobic triangles occur frequently in protein ASX motifs and have an unexpectedly high impact on model peptides. Thus, replacement of hydrophobic N’, N3, and N4 side chains with a single hydrophobic fragment covalently linked to all three residues and the effects on helical N-cap stabilization were investigated. This was achieved using CLIPs (Chemical Linkage of Peptides onto Scaffolds) chemistry, as described in Richelie et al., Angew. Chem. Int. Ed. 57, 501-505 (2018), incorporated herein by reference in its entirety. Chemoselective CLIPS reactions of electrophiles with Cys and Cys-derivative thiols is a peptidic equivalent of the ubiquitous click reactions in organic methodology.

[0184] CLIPs can feature electrophiles with two benzylic bromomethyl groups to form one ring, or three benzylic bromomethyl groups to form bicyclic systems. Among those electrophiles,l,3,5-tris(bromomethyl)benzene (TBMB) is privileged insofar as all three electrophilic sites are equivalent. Therefore, reactions with three inequivalent Cys residues generally produce only one major product. A peptide with three Cys residues spaced i-1, i+3, i+4 relative to Asp (i) in a nonhelical ASX motif was investigated for its propensity to form a constrained, Bicyclic, helical ASX Motif Mimics (BAMMs) on CLIPs with TBMB (FIG. 4A). Alkylation of the three Cys residues is concurrent with transformation of TBMB tribromide reagent into a TMB { 1,3,5- tris(methylene)benzene) fragment as shown in FIG. 4C.

[0185] Molecular dynamics (MD) simulations were used to test the hypothesis outlined above, and probe dependence on Cys stereochemistries. A 13-mer based on an ASX motif with a hydrophobic triangle (“VFW”, ie Ac-VDAAFWAAAAAAA-NH2, SEQ ID NO: 46) was subjected to MD (298 K, 1 bar, explicit water, using Desmond from Schrodinger) for 50 ns. Throughout, helix persistence times were quantitatively monitored for this control linear sequence using a custom procedure, and that simulation is represented by the first bar in FIG. 4B. The same procedure was repeated on eight 13-mers where natural hydrophobic triangles were replaced by synthetic bicyclic caps with different permutations of L-Cys and D-Cys. Peptide CCC with three L-Cys residues best stabilized helicity relative to the linear control VFW and all other Cys stereochemistry permutations. Specifically, CCC unwound in 21.3 ns; other data in FIG. 4B are normalized to this. Stereoisomer cCC (lower case denotes D-amino acid) was second slowest, while peptides with D-Cys at N3 and / or N4 unwound even faster. Helical conformations were expected to tolerate D-Cys at N’ more than N3 and / or N4 because N’ is outside the helix whereas the N3 and N4 are within. Based on these observations, LLL-Cys combinations were further investigated. -Example 6 - Circular Dichroism (CD) Studies and Calculation of Percent Helicity

[0186] For simplicity, ‘helicity’ or ‘helical’ in the following text all refers to a-type. For each peptide, mean residue ellipticities ([9] / (c*n), c = concentration of the peptide, n = number of amino acids in the peptide) were used to calculate % helicity, which is an empirical indicator of peptide helicity. Higher values of % helicity usually suggest the tested peptides are more helical, but does not necessarily denote a percentage of peptide’s length that is helical. For example, 100 % helicity does not mean the peptide is completely helical.

[0187] Percent helicity was calculated based on the equation: % helicity = [0]222 / [0]max, where [0]max = (-4400- + 250T)(l - k / n)] (n = number of amino acids in the peptide, T = 25.0 °C). The variable “k” refers to ‘the number of non-H-bonded peptide mainchain carbonyls in a carboxyamidated peptide when it is completely helical, hence for linear peptide, k was 3, and for bicyclic peptides, depending on the number of residues extended after N’ Cys, k could be 4 (no extension) or 5 (one residue extension, ie, N”).

[0188] Concentrations of the stock solution were determined by 288 nm absorption of Trp or 283 nm absorption of Tyr. Each sample was dissolved in 1 x PBS (pH 7.4), 10% TFE / PBS or 20% TFE / PBS with the final concentration as 20 pM. CD spectra were acquired using circular dichroism spectrometer (Chirascan) equipped with a temperature controller using 1 mm cell at a scan speed of 0.5 nm / sec at indicated temperature. Each sample was scanned three times and the averaged spectrum was smoothed.

[0189] Bicyclo 12-mer, WC*DAAC*C*AAAKA-NH2(SEQ ID NO: 32), was prepared to measure its helicity by circular dichroism (CD) spectroscopy. C* indicates L-Cys alkylated with TBMB to coincide with hydrophobic triangle at N’, N3 and N4. Uncertainty regarding CLIPs efficiencies to form the bicyclic N-caps was dispelled since crude product HPLC analyses showed only one significant peak.

[0190] Comparisons were then made for the bicyclo 12-mer relative to two linear controls: AAKA 12-mer, a negative without structural motifs, the linear LDLL 12-mer which has an ASX motif and hydrophobic triangle but no rings, and two monocyclic controls (FIG. 4C). The two 12- mer monocyclic controls have ring sizes corresponding to the BAMM bicycle, i.e., a wide N’-N4 linkage and a narrower one which connects N’-N3. BAMM bicycles contain three rings, but the third conceivable control was not employed because it would connect N3 and N4 which are adjacent and could not stabilize a helical turn.

[0191] Key CD data for peptides in this series in 10% TFE / PBS are depicted in FIG. 4D. FIG. 4D and data in the table in FIG. 8 compare relative percent helicities and molar ellipticity ratios (0222 / 0208) in 10%TFE / PBS and in PBS. These data show the bicyclic V-cap to be significantly more helical than all the controls described above, in both solvents. Conspicuously less helix induction by both monocyclic controls relative to the bicyclo 12-mer is intriguing because it represents the effect associated with the rigidity inherent in the bicyclic system. Moreover, the narrow N’-N3 monocyclic system represents an i - i+4 staple, so the bicyclic system is more helix-inducing than a closely related stapled system. CD experiments measured in 20% TFE / PBS show greater ellipticity at 222 nm and 222 / 208 ellipticity ratios for bicyclo 12-mer than typical oc -helices, this can be explained by the “curvature” effect previously noted for Ala-rich helical peptides. Throughout, CD studies were also performed at different concentrations to probe for aggregation of BAMM bicyclic peptides, and no evidence of this was observed.

[0192] Variable temperature CD (VT-CD) experiments (FIG. 4F) showed bicyclo 12-mer was significantly more helical at most temperatures. Thermodynamic data might be extracted via the accepted Van’t Hoff-like strategy (Greenfield, N. J. Nat. Protoc. 1, 2527-2535 (2006), incorporated herein by reference in its entirety), but this would be misleading because, unlike bicyclo 12-mer, the four control peptides were far from helical states even at low temperature in 10% TFE / PBS; these controls had much weaker helix-inducing effects than bicyclo 12-mer.

[0193] Two other bicyclic peptides were prepared to test effects of changing the Neap residue from Asp. For bicyclo-D / A 12-mer (WC*AAAC*C*AAAKA (SEQ ID NO:32)), substitution of the Neap Asp by Ala reduced the helicity in PBS by 49%. That change negates the ASX H-bonding pattern so, as expected, is undesirable for optimal helicity. By contrast, Asp to Asn substitutions had less predictable consequences because both residues form similar H-bonding patterns in ASX motifs. The Asp containing system was more helical based on the CD data. This can likely be attributed to the (negatively charged) Asp carboxylate being a better H-bond acceptor than the Asn amide side chai-.Example 7 - NMR Studies

[0194] NMR experiments (20% TFE-d3 / 5% D2O / 75% H2O) were performed on the LDLL 12- mer and bicyclo 12-mer. Enhanced Monte Carlo Multiple Minimum (MCMM) calculations (Glanders, G., et al., J. Comput.-Aided Mol. Des. 34, 231-252 (2020), incorporated herein by reference in its entirety) using NMR constraints gave 23 and 50 conformations within 3 kcaEmoF1of the minimum energy conformers, respectively (FIGS. 5A and 5D). Both show minimal divergence, indicating important (populated) conformers are essentially homogenous and exhibit triangulated hydrophobic packing; LDLL 12-mer via Van der Waals interactions, and bicyclo 12- mer via covalent linking (FIGS. 5B, 5C, and 5E).

[0195] Two abnormally high upfield chemical shifts observed for N’ Cys CaH and Neap Asp NH are consistent with the NMR-derived low-energy conformers: both hydrogens are in the TMBaromatic field (FIG. 5F). A larger upfield shift for N’ Cys CaH than for Neap Asp NH correlates with the observation that the former hydrogen is closer to the TMB than the latter.

[0196] Ramachandran plots for preferred conformers of the two helical peptides are in FIGS. 5G-5H. Dihedral angles of both Neap residues (Asp, blue dots) deviate from typical helical <p,\|r values as expected for the ‘Neap’ residue. Middle residues (red dots) of both peptides are mainly within oc-helix region, but dihedral angles are more concentrated around ideal helical cp, values in bicyclo 12-mer, indicating a more compact helical structure. Dihedral angles <p,V of the C- terminal A9 and W10 are outside the helical region for the linear peptide, but those C-terminal residues for the bicyclo 12-mer (K8 and A9) are less distinguishable from the mid-helix, indicating the helix induction in the synthetic mimic is stronger.

[0197] Bicyclo 12-mer’s NOE constraints are consistent with its amide coupling constants (Table 1, below) where consecutive coupling <6 Hz indicates helicity after Neap, while larger values for N’ and Neap, suggest Neap is the helix breaker. Thus the BAMM N-cap facilitates transition from non-helical ( •N’-Ncap) to helical residues (N1-N2 • • •), just as in natural ASX motifs.Table 1 : Coupling constants and correspondingof bicyclo 12-mer calculated from equation {J(0) = 6.98*(cos(0))2-(1.38*cos(0)) + 1.72,

[0198] FIG. 51 shows log H / D exchange rate ratios for random coil over test structures. N4 and N3 NHs in the LDLL 12-mer and bicyclo 12-mer are relatively protected from the aqueousenvironment, because both contain ASX motifs to shield NH atoms of N3 and N4. Protection factors for the BAMM peptide are higher, indicating the covalent bicyclic cage is more static and helix-inducing than the noncovalent hydrophobic triang-e.Example 8 - BAMM-containing Analogs Of Natural Interface Helices

[0199] Interface helices can interact with protein receptors in different ways: three representative situations are shown in FIGS 6A-6C. For the case where the A-terminus of the helix is partially buried (FIG. 6A), non-interacting residues, N3 and N4 of the helix, may be mutated and a dipeptide fragment can be added ahead of the helix to form a BAMM cap. Alternatively, if the native helix totally protrudes beyond the interface (FIG. 6B), then residues substituted to form the BAMM will all be in that region. Finally, in rare cases the ligand interface helix may be totally encapsulated by the receptor, as in FIG. 6C. In that case a BAMM-module can be added to the N- terminus and the mimic will be longer than then native form.

[0200] The foregoing has described BAMMs applied to Ala-rich 12-mers. Details relating to mimicry of five naturally occurring helices comprising 10, 12, 14, 19, and 27 amino acids, 1 - 5, respectively are discussed below. There was no evidence that peptides corresponding to any of these would be helical in solution and indeed some of them contained the notorious helix breakers Gly and Pro. Consequently, helicities of the peptide analogs of these interface helix were investigated, but with Gly and Pro mutated to Ala throughout. Despite these mutations, only one peptide control, the 19-mer (41, see below for discussion of numbering), showed any evidence of helicity.

[0201] The 10, 12, and 14-mers correspond to partially buried helices, therefore, BAMM mimics were synthesized that were two residues longer in each case, i.e., 12, 14, 16-mers. The native 19-mer corresponds to a totally buried case; to mimic this a BAMM module comprising six residues was added such that the total length of the mimic was 25 residues. The native 27-mer corresponds to the extended helix (ii) above. The five N-terminal residues that do not contact the receptor were replaced by a BAMM module of six residues, giving 28-residues in the mimic. Full sequence information for all the controls and the BAMM-containing mimics are in the table in FIG. 9.

[0202] Two unconstrained controls were made corresponding to each BAMM mimic. The first type corresponds to interface helices with a few substitutions for helix breakers, as describedabove; these are numbered 11, 21, 31 etc. The second type corresponds to sequences of BAMM mimics except N’, N3, and N4 were S-methyl Cys, thus controlling for necessary extensions and mutations from linear peptides to BAMM mimics; these are denoted Im, 2m, 3m etc. Attempts to use Cys rather than S-methyl Cys controls were impractical due to partial oxidation to disulfides during preparation, purification and different biophysical assays. The anticipated results were the BAMM mimics (IB, 2B, 3B etc) would be more helical and stable in serum than either of the two control types.

[0203] FIGS. 6D-6H are CD spectra for the peptides, in 10% TFE PBS. Briefly, helicities of the BAMM-containing mimics B are all significant, whereas most linear 1 and m controls show little helical character. Interestingly, CD spectra of uncyclized linear peptides 1 m and 3 m implied dominance of P structures, but IB and 3B significantly favored helical conformations upon cyclization to BAMM N-caps. FIG. 61 shows half-lives from serum stability studies monitored by analytical HPLC and LC-MS enabling degradation products to be detected. BAMM containing mimics (red) prevented degradation from the N-termini, and were more stable than the 1 and m controls in four of five cases. In series 5 (the longest peptides; 27 ~ 28-mers) BAMM mimic 5B shows a similar half-life as control 51; the BAMM unit may be too far removed from the C- terminus to prevent extensive degradation by C- and endo-peptidases. Throughout, the BAMM motif tends to be the end point of the degradation (observed by LC-MS), and in some cases intermediate hydrolysis steps leading to these were observed. Stability enhancements for the BAMMs relative to the controls were greatest for the shorter systems where the BAMM can interfere with peptidases most.

[0204] Series 3 peptides and mimics correspond to the length mid-point for the five native interface helices. They correspond to the helix of CDK2 interacting with cyclin E, which is a PPI of special interest. Currently, there are no reports of helical mimics for the CDK2*cyclin E interaction. For these reasons binding of the series 3 compounds to cyclin E was investigated (FIG. 6J). Two binding assays were performed, the first being direct quenching based on fluorescence from the Trp224 of cyclin E (FIG. 6K); Trp224 of cyclin E interacts with the CDK2 helix (FIG. 6J), so its fluorescence is particularly sensitive to binding. In the event, the Kd of the BAMM mimic (0.52 pM) was significantly better than either control (3.4 and 7.4 pM). The complementary binding assay was based on fluorescence polarization. This (and the cell uptake experiments) required dye-labeled analogs, so a series of compounds 31f, 3mf, and 3Bf wereprepared with an N-terminal FITC (fluorescein iso-thiocyanate). FITC labeled series 3 peptides, 31f, 3mf and 3Bf were prepared for assays associated with fluorescence. Their sequences and calculate binding affinities are in FIG. 10. With limit amount of cyclin E, the highest final concentration of the protein was set as 3 iiM in the assays. None of the three peptides were completely saturated. However, the BAMM mimic was more closer to the top boundary and the equation was able to give an estimate value. Both linear peptides were still far away from the top boundary, so the equation was unable to calculate Kd values for them. Nevertheless, both linear peptides showed less affinity than the BAMM capped one. Binding data for these labeled peptides (FIG. 10) shows the same trend as the fluorescence quenching assay. Moreover, the strength of the BAMM is comparable with mimics formed from H-bond surrogates. Improvements over linear controls available in literature were observed.

[0205] Serum stability for extracellular targets is a good indicator of bioavailability after injection into the blood stream, but intracellular targets (like CDK2*cyclin E) require uptake and retention of helical analogs into cells. Fluorescent compounds 31f, 3mf, and 3Bf were incubated with breast cancer cells (in 10% fetal bovine serum, 37 °C) which were then washed thoroughly and analyzed via fluorescence activated cell sorting (FACS), in accordance with procedures described in Mi, T., et al., ACS Cent. Set. 9, 300-306 (2023) and Mi, T., et al., J. Am. Chem. Soc. 146, 10331-10341 (2024), which are both incorporated by reference in their entirety).

[0206] Data for the uptake and retention studies are summarized in FIG. 6L. Enhanced fluorescence for the BAMM-containing mimics was observed, particularly after extended incubation. These observations indicate that BAMM enhances cellular uptake, and these results can be attributed to several factors. Greater uptake and retention of the BAMM with time is primarily impacted by its enhanced stability in serum. Other potential factors include effects of increased hydrophobicity due to the TMB group, or the impact of greater conformational rigidity.

[0207] CD suggested BAMM N-cap can efficiently enhance oc-helicity for biological sequences from 10-mer to 27-mer; serum stability assays suggested BAMM N-cap can prolong half-life of biological sequences, by preventing peptides from degrading from N-termini, which is proved by observing intact N-terminal fragments in all tested BAMM peptides. However, helixinducing effect and degradation resistance of BAMM N-cap gradually decrease as peptides grow longer, which is probably due to a more and more flexible C -termin-.Example 9 - In Vitro Peptide Stability in Human Serum

[0208] Male human serum (H-4522) was obtained from Sigma Aldrich (Missouri, USA). The tested peptide was mixed to make a final peptide concentration of 150 pM in 25 % human serum diluted in RPMI medium. Before the addition of the tested peptide, the diluted serum solution was temperature-equilibrated at 37 ± 1 °C for 15 min. The initial time is recorded as peptides were added. The mixture was incubated at 37 °C and an aliquot of 40 pL was removed at different time intervals. The aliquot was mixed with 160 pL cold methanol and incubated at 4 °C for 15 min before centrifuging for 10 min to precipitate serum protein. The supernatant was analyzed in analytical RP-HPLC to calculate the amount of tested peptides, and in LCMS to analyze degradation products. The eluted peptides were detected by absorbance at 210 nm and quantified by their peak areas relative to the initial peak areas (0 h). The assay for each peptide was performed twice.

[0209] FIGS. 13-20 show results from these serum stability studies; as monitored by analytical HPLC and LC-MS to enable detection of degradation products. BAMM containing mimics (red) prevented degradation from the N-termini, and stability enhancements relative to controls were greatest for shorter systems (1-3) where BAMM can block peptidases most effectively. Stability enhancements are less significant for longer peptides (4 and 5) where the BAMM unit is removed, hence less protective of the C-terminus with respect to degradation by C- and endo-peptidases. Throughout, BAMM motifs tend to be degradation end points (LC-MS); in some cases we were able to observe hydrolysis intermediates leading to them.

[0210] The peptides and mimics numbered 3 are longer than two, and shorter than the other two of the five native interface helices comprising 10, 12, 14, 19, and 27 amino acids, 1-5, so 3 was chosen for further studies. They correspond to a PPI of special interest: the helix of CDK2 interacting with cyclin E. Currently, there are no reports of helical mimics for the CDK2*cyclin E interface so we chose to look at series 3 binding cyclin E. Two binding assays were performed, the first being fluorescence quenching of cyclin E Trp224 as described in Lakowicz, J. R. Principles of Fluorescence Spectroscopy. 3rd edn, (Springer, 2006) and Yammine, A., et al., Bio Protoc 9, e3253 (2019), which are both incorporated by reference in their entirety). That residue interacts with the CDK2 helix, so its fluorescence is particularly binding sensitive. In the event,the BAMM mimic Kd(0.75 pM) was significantly better than either control (2.8 and 8.6 pM). A complementary binding assay based on fluorescence polarization (FP) was also performed. This required dye-labeled analogs, so 31f, 3mf, and 3Bf were prepared which bear A-terminal FITC (fluorescein Ao-thiocyanate) (see FIG. 10). Binding data for these labeled peptides (FIG. 10) shows the same trend as the fluorescence quenching assay. Moreover, BAMM-induced improvement of binding affinity appear to be comparable with those from / / -bond surrogate mimics. Direct comparisons could not be made with the literature data because the protein receptors are different, but improvements over linear controls were comparable (Table 2 be-ow).Table 2 - Comparison of HBS, peptidic strap and BAMM on mimicking natural helices.X: pent-4-enoic acid before coupling and olefin metathesisT*: a derivative of pent-4-enoic acid, where the side chain of Thr was at the a position of pent-4-enoic acid (“(S)-2- ((R)-l-hydroxycthyl)pcnt-4-cnoic acid”)G, W*. D*. A*: N-allyl amino acids required in HBS strategyDap: 'L-2.3-Diaminopropionic AcidIso-D: Isoaspartic acidFITC: fluorescein isothiocyanateb-A: P-Alanine

[0211] In conclusion, CD suggested BAMM A'-cap can efficiently enhance a-helicity for biological sequences from 10-mer to 27-mer; serum stability assays suggested BAMM V-cap can prolong half-life of biological sequences, by preventing peptides from degrading from A'-termini (intact BAMM-containing A -term in al fragments prevailed in degradation profiles). Helixinducing and degradation resistance of BAMM A-caps decrease as peptides grow longer, as expected.Example 10 - BAMMs and BSMs compared

[0212] Mi et al., ACS Cent. Sci. 9, 300-306 (2023), incorporated herein by reference in its entirety, discusses bioinformatic studies leading to the discovery of C-terminal hydrophobic triangles in Schellman loops giving synthetic C-caps, Bicyclic Schellman loop Mimics (BSMs). Bioinformatic studies of N-terminal hydrophobic triangles in ASX motifs described here feature similar approaches but give fundamentally different findings in the following ways (FIG. 32A- 32B): (i) the N-terminal hydrophobic triangles comprise four helical residues, N1 - N4, whereas the C-terminal ones have only three: C3 - Cl; (ii) N-terminal hydrophobic triangles tend towards isosceles, but at the C-terminus they are more closely equilateral; and, (iii) apical residues in C- hydrophobic triangles are more evenly spaced allowing their side chains to pack more symmetrically. Consequently, bioinformatic studies at N-termini were necessary. BAMMs could not have been designed by analogy with BSMs, because their residue dispositions, degree of incorporation into helices, and triangular shapes are different.

[0213] Further, it is contemplated that these observations account for VT CD data shown in FIG. 32D and impact a larger issue. FIG. 33C-33D shows the BSM-containing peptide is more stable at all temperatures than ones based on BAMM and the control comprising only natural amino acids; thus the rigidified C-capped system is more thermodynamically stable. Conversely, and perhaps counter-intuitively, fitting the VT CD data to a two state model (helical folded in equilibrium with more random unfolded) reveals the BAMM-containing mimic has a significantly higher melting temperature than the BSM and linear control; the latter two were seen to be significantly lower and almost the same. Thus, BAMMs are more helix-inducing but less - stabilizing than BSMs: despite being more random at high temperature, BAMM N-capped 12-mer folded into helical conformations faster and much earlier than linear and BSM C-capped 12-merpeptide. In conclusion, BAMMs and BSMs are complementary with respect to inducing and stabilizing helical conformation-.Example 10 - Degradation Kinetic Curves and RP-HPLC Spectra of Series 1 Peptides

[0214] The raw data of kinetic curves showed how fast tested peptides degraded over time, and also presented the increase of final degradation product of IB, which was named A1B (FIG. 14A). The percentage of A1B was calculated by comparing its peak areas with initial peak areas (0 h) of IB, and the progression from IB to A1B, through two likely intermediates, was monitored by HPLC (FIGS. 13B-13E). The cleavage product should have a smaller absorbance coefficient than the initial peptide, however, this method is sufficient to qualitatively show the kinetics and stability of the cleavage product.

[0215] Linear peptides 11 and Im were unstable, and quickly self-degraded upon dissolving in aqueous solution. This made their (Oh) HPLC peaks even smaller (data not shown). Nevertheless, due to the effect of self-degradation and protease degradation, both peptides disappeared within 4 hours with no obvious remnants observed (FIG. 14A). For 1 B, several intermediates were observed and finally converted to A1B (FIGS. 13B-13E). A1B did not show obvious degradation from 48 to 72 hours (FIG. 14B) indicating high serum stability of the bicyclic N-cap fragment. Chemical formula of degradation product can be speculated by mass spectra obtained from LCM-.Example 11 - Degradation Kinetic Curves and RP-HPLC Spectra of Series 2 Peptides

[0216] The raw data of kinetic curves showed how fast tested peptides degraded over time, and also presented the increase of final degradation product of 2B, which was named A2B (FIG. 15A). The percentage of A2B was calculated by comparing its peak areas with initial peak areas (0 h) of 2B, and the progression from 2B to A2B, through two likely intermediates, was monitored by HPLC (FIGS. 15B-15D). The cleavage product should have a smaller absorbance coefficient than the initial peptide, however, this method is sufficient to qualitatively show the kinetics and stability of the cleavage product.

[0217] For the linear peptides 21 and 2m, no obvious remnants were found and it showed plain curves with several minor background peaks after 6 hours (data not shown). Kinetic curves of series 2 peptides over 8 hours showed that linear peptides 21 and 2m degraded more rapidly than2B (FIG. 16A). For 2B, several intermediates were observed and finally converted to A2B (FIGS. 15B-15D). A2B did not show obvious degradation from 48 to 72 hours (FIG. 16B) indicating high serum stability of the bicyclic N-cap fragment. Chemical formula of degradation product can be speculated by mass spectra obtained from LCM-.Example 12 - Degradation Kinetic Curves and RP-HPLC Spectra of Series 3 Peptides

[0218] The raw data of kinetic curves showed how fast tested peptides degraded over time, and also presented the increase of degradation products of 3B. Contrary to IB and 2B, 3B showed a stable intermediate, A3B, which then slowly degraded to other smaller peptides, for example, A3B’ (FIG. 17A). The percentage of degradation products was calculated by comparing its peak areas with initial peak areas (0 h) of 3B, and the progression from 3B to A3B and A3B’, through two likely intermediates, was monitored by HPLC (FIGS. 17B-17E). The cleavage product should have a smaller absorbance coefficient than the initial peptide, however, this method is sufficient to qualitatively show the kinetics and stability of the cleavage product.

[0219] For linear peptides 31 and 3m, no obvious remnants were found and it showed plain curves with several minor background peaks after 8 hours (data not shown). Kinetic curves of series 3 peptides over 8 hours showed that linear peptides 31 and 3m degraded more rapidly than 3B (FIG. 18A). For 3B, it first primarily degraded to a stable intermediate, A3B, within 24 hours. A3B is highly stable in 25% human serum and then slowly degraded to other smaller peptides since 48 hours (FIG. 18B). All degradation products contained bicyclic fragments indicating high serum stability of the N-cap fragment. Chemical formula of degradation product can be speculated by mass spectra obtained from LCM-.Example 13 - Degradation Kinetic Curves and RP-HPLC Spectra of Series 4 Peptides

[0220] The raw data of kinetic curves showed how fast tested peptides degraded over time, and also presented the increase of degradation products of 4B (FIG. 19A). 4B was a long peptides with 25 amino acids. It yielded plenty of degradation intermediates without a dominant one, so it was complicated to track them and conclude a potential process of degradation. Therefore, for 4B only the degradation products in 24 hours were carefully analyzed by LCMS (FIG. 19B).

[0221] For linear peptides 41 and 4m, no obvious remnants were found and it showed plain curves with several minor background peaks after 24 hours (data not shown). Kinetic curves of series 4 peptides over 8 hours showed that linear peptides 41 and 4m degraded more rapidly than 4B (FIG. 19C).-Example 14 - Degradation Kinetic Curves and RP-HPLC Spectra of Series 5 Peptides

[0222] The raw data of kinetic curves showed how fast tested peptides degraded over time, and also presented the increase of degradation products of 5B. 5B was a long peptides with 28 amino acids. It yielded plenty of degradation intermediates without a dominant one (FIG. 20A). Therefore, for 5B only the degradation products in 24 hours were carefully analyzed by LCMS (FIG. 20B).

[0223] For linear peptides 51 and 5m, after 4 hours the original peptides have been completely degraded, and only a peak at 5.3 min was observed for both peptides (data not shown). It seemed stable after 24 hours. It was a random case because none of previous linear peptides showed any significant remnants after 24 hours. Kinetic curves of series 5 peptides over 8 hours showed that linear peptide 5m degraded more rapidly than 51 and 45B (FIG. 20C).Example 15 - Cell Culture and Cellular Uptake Assay

[0224] MDA-MB-231 and MCF-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) / F12 or DMEM containing 10% fetal bovine serum (FBS), respectively. Cells were grown in an incubator at 37 °C, humidified atmosphere containing 5% CO2. Cells were grown in T-75 culture flask until 90% confluency before splitting into next passage.

[0225] Cells were seeded into a 24-well plate with a density of 200,000 cells / well and allowed to adhere overnight before treated with different concentration of compounds. After incubation at 37 °C for certain fixed time, the cells were washed with PBS twice and treated with 100 pL trypsin solution per well over 5 min at 37 °C for detachment. 200 pL media were added to each well and transferred to 96 well plates, which were analyzed by flow cytometer Cytoflex. The FITC channel was used to detect FITC labeled peptides. Live single cells were gated, and then the fluorescent intensity was recorded and input into Graphpad Prism 10 for analysis and graphic presentation. 0.1 % DMSO vehicle was used as a negative control to be subtracted as background fluorescence.

[0226] FIGS. 22A-22C demonstrate that low fluorescence was detected for the linear peptide at 1 pM, indicating low cellular uptake. N-capped (BAMM) and C-capped (BSM) peptides at 1 pM showed greater uptake in cancer cells, whereas the dual-capped peptide exhibited significantly greater uptake in HepG2 cells. FIGS. 23A-23C show the same trend for 10 pM peptide concentrations.

[0227] FIGS. 24A-24E are confocal microscopy images of MCF-7 cells treated with test compounds at 4 hours. The N-capped and C-capped peptides show greater cellular uptake than the non-capped linear peptide. The dual-capped peptide shows significantly greater uptake than either of the single-capped peptides. FIGS. 24A-24E are confocal microscopy images of MCF-7 cells treated with test compounds at 4 hours. These images show the same trend; the dual-capped peptide.

[0228] As demonstrated above, N-cap and dual-cap modules impart a-helical conformations and increase serum stabilities. The N-caps and dual-caps disclosed herein can be obtained without using reagents that are expensive or leave trace heavy metal impurities. This is different from caps or staples prepared via ruthenium catalysis or palladium-mediated cross couplings. Trace heavy metal contaminants are unacceptable for good manufacturing practice (GMP) of clinical samples. Therefore, the N-cap and dual-cap modules disclosed herein are more accessible and have fewer disadvantages than H-bond surrogates and staples featuring unsaturated hydrocarbons. The capping moieties disclosed herein serve as a promising new strategy in drug discovery by reducing degradation, increasing cellular uptake and potentially targeting PPIs for novel treatment mechanisms-Example 16 - Dual-Capped Helical Interface Mimics

[0229] Disruption of protein-protein interactions (PPIs) (1) using synthetic molecules resembling interface segments is valuable for generation of probes and pharmaceutical leads. (2-8) “Helical interface mimicry” features helical interface regions. (9-11) It is useful to regard proteins with the interface helices as protein ligands, binding to their protein receptors. Stabilities of helical conformations in the absence of protein and affinities for the protein receptor are indicators of the effectiveness of helical mimics. The next challenge in probe development is serum stability; robust mimics are expected to have higher effective concentrations, reaching the target in vivo.Consequently, protein receptor binding and serum stabilities are two early stage benchmarks to compare probes.

[0230] Staples are characterized by joining side chains of at least two amino acids in the helical region, i.e., having a-helical cp,\|z dihedral angles of ~-60,-40°. Stapling of helices (e.g., Figure la) prevails in helical mimicry. The field began with staples comprising amides linked by natural amino acids with carboxylic acid and amine side chains, including single sequences with two overlapping or three distinct staples. Now it has expanded to include many different compositions. A milestone was formation of hydrocarbon staples via alkene metathesis, typically (FIG. 34A) via pentenyl-Gly and -Ala derivatives. This is probably the most widely applied stapling method; it is arguably the current gold standard for helical mimicry.

[0231] Synthetic capping motifs are distinct from staples. They comprise modifications to amino acids immediately outside the helical motif, i.e., ones for which cp,\| / deviates significantly from -60,-40°. Capping methodologies have been employed significantly less often than stapling. Development of synthetic caps has been almost exclusively for N-termini. Contemporary methods for N-capping involve amino acid side chains joined via amide bonds (e.g., FIG. 34B right: an iso-Asp amide linked to diaminopropionic acid, Dap), (or N-allyl amino acid substrates for alkene metathesis to give alkenes in place of hydrogen bonds, i.e., H-bond surrogates (FIG. 34B left: N-allyl amino acid and N-terminal 4-pentenoic acid joined).

[0232] Progress on capped helical interface mimics is retarded by (i) lack of generalizable and helix-inducing peptidomimetics capped at the C-terminus; (ii) capped mimics comprising only one monocyclic ring resulting in suboptimal helical rigidities; and (iii) inconvenient requirements for modified amino acid building blocks (e.g., N-allyl protected amino acids {H-bond surrogates}). Consequently, the literature to date emphasizes new stapling methods and applications of hydrocarbon staples in helical mimicry. There is less research on synthetic N-caps and hardly anything on C-caps. Studies featuring side-by-side comparisons of helical interface mimics are rare.

[0233] In earlier Examples, N-terminus caps are described. Specifically, Bicyclic ASX Motif Mimics (B AMMs) enforce helicities and reorient the N-terminus. Bicyclic Schellman loop Mimics (BSMs), described Mi et al., ACS Cent. Sci. 9, 300-306 (2023), incorporated herein by referencein its entirety, enforce helicities and reorient the C-terminus. Consequently, it is now possible to “dual cap” peptides at both termini. In this Example, the hypothesis that mimics with two bicyclic caps would be exceptionally biased to helical conformations and hence would be superior binders to protein receptor targets was tested. Capping at both ends was also predicted to impart stability in serum, and, possibly, cell uptake. This would be significant because optimization of helicity, protein binding, stability in serum, and cell uptake are the chemical challenges in this area, after which the focus turns to pharmacokinetics. The specific aims of Example were to explore those issues by comparing a dual-capped peptide (“dual”) with the corresponding wild type (“linear”), C- and N-capped (“BSM” and “BAMM”), and two hydrocarbon staple controls (“staples 1 and 2”). The Experiments described herein demonstrate generalizable bicyclic dual capping methodology for the first time and compare it to these gold standard helical staple systems.

[0234] Tens of thousands of PPIs contain helical interface segments. No one helical mimic design would work uniformly well in each case, and it is logistically impossible to research a statistically significant sample, so PPI of interest was chosen. The wild-type C-helix sequence of CDK2 binding to cyclin E was selected because: (i) helical mimicry for CDK2 cyclin E is new; (ii) it is illustrative of PPIs which lead to phosphorylation of retinoblastoma protein (Rb, phosphorylation of which to pRb “lifts the suppression handbrake” and leads to uncontrolled cell cycling and growth in many tumor types; and (iii) the 13-residue CDK2 C-helix system is not long enough to fold into a helix in aqueous solution but has sufficient residues to accommodate two capping motifs. Staples 1 and 2 are typical i-i+4 systems. The positioning of their hydrocarbon linkers was chosen so they would not impede binding to cyclin E-Example 17 - Molecular Dynamics (MD) to Explore Dual Capping Feasibility

[0235] FIGS 35A-35B shows Ala-rich sequences (“linear-a”, “BAMM-a”, “BSM-a”, and “dual-a”, all 17-mers; FIG. 35 A), assembled into ideal a-helical conformations and then subjected to MD (explicit water, 300 K, using Desmond over 200 ns; FIG. 35B). Resistance to unfolding in these experiments is an indicator of helical stability. Root mean square deviations (RMSD) from starting helical conformations during MD as a function of time are representative of helical robustness; low RMSDs indicate stabilized helical conformations. Observations were: (i) rapid,irreversible, loss of helicity from N- and C-termini for linear-a; (ii) loss of helicity at uncapped termini for BAMM-a and BSM-a; and (iii) persistent helicity for dual-a. Dual-a only occasionally, and reversibly, lost helicity until ~ 175 ns, when all the systems were already relatively disordered (FIG. 35B). Another three peptides with previously known N-caps or a hydrocarbon staple were also studied by MD simulations (FIG. 35C); they were also affected by loss of helicity from the unconstrained termini and hence were less helix-stabilizing than dual-a.Example 18 - Synthesis of Linear Peptides

[0236] The linear control peptides were synthesized using standard Fmoc peptide synthesis protocols as generally described in Example 3 and shown in FIG. 11 above. Specifically, as shown in FIG. 48, the linear control peptides were synthesized on TentaGel S RAM resin (capacity = 0.22 mmol / g) on a LibertyBlue peptide synthesizer using standard Fmoc peptide synthesis protocols. Couplings were carried out using 5 equivalents (equiv) of Fmoc-protected amino acid, 10 equiv of Oxyma, 10 equiv of N,N'-Diisopropylcarbodiimide (DIC). Coupling reactions were allowed to proceed for 2 minutes in dimethylformamide (DMF) at 90 °C, after which Fmoc deprotection was carried out with 20% (vol / vol) piperidine in DMF at 90 °C for 1 minutes. Upon completion of the peptide sequence assembly on resin and deprotection of the final Fmoc group, the N-terminal amine was acetylated by treatment with 25% (vol) acetic anhydride and 3.5% (vol) di isopropyl ethyl amine (DIPEA) in DMF. Cleavage and deprotection from the solid support was carried out using trifluoroacetic acid (TFA) / H2O / triisopropylsilane (TIPS) (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature. The resin was then filtered and washed with excess TFA. The isolated TFA solution was then evaporated, and the peptide was precipitated by adding cold diethyl ether. The peptide suspension in ether was centrifuged to pellet followed by decanting the ether. The pellet was then washed twice with cold diethyl ether. Purification of the resulting peptides was achieved by high performance liquid chromatography (HPLC) on a reversed-phase C8 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a Cl 8 column.Example 19 - Synthesis of Bicyclic C-Capped Peptides

[0237] FIG. 49 provides a schematic of the synthesis of bicyclic C-terminal capped peptides (e g., peptides having a BSM cap at the C-terminus). Initially, linear peptides were synthesized using standard Fmoc peptide synthesis protocols as described in Example 18 and Example 3 (FIGS. 11 and 48 ) on TentaGel S RAM resin (capacity = 0.22 mmol / g) on a LibertyBlue peptide synthesizer. The three cystines were placed at C3, Ccap and C” near the C-terminus. Couplings were carried out using 5 equivalents (equiv) of Fmoc-protected amino acid, 10 equiv of Oxyma, 10 equiv of DIC. Coupling reactions were allowed to proceed for 2 minutes in dimethylformamide (DMF) at 90 °C, after which Fmoc deprotection was carried out with 20% (vol / vol) piperidine in DMF at 90 °C for 1 minutes. Upon completion of the peptide sequence assembly on resin and deprotection of the final Fmoc group, the N-terminal amine was acetylated by treatment with 25% (vol) acetic anhydride and 3.5% (vol) DIPEA in DMF. Cleavage and deprotection from the solid support was carried out using trifluoroacetic acid (TFA) / H2O / triisopropylsilane (TIPS) (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature. The resin was then filtered and washed with excess TFA. The isolated TFA solution was then evaporated, and the peptide was precipitated by adding cold diethyl ether. The peptide suspension in ether was centrifuged to pellet followed by decanting the ether. The pellet was then washed twice with cold diethyl ether and dissolved in 1 : 1 mixture of aqueous buffer (20mMI4HCO3) and ACN to make final concentration around 1 mM. 1.5 equiv of TBMB was added and the solution was stirred for 15 min at room temperature. Purification of the resulting peptides was achieved by high performance liquid chromatography (HPLC) on a reversed-phase C8 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a C 18 column.Example 20 - Synthesis of Bicyclic N-Capped Peptides

[0238] FIG. 50 provides a schematic of a protocol to synthesize bicyclic N-terminal capped peptides (e.g.., peptides capped at the N-terminus with BAMM). Initially, linear peptides were synthesized using standard Fmoc peptide synthesis protocols as described in Examples 3 and 18 and shown in FIGs. 11 and 48, on TentaGel S RAM resin (capacity = 0.22 mmol / g) on a LibertyBlue peptide synthesizer. The three cystines were placed at N’, N3 and N4 at the N- terminus. Couplings were carried out using 5 equivalents (equiv) of Fmoc-protected amino acid, 10 equiv of Oxyma, 10 equiv of DIC. Coupling reactions were allowed to proceed for 2 minutes in DMF at 90 °C under microwave, after which Fmoc deprotection was carried out with 20%(vol / vol) piperidine in DMF at 90 °C for 1 minutes. Upon completion of the peptide sequence assembly on resin and deprotection of the final Fmoc group, cleavage and deprotection from the solid support was carried out using TFA / H2O / TIPS (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature (longer time is required for peptides with Arg). The resin was then filtered and washed with excess TFA. The isolated TFA solution was then evaporated, and the peptide was precipitated by adding cold diethyl ether. The peptide suspension in ether was centrifuged to pellet followed by decanting the ether. The pellet was then washed twice with cold diethyl ether and dissolved in 1 : 1 mixture of aqueous buffer (20mM NH4HCO3) and ACN to make final concentration around 1 mM. 1.5 equiv of TBMB was added and the solution was stirred for 15 min at room temperature. Purification of the resulting peptides was achieved by high performance liquid chromatography (HPLC) on a reversed-phase C18 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a Cl 8 column.Example 21 - Synthesis of i - i+4 Hydrocarbon Stapled Peptides

[0239] FIG. 51 provides a schematic of the synthesis of i - i+4 Hydrocarbon Stapled Peptides. First, linear peptides were synthesized using standard Fmoc peptide synthesis protocols as described in Examples 3 and 18 (FIGs. 11 and 48)) on TentaGel S RAM resin (capacity = 0.22 mmol / g) on a LibertyBlue peptide synthesizer. (S)-2-(4-pentenyl)Ala (pA) was used at chosen i and i+4 positions to mutate original non-interactive amino acids. Couplings were carried out using 5 equivalents (equiv) of Fmoc-protected amino acid, 10 equiv of Oxyma, 10 equiv of DIC. Coupling reactions were allowed to proceed for 2 minutes in DMF at 90 °C under microwave, after which Fmoc deprotection was carried out with 20% (vol / vol) piperidine in DMF at 90 °C for 1 minutes. Double coupling was conducted for the coupling of (S)-2-(4-pentenyl)Ala (pA) and the next residue of it. Upon completion of the peptide sequence assembly on resin, olefin metathesis were done following the published protocoll. Cleavage and deprotection from the solid support was carried out using TFA / H2O / TIPS (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature (longer time is required for peptides with Arg). The resin was then filtered and washed with excess TFA. The isolated TFA solution was then evaporated, and the peptide was precipitated by adding cold diethyl ether. The peptide suspension in ether was centrifuged to pellet followed by decanting the ether. Purification of the resulting peptides was achieved by high performance liquidchromatography (HPLC) on a reversed-phase Cl 8 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a C18-column.Example 22 - Synthesis of a Dual-Capped System

[0240] An efficient synthesis route for dual-capped peptidomimetics was critical. Initially, the most directly conceivable approach (one-pot cyclization; FIG. 35D, top) was attempted. A peptide precursor was made on a solid phase, deprotected, and cleaved into solution to give a hexa-Cys system then reacted with l,3,5-tri(bromomethylene)benzene (TBMB). Specifically, a linear peptide bearing six Cys was synthesized following the protocols in Examples 3 and 18, above. After cleavage from resin, it was immediately dissolved in a 1 : 1 mixture of aqueous buffeI20mM NH4HCO3) and ACN, and then 3 eq. TBMB was added to the solution. After 15 min, the reaction mixture was checked by LCMS, and analytical HPLC (25 min from 10% acetonitrile {0.1% TFA} + 90% H2O {0.1% TFA} to 90% acetonitrile {0.1% TFA} + 10% H2O {0.1% TFA}, plus 5 min wash at 10% acetonitrile {0.1% TFA} + 90% H2O {0.1% TFA}, same conditions for analytical HPLC traces in this section). As shown in FIG. 53, many products were formed.

[0241] A second strategy explored relied on a solid phase CLIPS reaction based on a protocol described in Jo, H. et al. J. Am. Chem. Soc. 134, 17704-17713, which is incorporated herein by reference in its entirety. Instead of Cys(Trt), Cys(Mmt) was used to facilitate selective on-bead deprotection. Specifically, a solution of 1% TFA / DCM was prepared. 2 mL solution was added to the syringe, shaking for 2 min, then drained. This procedure was repeated, and the solutions normally would change from yellow, to orange, to red, then back to orange, and finally to light yellow after around 10 to 15 rounds of deprotections. After that, the beads are washed with several rounds of DCM and DMF. For solid phase cyclization, a 3 mL DMF solution with 2 eq. TBMB and 10 eq. DIPEA are prepared, then added to the syringe, and reacted for 1 hour, followed by thorough wash with DMF and DCM before the next step.

[0242] This protocol was applied on the syntheses of solid-phase C-cap BSM and N-cap BAMM, respectively, to evaluate CLIPS efficiency on different helical termini. HPLC was used to evaluate crude peptides (after cleaving from the resin) and to evaluate the purity of the resulting product. It was found that clear differences were observed depending on whether BAMM or BSMwere first synthesized on the solid phase. Specifically, when BAMM fragments were immobilized first on the solid phase (FIG. 35D, BAMM first; middle), many fragments were observed (FIG. 54B). In contrast, surprisingly, when BSM was immobilized first on the solid phase (FIG. 35D, bottom, and FIG. 35F,), better results were observed (FIG. 54A). In other words, clean transitions from the linear to bicyclic capped peptide were observed for the C-terminal CLIPS, but not so for N-terminal CLIPS. This may be rationalized below. The peptide sequences were linked to resin on their C-termini, so C-terminal Cys are less flexible, and therefore less likely to contact Cys of another chain to cause inter-chain cross-linking. On the other hand, the N-terminal Cys are away from the resin, making them more flexible and therefore more prone to contact Cys of another chain, resulting in messy cross-linking products upon addition of CLIPS reagents (TBMB). This is shown in FIG. 54C. The results from these experiments, guided the inventors to a route which synthesized the bicyclic caps at different stages. Specifically, the bicyclic C-cap, BSM, was first synthesized on solid phase, while the bicyclic N-cap, BAMM, was later synthesized in solution.

[0243] In brief, the C-terminal sequence of the peptidomimetic was first assembled on the solid phase to give the BSM fragment after selective removal of Cys(S-Mmt) (monomethoxy trityl) protection then reaction with TBMB by solid phase CLIPS (Chemical Linkage of Peptides onto Scaffolds) reaction; formation of one predominant product was verified by HPLC. Solid phase synthesis was then continued, three Cys(S-Trt) were incorporated, and the whole system was simultaneously deprotected, cleaved from the resin, and then finally reacted again with TBMB but in solution to form the BAMM fragment. This successful strategy separates the formation of two bicyclic caps into noncompeting stages and gives crude material comprising only one major peptide product (FIG. 35E), which can be conveniently purified via prep HPLC.

[0244] In view with the foregoing, FIG. 52 provides a scheme for the method for dual-capped peptide synthesis (BSM on solid, BAMM in solution). The seven residues at the C-termini, including six residues forming BSM and the one before it, were first coupled on the resin, either manually or automatically using peptide synthesizer. Here Cys were in the form of Cys(Mmt). The Fmoc group was intact for the last residue, and then selective deprotection of Mmt followed by solid phase CLIPS was conducted to form BSM C-cap on solid phase. After thorough wash by DCM and DMF, solid phase peptide synthesis was continued to grow the rest amino acids on the short C-terminal sequence. Cys at the N-termini were in the form of Cys(Trt). After final cleavagefrom resin and precipitation in ether, peptides were dissolved in solution for solution phase CLIPS to build BAMM N-cap. Purification of the resulting peptides was achieved by high performance liquid chromatography (HPLC) on a reversed-phase C18 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a Cl 8 column. FIGS. 55A and 55B show HPLC results for intermediates (BSM on solid (FIG. 55A) and BAMM in solution (-IG. 55B).Example 23 - Helicities

[0245] FIG. 36A-36B shows CDK2 cyclin E (PDB entry 1W98) and how sequences of linear, BSM, ’AM, dual, and staples 1 / 2 relate to the CDK2 C-helix. Pink residues in FIG. 36A (I, S, E) are hot spots from CDK2 C-helix binding cyclin E as determined by solvent accessible surface area (SASA) calculations. All six systems in FIG. 36B encompass these hot spots.

[0246] BAMMs and BSMs are not simply pseudo-mirror images; they carefully parallel ASX motifs and Schellman loops in natural helical caps of proteins. BAMM units, CDXXCC (-N'- Ncap-Nl-N2-N3-N4-, where the helix starts at Nl, X is any amino acid, SEQ ID NO: 1), incorporate two nonhelical residues (CD , -N'-Ncap-; throughout “C” represents Cys capped with TMB), while BSM units, CXXCGC (-C3-C2-Cl-Ccap-C'-C"-, where the helix stops at Cl , SEQ ID NO: 6) incorporate three (■• CGC, -Ccap-C'-C'-). We expect all residues in the linear sequence to be helical, so BAMM is longer than linear by two residues (the N-terminal C and D), BSM is longer by three (the C-terminal CGC sequence), and dual is longer by a total of five (CD ■ • ■ CGC). Stapled peptides- 1 / 2 have the same number of amino acids as the linear control.

[0247] Accurate concentration measurements are important when comparing the peptide and peptidomimetic helicities. Thus, a Tyr (Y; not present in the CDK2 C-helix) was included so concentrations of tested peptides could be calculated by UV absorbance at 275 nm. However, UV spectra of prepared peptides showed linear and stapled peptides had similar shapes but bicyclic capped peptides were different: their UV intensities increased dramatically before 270 nm (FIG. 36C). This suggested the additional trithiol-alkylated TMB group could significantly contribute to the absorbance around that area, so a calibration for this module was required. Consequently, TMB-3C was prepared for this purpose, and its UV spectra, as well as extinction coefficient at 275nm, were quantified by the Beer-Lambert law (FIG. 36D). This gives a value of 555 (M-l cm- 1), more than one-third of the value for Tyr: 1455 (M-l cm-1); hence, it is not negligible.

[0248] Concentration dependent circular dichroism (CD) studies were conducted for dual and stapled peptides in PBS. Their mean-residue ellipticities at 222 nm gradually increased since 5 pM and finally reached stable states at higher concentrations: dual, 13 pM; staple-1, 17 pM; staple-2, 22 pM (FIGS. 41A-41D). These concentrations were used to obtain their CD spectra as shown in FIGS. 36E-36F. The observation suggested a helix-initiation process for these peptides, probably by aggregation or formation of oligomeric coiled coils. Similar phenomena were not observed for linear and monocapped peptides which were less helical.

[0249] All six systems were also studied in different ratios of trifluoroethanol BS (up to 60% for linear and 50% for the others, FIGS. 42A-42F) to reach experimentally maximal helical states (as described by Roccatano et al., PNAS (2002), 99 (19): 12179-1218 and Hicks et al., Folding and Design 1997, 2(3), 149-158, which are incorporated herein by reference in their entirety) and obtain their maximal 222 nm ellipticities [9]max; thus, the experimental percent helicity can be calculated by [9]222 mPBs / [9]max(FIG. 36G).

[0250] In PBS at 25 °C, BSM (red) and BAMM (blue) were similarly helical and more so than linear, which has little helicity (222:208 ratios, and 222 nm ellipticities are relevant; (FIG. 36E and Table 3). This suggested one cap was enough to measurably induce helicity. Dual, however, had more than 20% improvement of percent helicities over BAMM and BSM. Compared with two staple controls, dual had higher percent helicity than staple-2 but lower than staple-1 (FIGS. 36F- 36G). CD shape is also relevant; the 222 / 208 ratios indicate dual could be helical, but this is not definitive due to the potential influence of TMB on CD shapes.Table 3 : Helical Propensities, Binding Affinities, and Serum Stabilities for the FeaturedPeptidesaMRE: mean-residue ellipticity.b95% confidence interval in parentheses.

[0251] Aggregation of dual peaks was observed via CD as described earlier. Its multimeric state at 0.4 mM was confirmed by analytical ultracentrifugation (AUC) in 20% DMSO / PBS (DMSO cosolvent for solubilization; concentrations are necessarily high in this technique). Other work indicates native structures are not perceivably impacted at similar DMSO concentrations. (56-59) In 20% DMSO / PBS, 90% dual oligomerized into a mixture of dimer, tetramer, and hexamer (1 :4:3) (Table 4). Dimer / tetramer and trimer / hexamer peaks were also observed in the MS spectra of the dual samples (FIG. 43C and characterization).Table 4: SV-AUC measurement results of C-helix dualExample 24 - Conformation of Dual by NMR

[0252] NMR data for dual unambiguously showed it adopts one predominant helical conformation in 35% TFE / H2O. That solvent ratio was chosen by (i) titrating TFE into a PBS solution of 17 pM dual to establish the minimum TFE to eliminate aggregation (FIGS. 44A-44B) and (ii) comparing peak separations in ID 1H-1H spectra of 4 mM dual in 20-35% TFE / H2O (FIGS. 45A-45C). Specifically, three solutions of 4 mM C-helix dual were prepared: 1) 20% TFE / H2O (30 LLL D2O, 120 pL TFE-d3 and 450 pL H2O), 2) 30% TFE / H2O (30 pL D2O, 180 pL TFE-d3 and 390 pL H2O), and 3) 35% TFE / H2O (30 pL D2O, 210 pL TFE-d3 and 360 pL H2O) and the fingerprint regions (6.5-9 ppm) in these 1D-H-H spectra were compared as shown in FIGS. 45A-45C. These experiments point to a threshold of 30% TFE, so 35% TFE was used for the NMR experiments to allow for potential deviations in these measurements.

[0253] Helical residues are recognized by chemical shift index (CSI) studies such as described in Wishart et al., Biochemistry ( 1992), 31 (6): 1647- 1651 , which is incorporated herein by reference in its entirety. Dual was robustly helical by this metric because nearly all the putative helical residues (A-Y in green) have A8 values <-0.1 ppm (FIG. 37A), the only exception being LI and L2 (leucines have been noted to give high A8s in reported helical peptides / peptidomimetics as described in Wang et al., Journal of the American Chemical Society (2006), 128 (28), 9248-9256 and Barthe et al., Protein Sci. 2000, 9 (5), 942- 955, which are both incorporated herein by reference in their entirety). Stark differences were observed between A8s for the putative helical region in comparison to the nonhelical residues beyond the N- and C-caps (blue and red, respectively, in FIG. 37A). Residues Cl and C4 had high negative A8s, consistent with shielding of their CaH atoms in the TMB anisotropic field in BSMs and BAMMs (FIGS. 46A-46B).

[0254] Dual was also shown to be unambiguously helical after conformational sampling constrained by distances deduced by 2D NMR. Only one cluster was observed within 3 kcal moF1of the global minimum and all conformers under this threshold overlaid tightly (FIG. 37B). A Ramachandran presentation (FIG. 37C) shows putative helical residues are in the ideal {right-handed} a-helix region (A-Y in green), while the nonhelical ones are outside it, as expected (Abterminal D {blue}, C-terminal G {red}, and C6 {magenta}). By definition, the peptide fragment projecting from the C-terminus must “turn back” on the helix in Schellman loops; consistent with this, the C residues (Gly in this case) are uniformly in the left-handed region on Ramachandran plots.

[0255] FIG. 37D shows the lowest-energy conformation of dual. Expansions show a natural N- cap (ASX motif) and a natural C-cap (Schellman loop) are constrained within the BAMM and BSM bicycles. These serve as two helix-inducing nuclei at both termini to constantly enforce helicity for the pepti-omimetics.Example 25 - Binding to Cyclins

[0256] Fluorescence polarization and quenching assays for binding of the featured compounds with cyclin E (FP and FQ, FIGS. 38A-38C and FIGS. 47A-47D) showed the same trends. Linear bound weakly, BAMM, and BSM had lower Kd (3.96, 0.51, 1.61 pM, respectively, FQ), while staples 1, 2 and dual had the best affinities (0.10, 0.14, 0.07, pM, respectively, FQ). CDK2 is known to bind predominantly to cyclins A and E. Cyclin Al, unlike E, does not have an interface Trp, so analogous FQ assays are not possible, but FP using fluorescein labeled peptides (dual-f, etc.) was performed. In these FP experiments, dual showed similar affinities for cyclins E (center) and Al (right). Staple 1 bound cyclin E with higher affinity and Al with 10-fold lower affinities FIGS. 38B-38C. Table 5, below, provides sequences of fluorescently labeled peptides.Table 5: Sequences of fluorescent labeled peptides.Example 26 - Serum Stabilities

[0257] Further to Examples 9-14 above, the test compounds were separately incubated with 25% human serum at 37 °C; residual starting materials were monitored as a function of time, and degradation products were characterized using LC-MS (FIG. 38D). Linear rapidly degraded to its constituent amino acids, whereas BSM and BAMM hydrolyzed more slowly and formed stable byproducts comprising the BSM bicycle or the BAMM bicycle with three residues (FIG. 38E). Staples 1 and 2 had overall higher stabilities but lesser protection than BAMM at the N- and BSM at the C-terminus. This is proved by their identified metabolic products where proteolysis happened at both termini. Besides terminal degradation, we also observed ring-opening products by hydrolysis of amide bonds within the hydrocarbon macrocycles in two stapled peptides FIGS 43D-43E). This suggested that the hydrocarbon macrocycles had some flexibility to adapt to the cleavage sites of proteases and could be cut from the middle.

[0258] Dual unambiguously was the most stable in the series: it had the longest half-life, tl / 2 > 49* the linear control, and >5.6x that of the next most stable peptidomimetic, staple-2. Ultimately, oxidation (sulfoxides, M++16) but not proteolysis was observed (LC-MS) in these experiments, probably due to oxygen or reactive oxidative species (FIGS. 38F-38G). These dualstaple differences are important because drug approval often depends on characterization and toxicity assays for dominant metabolites; this would be less arduous for dual metabolites than staples.Example 27- Cell Uptake

[0259] Further to Example 15 above, FITC-conjugated derivatives of the six peptides were prepared, and relative levels of uptake of these compounds into three cell lines were measured via fluorescence activated cell sorting (FACS; FIGS. 39A-39C). Specifically, referring to FIGs. 48- 52, after deprotection of last Fmoc on solid phase and thorough wash with DMF and DCM, a 3 mL DMF solution with 7 eq. FITC and 14 eq. DIPEA was added to the syringe at room temperature for 16 hours. After another round of thorough wash with DMF and DCM, FITC-peptides were cleaved from the resin by TFA / H2O / TIPS (95 / 2.5 / 2.5 vol / vol) for 3 hours at room temperature. Linear (black line) showed minimal uptake, while BAMM (blue) and BSM (red) permeated slightly better. However, staple-1 (green), -2 (gold), and dual (pink) were taken up significantly more. Based on the fluorescence intensity in FIGS. 39A-39C, it appears staple-1 was taken up ~2* better than dual, but the actual difference is less considering their difference in quantum yields shown in FIG. 39D. Relative levels of cyclin E expression in the three cell lines correlated to the observed uptake levels (FIGS. 39E-39F). This is a possible indicator of protein-target driven cell uptake; i.e., cyclin E tends to hold the compounds in-the cells.Example 28 - Dual capped peptides have improved properties compared to monocapped systems

[0260] The Examples above demonstrate that dual capped peptides have better helicity, serum stability, cyclin E binding, and cell uptake when compared with the monocapped systems, BAMM and BSM. NMR studies showed that dual capped peptides are unambiguously helical in a TFE / H2O medium (FIGS. 37A-37D). FIGS 40A-40B graphically summarizes findings when dual was also compared to two gold standard stapled helices; all three gave good helical inductions, binding affinities to cyclin E, and cell uptake, and differences within that series were relatively small. However, proteolytic stabilities in serum were conspicuously higher for dual than staples 1 and 2.

[0261] Dual-capped helical peptidomimetics have no constraint on amino acid compositions or peptide length. They are more conveniently prepared than hydrocarbon-stapled peptides, because only cheap, orthogonally protected Cys as building blocks are required. Further, they should not be contaminated with heavy metal residues such as ruthenium (used to staple or cap via metathesis reactions). Consequently, if no other modifications were made, dual peptidomimeticwould be the first-choice peptidomimetic for helix mimicry. Moreover, dual-capping and staples are orthogonal helix-mimicry strategies, so it is possible to apply both on long and complicated sequences.

[0262] FIG. 40B shows three situations of interface helices binding their receptors (left) and how to install BAMM A-caps on these helices, respectively (right, blue for added or mutated fragments). Helices in protein ligands can (1) be totally enclosed by the receptor, (2) be exposed to solvent on at least one side, or (3) extend beyond the receptor. Situations in (1) are rare; the design strategy for these might be to install an additional BAMM A-cap at the A-terminus of the helix. More commonly, ligand helices bind to receptors using one face and leave the other exposed as in (2); then two TV-terminal, solvent-exposed residues at N3 and N4 may be mutated to Cys. Posthelical residues N' (Cys) and Neap (Asp) could be added at the A-terminus. The three Cys at N', N3, and N4 could then react with TBMB to form a BAMM A-cap. In situations such as (3), the natural overhangs may be directly replaced by BAMM A-caps. Similar situations may happen at peptide C-termini, and BSM C-caps could be installed via similar designs; hence, there are ways to incorporate BAMM and BSM cassettes to nearly all interface helices.

[0263] In summary, it is now possible to dual cap helical mimics, and the advantages of dual capping as presented are numerous.* * *

[0264] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

WHAT IS CLAIMED IS:

1. A dual capped peptide comprising a structure of Formula I:wherein [Xaa] is any natural or unnatural amino acid and q is an integer from 1 to 40.

2. The dual capped peptide of claim 1, wherein [Xaa]qis a peptide comprising at least one helical domain.

3. The dual capped peptide of claim 1, wherein the dual capped peptide does not comprise a linkage between the synthetic N-cap and the synthetic C-cap other than [Xaa]q.

4. The dual capped peptide of claim 1, wherein the synthetic N-cap and / or the synthetic C-cap are bicyclic, tricyclic or have a higher order cyclic structure.

5. The dual capped peptide of claim 1, wherein the synthetic N-cap is represented by formula II:II whereinU is S, O, or CH2;V is H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CNfe;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

6. The dual capped peptide of claim 5, wherein AA1is D- or L-aspartic acid.

7. The dual capped peptide of claim 5, wherein Y1, Y2, and Y3are CH2.

8. The dual capped peptide of claim 5, wherein X1, X2, and X3are S.

9. The dual capped peptide of claim 5, wherein X1, X2, and X3are S and Y1, Y2, and Y3are CH2.

10. The dual capped peptide of claim 5, wherein W isand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

11. The dual capped peptide of claim 5, wherein Z is H, alkyl, or an amine protecting group.

12. The dual capped peptide of claim 5, wherein the synthetic N-cap further comprises at least one2H,3H,nC,13N, or15O isotope.

13. The dual capped peptide of claim 1, wherein the C-cap is represented by formula III:whereinB is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is independently H or alkyl;W isX4, X5, and X6are each independently S, O, Se, or NV;Y4, Y5, and Y6are each independently CH2, (CTfc) , or CMez;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

14. The dual capped peptide of claim 13, wherein Y4, Y5, and Y6are CH2.

15. The dual capped peptide of claim 13, wherein X4, X5, and X6are S.

16. The dual capped peptide of claim 13, wherein X4, X3, and X6are S and Y4, Y5, and Y6are CH2.

17. The dual capped peptide of claim 13, wherein AA6is glycine.

18. The dual capped peptide of claim 13, whereinand A1, A2, and A3are each independently hydrogen, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

19. The dual capped peptide of claim 13, wherein R is H, alkyl, aryl, or a carboxy protecting group.

20. The dual capped peptide of claim 13, wherein the C-cap further comprises at least one2H,3H,nC,13N, or13O isotope.

21. The dual capped peptide of claim 1, wherein the dual capped peptide is represented by formulaLA:whereinB is O, S, NH, NR, or a bond; each U is independently S, O, or CH?; each V is independently H or alkyl; each W is independentlyX1, X2, X3, X4, X5, and X6are each independently S, O, Se, or NV;Y1, Y2, Y3, Y4, Y5, and Y6are each independently CH2, (CH?) , or CMe?;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, AA3, AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond; n is 1, 2, or 3;Xaa is any natural amino acid or any unnatural amino acid; and q is an integer from 1 to 40.

22. The peptide of claim 21, wherein Y1, Y2, and Y3are CH2.

23. The peptide of claim 21, wherein Y4, Y5, and Y6are CH2.

24. The peptide of claim 21, wherein Y1, Y2, Y3, Y4, Y3, and Y6are CH2.

25. The peptide of claim 21, wherein X1, X2, and X3are S.

26. The peptide of claim 21, wherein X4, X5, and X6are S.

27. The peptide of claim 21, wherein X1, X2, X3, X4, X5, and X6are S.

28. The peptide of claim 21, wherein X1, X2, and X3are S and Y1, Y2, and Y3are CH2.

29. The peptide of claim 21, wherein X4, X5, and X6are S and Y4, Y5, and Y6are CH2.

30. The peptide of claim 21, wherein X1, X2, X3, X4, X5, and X6are S and Y1, Y2, Y3, Y4, Y5, and Y6are CH2.

31. The peptide of claim 21, wherein eachand A1, A2, and A3are each independently hydrogen, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

32. The peptide of claim 21, wherein R is H, alkyl, aryl, or a carboxy protecting group and Z is H, alkyl, or an amine protecting group.

33. An N-capped peptide of formula IV:IV wherein each U is independently S, O, or CH2; each V is independently H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; n is 1, 2, or 3;Xaa is any natural amino acid or any unnatural amino acid; and q is an integer from 1 to 40.

34. The N-capped peptide of claim 33, wherein AA1is D- or L-aspartic acid.

35. The N-capped peptide of claim 33, wherein Y1, Y2, and Y3are CH2.

36. The N-capped peptide of claim 33, wherein X1, X2, and X3are S.

37. The N-capped peptide of claim 33, wherein X1, X2, and X3are S and Y1, Y2, and Y3are CH2.

38. The N-capped peptide of claim 33, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

39. The N-capped peptide of claim 33, wherein Z is H, alkyl, or an amine protecting group.

40. The N-capped peptide of claim 33, wherein the peptide further comprises at least one2H,3H,nC,13N, or15O isotope.

41. A capping moiety for capping the N-terminal region of a peptide, comprising a compound of formula II:wherein each U is independently S, O, or CH2; each V is independently H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

42. The capping moiety of claim 41, wherein Z is H, alkyl, or an amine protecting group.

43. A method of imparting helicity to at least a portion of an N-terminal region of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide:wherein each U is independently S, O, or CH2; each V is independently H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

44. A method of reducing systemic degradation of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide:wherein each U is independently S, O, or CH2; each V is independently H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

45. A method of increasing cellular uptake of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide:wherein each U is independently S, O, or CH2; each V is independently H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

46. The method of any one of claims 43 to 45 , wherein Z is H, alkyl, or an amine protecting group.

47. The method of any one of claims 43 to 45 , wherein X1, X2, and X3are each S.

48. The method of any one of claims 43 to 45 , wherein Y1, Y2, and Y3are each CH2.

49. The method of any one of claims 43 to 45 , wherein AA1is D- or L-aspartic acid.

50. The method of any one of claims 43 to 45, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

51. A method of imparting helicity to at least a portion of an N-terminal region of a peptide and to at least a portion of a C-terminal region of the peptide, comprising attaching a compound of formula II to an N-terminus of the peptidewherein each U is independently S, O, or CH2; each V is independently H or alkyl; W isare each independently S, 0, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3; and attaching a compound of formula III:to a C-terminus of the peptide; wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is independently H or alkyl;;X4, X5, and X6are each independently S, O, Se, or NV; Y4, Y5, and Y6are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non- natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

52. A method of reducing systemic degradation of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptidewherein each U is independently S, O, or CH2; each V is independently H or alkyl; W isare each independently S, O, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3; and attaching a compound of formula III to a C-terminus of the peptide:wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is;X4, X5, and X6are each independently S, O, Se, or NV; Y4, Y5, and Y6are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one nonnatural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

53. A method of increasing cellular uptake of a peptide, comprising attaching a compound of formula II to an N-terminus of the peptide:wherein each U is independently S, O, or CH2; each V is independently H or alkyl; W isare each independently S, O, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CH )?, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3; and attaching a compound of formula III to a C-terminus of the peptide: .Ill wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is;X4, X5, and X6are each independently S, O, Se, or NV; Y4, Y5, and Y6are each independently CH2, (CH2)2, or CMe2; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non- natural amino acid; AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.- I l l -54. The method of any one of claims 51 to 53, wherein Z is H, alkyl, or an amine protecting group and R is R is H, alkyl, aryl, or a carboxy protecting group.

55. The method of any one of claims 51 to 53, wherein X1, X2, X3, X4, X , and X6are each S.

56. The method of any one of claims 51 to 53, wherein Y1, Y2, Y3, Y4, Y5, and Y6are each CH2.

57. The method of any one of claims 51 to 53, wherein AA1is D- or L-aspartic acid and AA6is glycine.

58. The method of any one of claims 51 to 53, wherein eachand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

59. A method of synthesizing an N -capped peptide, the method comprising: reacting a peptide of Formula IV-A:wherein each U is independently S, O, or CH2; each V is independently H or alkyl;K1, K2, and K3are each independently SH, OH, SeH, NH2, or NHVY1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one nonnatural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; with acompound selected from the group consisting ofan ;wherein: n is 1, 2, or 3; L1, L2, andL3are each independently selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate, and A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl.

60. A method of synthesizing a dual-capped peptide of Formula I-A, the method comprising:(a) reacting an immobilized peptide of Formula III-A:wherein each U is independently S, O, or CH2; each V is independently H or alkyl, K4, K5, and K6are each independently SH, OH, SeH, NH2, or NHV; Y4, Y3, and Y6are each independently CH2, (CH2)2, or CMe2; AA4, AA5, and AA6are each independently any natural amino acid, unnatural aminoacid, or a bond; with a compound selected from the group consisting of:;wherein: n is 1, 2, or 3; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; and L1, L2, and L3are each independently selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate to form an immobilized cyclized peptide of Formula III-B:, , ,are as previously defined;(b) ligating the immobilized cyclized peptide of Formula III-B to a second peptide comprising Formula IV- A:wherein U and V are as previously defined; K1, K2, and K3are each independently SH, OH, SeH, NH2, or NHVY1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; AA1, AA2and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; to form an immobilized peptide of Formula I-A-a:wherein U, Z, V, W, AA1, AA2, AA3, AA4, AA5, AA6, K1, K2, K3, X4, X5, X6, Y1, Y2, Y3, Y4, Y5, Y6, Xaa , and q are as previously defined;(c) cleaving the peptide of Formula (I-A-a) to form a non-immobilized peptide of Formula I-A-b:A-b, wherein B is O, S, NH, NR, or a bond; R is alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid; and Z, U, V, K1, K2, K3, X4, X5, X6, Y1, Y2, Y3, Y4, Y5, Y6, AA1, AA2, AA3, AA4, AA5, AA6, Xaa, n and q are as previously defined;(d) reacting the peptide of Formula I-A-b with a compound selected from the group consisting of:3; A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; and L1, L2, and L3are each independently selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate, to form a dual capped peptide of Formula I-A:LA.

61. A compound comprising the structure of Formula V:wherein each U is independently S, O, or CH2; each V is independently H or alkyl;W isX1, X2, and X3are each independently S, O, Se, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CNfe;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non- natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;B is O, S, NH, NR, or a bond;R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, and AA3are each independently any natural amino acid, any unnatural amino acid, or a bond; and n is 1, 2, or 3.

62. The compound of claim 61, wherein R is H, alkyl, aryl, or a carboxy protecting group and Z is H, alkyl, or an amine protecting group.

63. A method of imparting helicity to at least a portion of an N-terminal region of a peptide and to at least a portion of a C-terminal region of the peptide, the method comprising attaching a synthetic N-terminal cap to the N-terminal region of the peptide and a synthetic C-terminal cap to the C-terminal region of the peptide.

64. A method of reducing systemic degradation of a peptide, the method comprising attaching a synthetic N-terminal cap to an N-terminal region of the peptide and a synthetic C-terminal cap to a C-terminal region of the peptide.

65. A method of increasing cellular uptake of a peptide, the method comprising attaching a synthetic N-terminal cap to an N-terminal region of the peptide and a synthetic C-terminal cap to a C-terminal region of the peptide.

66. The method of any one of claims 63 to 65, wherein the synthetic N-terminal cap and / or the synthetic C-terminal cap are bicyclic, tricyclic, or have a higher order cyclic structure.

67. A compound comprising, consisting essentially of, or consisting of a synthetic N-cap covalently linked to a synthetic C-cap.

68. The compound of claim 67, comprising the structure of Formula VwhereinB is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V is independently H or alkyl; each W is independentlyX1, X2, X3, X4, X5, and X6are each independently S, O, Se, or NV;Y1, Y2, Y3, Y4, Y\ and Y6are each independently CH2, (CH )?, or CMe?;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl;R is H, alkyl, aryl, a carboxy protecting group, at least one natural amino acid, at least one non-natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;Z is H, alkyl, an amine protecting group, at least one natural amino acid, at least one non- natural amino acid, or a combination of at least one natural amino acid and at least one non-natural amino acid;AA1, AA2, AA3. AA4, AA5, and AA6are each independently any natural amino acid, any unnatural amino acid, or a bond; n is 1, 2, or 3; andQ is a bond or alkyl.

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