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

Abstract

Embodiments of the disclosure include compositions comprising peptides with C-terminal capping motifs, and methods for making the compositions. The capping motifs impart helicity to C-termini of peptides that otherwise do not exhibit helicity. The helix capping motifs are conformationally rigid, stable in serum, 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 / 519,474 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.P0005WO_SequenceListing.xml and is about 57,422 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] Proteins are genetically programmed to enact specific cellular functions in response to external stimuli, and perform essential functions such as catalysis and the transport of molecules and ions. In additional to specific functions, proteins also interact through broader and highly organized protein networks, which relay protein function cell-wide via interactions between protein nodes. The communicative interactions between proteins are referred to as protein-protein interactions (PPIs). In essence, PPIs are an interaction of two identical or dissimilar proteins at their domain interfaces that regulates the function of the protein complex.

[0006] Protein-protein interactions are involved in numerous cell signaling pathways. Modulation of these signaling pathways provides numerous new opportunities for drug discovery. Chemists can contribute to understanding cell biology, and the biomedicinal consequences of the PPIs by designing macrocyclic peptides that resemble interface hot segments, continuous epitopes that provide the majority of the protein-protein binding energy.

[0007] Of the different protein epitopes, interface helical mimicry is well-studied and some probes of this type have even progressed to clinical trials. Strategies for designing helical peptidomimetics include stapling, and capping. Many stapling methods have been investigated in depth, and studies on N-cap mimics are common. However, only two studies have focused on synthetic C-cap mimics. The first study involved incorporating a 1,3-xylene linker between residue C3 and C” at helix C-terminus with moderate helix inducing effect. The second study involved the use of a dual-capped peptide (FIG. 2E) that includes only 9 helical residues, thereby limiting its practical application in mimicking longer helices. Novel protein C-caps can impart improved helix-inducing effects over existing caps and staples.

[0008] Thus, there is a need for novel protein C-caps with improved functionality that can act as modulators of PPIs and provide new avenues for drug discovery.SUMMARY

[0009] Some embodiments of the disclosure are directed to C-capped peptides comprising a synthetic C-cap. In some embodiments, the synthetic C-cap is bicyclic, tricyclic, or has a higher order cyclic structure.

[0010] Some embodiments of the disclosure are directed to a C-capped peptide of formula I: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 isX1, X2, and X3are each independently S, O, Se, NH, or NV, Y1, Y2, and Y3are 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, unnatural amino acid, or a bond, Xaa is any natural or unnatural amino acid, and q is an integer from 1 to 40.

[0011] In some embodiments, Y1, Y2, and Y3are CH2. In some embodiments, X1, X2, and X3are S. in certain embodiments, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In some embodiments, AA1is glycine. In some embodiments, the peptide contains at least one2H,3H,nC,13N, or15O isotope. In some embodiments,and A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl. In some aspects, each ofA1, A2, and A3are H. . In some embodiments, R is H, alkyl, ary or a carboxy protecting group, In some embodiments, the peptide comprises an amino acid sequence of C*XXC*GC* (SEQ ID NO: 1), wherein each C* is L-Cys alkylated with 1,3,5 trimethylenebenzene (TMB) and each X is independently any natural or unnatural amino acid. In some instances, each X is independently glutamate, tyrosine, lysine, or isoleucine. For instance, in some aspects, the peptide comprises , an amino acid sequence of C*EYC*GC* (SEQ ID NO: 2), C*YKC*GC* (SEQ ID NO: 3) or C*IYC*GC* (SEQ ID NO: 4), wherein each C* is L-Cys alkylated with 1,3,5 trimethylenebenzene (TMB).

[0012] Some embodiments of the disclosure are directed to a method of synthesizing a C- capped peptide, comprising reacting a peptide of formula II: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; K1, K2, and K3are each independently SH, OH, SeH, NH2, or NHV, Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2, 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, unnatural amino acid, or a bond, 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 of: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). In some embodiments, the method comprises reacting the peptide of Formula II with l,3,5-tris(bromomethyl)benzene (TBMB).

[0013] Some embodiments of the disclosure are directed to a capping moiety for capping a C- terminal region of a peptide, comprising a compound of formula III., 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;; X1, X2, and X3are each independently S, O, Se, NH, or NV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; , A1, A2and 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, unnatural amino acid, or a bond;and n is 1, 2, or 3. In some aspects, X1, X2, and X3are each S, Y1, Y2, and Y3are each CH2, and / or

[0014] A compound of formula III can be attached to an existing peptide using native ligation methods, for example. Some embodiments of the disclosure are directed to a method of imparting helicity to at least a portion of a C-terminal region of a peptide, comprising attaching a compound of formula III, as defined herein, to a C-terminus of the peptide. Some embodiments of the disclosure are directed to a method of reducing systemic degradation of a peptide, comprising attaching a compound of formula III to a C-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 III, as defined herein, to a C-terminus of the peptide.

[0015] Further aspects of the disclosure are directed to methods of imparting helicity to at least a portion of a C-terminal region of a peptide, the methods comprising attaching a synthetic C- terminal cap to the C-terminal region of the peptide. Also provided are methods of reducing systemic degradation of a peptide, the methods comprising attaching a synthetic C-terminal cap to a C-terminal region of the peptide. Also provided are methods of increasing cellular uptake of a peptide, the methods comprising attaching a synthetic C-terminal cap to a C-terminal region of the peptide. In any of these methods, the synthetic C-terminal cap may be bicyclic, tricyclic, or have a higher order cyclic structure.

[0016] 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.

[0017] 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 modificationswithin 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

[0018] 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 better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0019] FIGS. 1A-1E: Structural features of Schellman loops and synthetic mimics. FIG. 1A Common Schellman loops (from PDB 10MH) have H-bonds to the C2 and C3 carbonyls (‘m’ refers to ‘mainchain’ in mC3, mC”, etc.). FIG. IB wide forms (from PDB 1 AGX) feature H-bonds to the C3 and C4 carbonyls. FIG. 1C Common forms are known to be biased towards forming a hydrophobic patch between C” and C3 side chains (from PDB 1ACO; ‘s’ refers to ‘sidechain’ in sC3 and sC”.). FIG. ID Schellman loop mimics previously made by incorporating an amino isobutyric acid residue (Aib) at C’ (from CSD XESNAK). FIG. IE Schellman loop mimics previously made by forming large macrocyclic rings (from PDB 6ANF).

[0020] FIG. 2: Diagram depicting data collected from Schellman Loop Finder approach.

[0021] FIGS 3A-3C Prevalent residues at C3 (FIG. 3A), C” (FIG. 3B), and Ccap (FIG. 3C) in common Schellman loops. ‘C-cap’ represents the whole motif, whereas ‘Ccap’ represents the capping residue.

[0022] FIGS. 4A-4E: FIG. 4A Diagram of a helical peptide indicating that three close contacts form a { 1,1,1 } hydrophobic triangle. FIG. 4B Proportions of helical, common Schellman loops with hydrophobic C3 and C” that have no, one (patches), two or three (triangles) hydrophobic interactions. FIGS 5C-5E Illustrative helical common Schellman loops in proteins. FIG. 4C two close contacts, { 1,1,0} from PDB 4WJ3. FIG. 4D { 1,0,1 } from PDB 4WJI. FIG. 4E compact (three) hydrophobic interactions (from PDB 5DLB). Prevalent amino acids at each position illustrated. Extended triangles of the form {0,1,1 } are rare (<0.5%; not shown).

[0023] FIGS 5A-5B: FIG. 5A is a diagram representing the approach for replacing three hydrophobic triangle residues with Cys then cyclizing with TBMB to form bicyclic Schellman loop mimics. FIG. 5B Deviation of non-H backbone atoms in the 12-mer model peptide from an ideal starting conformation coordinates comprising an a-helix and a Schellman C-cap with the BSM modification in MD at 300 K.

[0024] FIGS. 6A-6D: Diagram illustrating structures of the three controls (FIG. 6A, FIG. 6B, and FIG. 6C), and the featured bicyclo 12-mer (FIG. 6D).

[0025] FIGS 7A-7C: FIG. 7A CD spectra of the linear, wide monocyclic, tight monocyclic controls and bicyclo 12-mer in PBS buffer at 25 °C. FIG. 7B Variation of molar ellipticities at 222 nm with temperature in PBS. FIG. 7C CD spectra of the peptide and peptidomimetics at 85 °C in PBS.

[0026] FIGS. 8A-8B: FIG. 8A Characteristic NOE cross peaks expected for a typical common Schellman loop. FIG. 8B NOE cross peaks observed experimentally for bicyclo 12-mer.

[0027] FIGS. 9A-9C: FIG. 9A All 71 low energy conformers generated by MD simulations. FIG. 9B lowest energy conformer which is also the representative of that set. FIG. 9C Diagram of conformation showing five H-atoms that are likely to be shielded by the TMB group.

[0028] FIGS. 10A-10B: FIG. 10A Ramachandran plot for 71 low-energy conformers of bicyclo 12-mer. FIG. 10B Protection factors for the NH in bicyclo 12-mer, where the letters above the data points denote residues in the sequence, while the x-axis shows their positions in the helixcap motif.

[0029] FIGS. 11A-11B: CD spectra compared for two fragments of peptides which occur in proteins with the corresponding BSM mimics (FIG. 11A-BSM-1; FIG. 11B-BSM-2) in PBS buffer at 25 °C.

[0030] FIG. 12 is a reaction scheme for the synthesis of linear peptides.

[0031] FIG. 13 is a reaction scheme for the synthesis of bicyclic C-capped peptides.DETAILED DESCRIPTIONExamples of Definitions

[0032] 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.

[0033] 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.

[0034] 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.”

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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, -CH3(Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr), -CH(CH3)2(z o-Pr), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3(.scc-butyl), -CH2CH(CH3)2 (z o-butyl), -C(CH3)3( / e / V-butyl), - CH2C(CH3)3 (neo-pentyl), are all non-limiting examples of alkyl groups. The alkyl groups employed herein can be substituted or unsubstituted.

[0040] 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.

[0041] 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)CH3(acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3,-C(O)CH2CeH5, and -C(O)(imidazolyl) are non-limiting examples of acyl groups.

[0042] 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.

[0043] 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 arenot limited to: -OH, oxo (=0), -Cl, -F, Br, Ci- alkyl, phenyl, benzyl, -NH2, -NH(Ci-4alkyl), -N(Ci- 4alkyl)2, -NO2, -S(Ci-4alkyl), -SO2(Ci-4alkyl), -CO2(Ci-4alkyl), and -O(Ci-4alkyl).

[0044] Examples of amine protecting groups include tert-butyl oxy 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)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.

[0045] 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), carbamoylmethyl (Cam), (2-phenyl-2-trimethylsiylyl)ethyl (PTMSE), 2-(trimethylsilyl)isopropyl (Tmsi), 2,2,2-trichloroethyl (Tee), p-hydroxyphenacyl (pHP), 1,1 -dimethyl allyl (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.

[0046] 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.

[0047] 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

[0048] In certain embodiments, the disclosure provides synthetic caps for peptides. In particular embodiments, the synthetic cap caps the C-terminus of a peptide.

[0049] In some embodiments, methods of synthesizing C-capped peptides are provided herein.

[0050] In some embodiments, compounds of the disclosure for use in the methods comprise a peptide that comprises a C-cap.Embodiments of C-capped Peptides

[0051] In particular embodiments, the disclosure encompasses a C-capped peptide of formula I:Wherein B is O, S, NH, NR, or a bond; each U is independently S, O, or CH2; each V isX2, and X3are each independently S, O, Se, NH, or NV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; A1, A2and 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; n is 1, 2, or 3, Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40.

[0052] In certain aspects, Y1, Y2, and Y3are CH2. In some aspects, X1, X2, and X3are S. In some aspects, X1, X2, and X3are S and Y1, Y2, and Y3are CH2. In still other aspects, AA1is glycine. In still other aspects,and A1, A2, and A3are each independentlyH, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

[0053] In various aspects, the C-cap further comprises at least one2H,3H,nC,13N, or15O isotope.

[0054] In certain aspects, the disclosure encompasses an isolated C-cap of Formula IV: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 isare each independently S, O, Se, orNV; Y4, Y5, and Y6are each independently CH2, (CH2)2, or CNfe; each R is independently 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.

[0055] 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 oxy thenyl 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-(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.Embodiments of Synthesizing a C-capped peptide

[0056] In certain embodiments, the disclosure encompasses methods of synthesizing a C- capped peptide, comprising reacting a peptide of formula II: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; K1, K2, and K3are each independently SH, OH, SeH, NH2, or NHV; Y1, Y2, and Y3are each independently CH2, (CH2)2, or CMe2; 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, unnatural amino acid, or a bond; Xaa is any natural or unnatural amino acid; and q is an integer from 1 to 40; with a compound selected from the group1, 2, or 3; A1, A2and A3are each independently hydrogen, deuterium, halogen or an unsubstituted alkyl; and L1, L2, and L3are each independently a leaving group (e.g., selected from F, Cl, Br, I, mesylate, triflate, and tosylate, and optionally further selected from Cl, Br, I, mesylate, triflate, and tosylate,). In an aspect, L1, L2, and L3can be any leaving group. In an aspect, the peptide of Formula II is reacted with l,3,5-tris(bromomethyl)benzene.

[0057] In some aspects, K1, K2, and K3are each SH. In some aspects, Y1, Y2, and Y3are each CH2. In some aspects, K1, K2, and K3are each SH and Y1, Y2, and Y3are each CH2. In some aspects, the peptide of formula II comprises CXXCGC (SEQ ID NO: 5), wherein each X is anynatural or unnatural amino acid. In some aspects, the peptide of formula II comprises CEYCGC (SEQ ID NO: 6), CYKCGC (SEQ ID NO: 7), or CIYCGC (SEQ ID NO: 8).

[0058] In some aspects, LI, L2, and L3 are each Br.Embodiments of Capping Moieties

[0059] In certain embodiments, the present disclosure is directed to a synthetic C-cap. In an aspect, the synthetic C-cap is bicyclic, tricyclic, or has a higher order cyclic structure. In an aspect, the synthetic C-cap is bicyclic. In an aspect, the synthetic C-cap is tricyclic. In certain embodiments, the present disclosure is directed to a capping moiety for capping a C-terminal region of a peptide, comprising a compound of Formula III: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; X1, X2, and X3are each independently S, O, Se, NH, or NV; Y1, Y2, and Y3are 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 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, unnatural amino acid, or a bond; and each n is independently 1, 2, or 3.

[0060] In some embodiments, X1, X2, and X3are each S and / or Y1, Y2, and Y3are each CH2.

[0061] 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.

[0062] In some embodiments, the disclosure encompasses an isolated C-cap comprising a structure of Formula IV:O, or CH2; each V is independently H or alkyl;orare each independently S, O, Se, or NV; Y1, Y2, and Y3are each independently CH2, (CFF 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; andn is 1, 2, or 3. In some embodiments, Z is H, alkyl, or an amine protecting group and R is H, alkyl, aryl, or a carboxy protecting group.Embodiments of Uses of the Compounds

[0063] In particular embodiments, the disclosure encompasses methods of use of the peptides disclosed herein.

[0064] Embodiments of the disclosure include methods of imparting helicity to a peptide by attaching a synthetic C-cap to the peptide. In an aspect, the methods of imparting helicity comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide.

[0065] Embodiments of the disclosure include methods of increasing helicity of a peptide by attaching a synthetic C-cap to the peptide. In an aspect, the methods of increasing helicity comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide.

[0066] Embodiments of the disclosure include methods of locking helicity of a peptide by attaching a synthetic C-cap to the peptide. In an aspect, the methods of locking helicity of a peptide comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide.

[0067] Embodiments of the disclosure include methods of decreasing cellular degradation of a peptide by attaching a synthetic C-cap to the peptide. In some aspects, 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. In an aspect, the methods of decreasing cellular degradation of a peptide comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide

[0068] Embodiments of the disclosure include methods of increasing the half-life of a peptide by attaching a synthetic C-cap to the peptide. In some aspects, 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. In an aspect, the methods of increasing the half-life of a peptide comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide

[0069] Embodiments of the disclosure include methods of increasing the cellular uptake of a peptide by attaching a synthetic C-cap to the peptide. In some aspects, 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. In an aspect, the methods of increasing the cellular uptake of a peptide comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide.

[0070] Embodiments of the disclosure include methods of interrupting protein-protein interactions comprising exposing a protein-protein complex to an C-capped peptide. In an aspect, the C-capped peptide comprises a compound of formula I, as provided herein.

[0071] Embodiments of the disclosure include methods of binding a macromolecule, and in particular a protein, comprising exposing the protein to an C-capped peptide. In an aspect, the C- capped peptide comprises a compound of formula I, as provided herein.

[0072] Embodiments of the disclosure include methods of interrupting protein-protein interactions comprising exposing a protein-protein complex to an C-capped peptide. In an aspect, the C-capped peptide comprises a compound of formula I, as provided herein.

[0073] Embodiments of the disclosure include methods of improving at least one pharmacokinetic property of a peptide comprising attaching a synthetic C-cap to the peptide. In an aspect, the methods of improving at least one pharmacokinetic property of a peptide comprise attaching a compound of formula III, as provided herein, to a C-terminus of the peptide.EXAMPLES

[0074] 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 many changes 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.Bioinformatics Workflow

[0075] Disclosed herein is a convenient and generalizable approach to smaller, inherently more rigid bicyclic C-capped helical mimics. The approach evolved from bioinformatic data collected on the most common C-cap motifs in proteins: Schellman loops (FIG. 1). FIG. 2 outlines the bioinformatic workflow for the experimental approach disclosed herein. A custom script, Schellman Loop Finder, was developed and used to search for and analyze common and wide Schellman loops in proteins in a two-branch approach. The first (FIG. 2, top) established a complete Schellman loop database which enabled searching the motifs for any particular PDB entry. The other branch (FIG. 2, bottom) involved removal of duplicate chains to enable counting of nonredundant Schellman loops for statistical analyses. Key data on common forms (>95% of the total, FIG. 1A) are presented here and corresponding data on wide forms (FIG. IB).

[0076] FIGS. 3A-3C highlight most popular residues at C3 (FIG. 3A), C” (FIG. 3B) and Ccap (FIG. 3C) in helical common Schellman loops. Dominance of hydrophobic residues at C3 and C” was predictable because these tend to pack into hydrophobic patches. Preference of hydrophobic residues at Ccap was observed in an early study on a small dataset (160 PDB entries containing 431 helices) without detailed analyses of how they might pack, and no studies have followed up on this point. As a result, a deeper study using a complete set of unique Schellman loops was facilitated to better understand packings of hydrophobic sidechains at C3, C” and Ccap in common Schellman loops.

[0077] A filtering script to identify potential hydrophobic contacts was developed. The script requires that the nearest carbon atoms between two side chains (C3, Ccap or C”) be less than 4.5 A to ensure hydrophobic interactions. ‘Patches’ were previously defined by one close interaction: C3 - C”. However, the present analyses uses a broader definition to include C3 - Ccap and C” - Ccap because these are also interactions between two sidechains. Data interpretation was facilitated by denoting the three possible interactions, C3 - C”, C3 - Ccap, and Ccap - C”, in binary nomenclature {X,X,X}. Thus there are eight possibilities from {0,0,0}- { 1,1, 1 } where 1 refers to potential interactions and 0 means none. A score of 1 in any position defines a patch. Hydrophobic triangles, a term proposed by us, are for those Schellman loops with at least two patches among C3, Ccap and C”, because in such cases all of the three residues participate in the triangulatedhydrophobic clusters. FIG. 4A illustrates { 1,1,1 }: a typical hydrophobic triangle with all three possible hydrophobic interactions.

[0078] Of all Schellman loops with hydrophobic C3 and C” (38935), FIG. 4B shows: (i) only 3% had no interactions; (ii) thus 97% had at least a patch; and, (iii) 60%, had a triangle. Therefore, hydrophobic triangles comprise the majority of the data set. FIGS. 4C-4E illustrate hydrophobic triangles can be extended where the three residues are inclined to be stacked, and within this group there are three possibilities: { 1,0,1 } (91% among all hydrophobic triangles; most frequently Ala, Leu, Leu, and Leu, Leu, Leu slightly less), { 1,1,0} (2.9%; Leu, Leu, Ala) or, conceivably {0,1,1 } (not shown, <0.5%). Hydrophobic triangles can also be compact { 1,1,1 } (5.4%; most frequently Leu, Leu, He and Leu, Leu, Leu slightly less). Leucine prevails over all other hydrophobic residues in these packing arrangements vaguely reminiscent of leucine zippers. Hydrophobic triangles stabilize Schellman loops by insulating the inner backbone conformations and intra-molecular H- bonds important for helix-induction.

[0079] Conformations of peptide fragments isolated from their native protein environments can be different to those in the parent protein, especially if the fragment contains a hydrophobic motif which could pack against similar regions when buried inside the protein. To be sure that is applicable in this particular case, hydrophobic triangles from the data set above were examined. 87 % were found to be fully buried and only 2 % were completely at the solvent accessible surface of the parent proteins.

[0080] It follows from the analysis above that triangles in isolated model peptides would be exposed to solvent, and may not adopt the desired conformation. Nevertheless, short peptides were synthesized with hydrophobic triangles to test if these motifs could measurably enhance helicities for isolated helical fragments in solution. Specifically, peptides having the most abundant residue combinations in compact triangles were prepared: AC-WAAAKAAAAKAXAAXGX-NH2 (SEQ ID NO: 9, X’ s denote these key hydrophobic residues). Peptides using Leu, Leu, Leu or Leu, Leu, He as triangles presented enhanced helicities versus a control peptide with no Schellman loops or triangles, but these effects were small as anticipated. Thus hydrophobic triangles could show a small but measurable impact on linear peptide fragments. Hydrophobic triangles might be mimicked in constrained helical mimics, where the conformational restrictions might overcomethe unfavorable hydrophilic-hydrophobic solvent interactions. Pursuit of that strategy led to the design of the mimics referred to herein as “BSMs”.

[0081] BSMs manifest a key innovation of the data disclosed herein, unification of the assembled hydrophobic triangles into a single trimethyl-benzenoid ring (FIG. 5A). That mimic design has C3, Ccap, and C” replaced by three Cys, then capped with TBMB (1,3,5- tri [ bromomethylene [ benzene) to enforce the structural integrity of the C-cap conformations. Thus we aspired to make Bicyclic Schellman loop Mimics (BSMs; these are, as one referee described them, “stapled Schellman loops”). TMB (l,3,5-tri{methylene}benzene) is an ideal framework for compact mimics because its three methylene groups are spatially equidistant like the comers of the near-equilateral hydrophobic triangle in these { 1,1,1 } situations.Molecular Dynamics

[0082] A starting point was required to test the conformational rigidifying effect of BSMs via molecular dynamics (MD). Consequently, we virtually amalgamated coordinates of an ideal a- helix and a BSM C-cap to form the 12-mer peptidomimetic in FIG. 5B (bold Cs denote Cys capped by a TMB fragment). MD simulations were executed over 1 ps (explicit water box at 300K); this relatively long simulation allows the mimics to reach stable conformations. Root mean square deviations (RMSD) of non-H backbone atoms relative to the ‘perfect’ starting conformation were quantitated as a function of time to track unwinding of this ideal structure. In the experiment, the unconstrained A-terminus (upper portion of trace in FIG. 5B) continually flips in and out of the ideal helical conformation, but the bicyclic C-cap integrity dominated throughout after fluctuation in the first 150 ns (lower portion of trace in FIG. 5B). These observations on a simulated system suggested the BSM framework is rigid and stable.Synthesis of Peptides

[0083] CLIPS (Chemical Linkage of Peptides onto Scaffolds) is emerging as the dominant click reaction in peptide chemistry (as described in Timmerman, P. et al., ChemBioChem. 2005, 6 (5), 821- 824, and Richelie, G. Angew. Chem., Int. Ed. 2018, 57, 501- 505, which are incorporated by reference herein in their entirety) and thistechnique was employed herein. Crude tri-Cys peptide precursors could reacted with TBMB at 25 °C to give mainly (typically >90%) desired products within 15 mins.

[0084] Three control compounds were made to compare with the featured BSM-capped peptide, i.e., “bicyclo 12-mer” (FIG. 6C): (i) a linear control from natural amino acids (FIG. 6A); (ii) a monocyclic one representing Cys-to-Ala substitution of the second Cys in the featured compound, then cyclization with l,3-di(bromomethylene)benzene, i.e., “wide monocyclic control” (FIG. 6B); and, (iii) another monocyclic control representing Cys-to-Ala substitution of the third Cys then cyclization with the same electrophile, i.e., “tight monocyclic control” (FIG. 6D). These two monocyclic controls are mimics of C3-C" and C3-Ccap hydrophobic patches, respectively, and can be used to examine if the bicyclic triangle mimic is truly superior to monocyclic patch mimics. The fourth conceivable control (a monocycle encompassing the C-terminal CGC) was not considered because those three residues in the parent Schellman loop are outside the helical region and would not be expected to exhibit helicity.

[0085] Linear control peptides were synthesized using standard Fmoc peptide synthesis protocols (FIG. 12) 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 DIC. Coupling reactions were allowed to proceed for 2 minutes in 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 TFA / H2O / 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 ESLMS and analytical HPLC on a Cl 8 column.

[0086] To generate the bicyclic capped peptides, as shown in FIG. 13, linear peptides were first synthesized as described above and shown in FIG. 12 (i.e., using standard Fmoc peptide synthesis protocols on TentaGel S RAM resin (capacity = 0.22 mmol / g) either on a LibertyBlue peptide synthesizer or manually. Then, 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 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 TFA / H2O / 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 (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 C8 column to yield peptide with purity >95%. Purity and identity were assessed using ESI-MS and analytical HPLC on a Cl 8 column.Circular Dichroism (CD) Studies and Calculation of Percent Helicity

[0087] 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.

[0088] Percent helicity was calculated based on the equation: % helicity = [0]222 / [0]max, where [0]max = (-44000 + 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”).

[0089] Circular dichroism (CD) spectra of the BSM-containing bicyclo 12-mer and the three controls were recorded to evaluate their helicities. At 25 °C, the bicyclo 12-mer was significantly more helical than the others (FIG. 7A). This was confirmed by two helical indicators: percent helicity and ellipticity ratio (Table 1). Both monocyclic controls had about the same helicities: more than the linear, but less than the bicyclic. Differences between bicyclo 12-mer and the three controls were even more conspicuous in the variable temperature studies (VT-CD; FIG. 7B); the featured compound clearly had a more robust helicity. At high temperature, molecules are usually more unordered. This is the case for three controls: their 222 nm ellipticities were converged to around -5000 (FIG. 7B) and corresponding CD spectra at 85 °C resembled random coils (FIG. 7C). However, bicyclo 12-mer had twice 222 nm ellipticities than controls as temperature rose, and its CD spectrum at 85 °C still had significant a-helix characteristics. This shows the helixinducing BSM C-cap has surprisingly high thermal stability.Table 1 Ellipticity ratios 222 / 208 nm, and percent helicity for four 12-mer peptides at 25 °C in PBS buffer.

[0090] Additional circular dichroism (CD) spectra of linear and bicyclic 12-mers and 17-mers were conducted. Specifically, in one experiment, different 17-mer linear peptides were prepared in PBS buffer (pH 7.4, 10 mM) and tested at 25°C. The resulting helicities of these peptides are shown in Table 2, below. In this experiment, Trp was intentionally moved to the front in those linear peptides with hydrophobic residues to avoid extra interactions among Trp, C3, Ccap and C”. Comparing with the control, helicities of modified linear peptides had some changes, but notsignificant. All of them had slightly higher 0222 / 0205 and some had slightly higher % helicities. This implies that simply adding hydrophobic residues at C3, Ccap and C” on linear peptides only had limited effect.Table: 2 : Helicities of synthesized linear 17-mers in PBS buffer.

[0091] In another experiment, bicyclic 17-mer was synthesized, dissolved in PBS buffer (pH 7.4, 10 mM) and tested at 25°C. In order to test if the Gly at C’ is still crucial for helicity in the bicyclic system, a negative control peptide (bicyclic-G / A 17-mer) was also synthesized and tested. The helicities of these peptides are shown in Table 3 below. Comparing to linear peptides, a similar trend as seen with the 12-mer series appeared in this 17-mer series. That is, bicyclic 17-mer is more helical than linear ones considering both % helicities and 0223 / 0204 (1.25 -1.75 for ideal canonical a-helix). The huge drop of helicity (32%) from bicyclo 17-mer to bicyclic-G / A 17-mer indicated that Gly at C’ is crucial and required for maximal function of the synthetic cap.Table 3: Helicities of the synthesized bicyclic 17-mers in PBS buffer.C is L-Cys alky lated with TMB (1,3,5-trimethylenebenzene)

[0092] In another experiment, the four synthesized 12-mers (AAKA 12-mer, [C3-Ccap] 12- mer, [C3-C”] 12-mer and bicyclo 12-mer) were dissolved in 20% TFE / PBS buffer (pH 7.4, 10 mM) and tested at 25 °C. The helicities of these 12-mers are shown in Table 4 below. It was foundthat helicities of 12-mers were all improved in 20% TFE / PBS, but bicyclo 12-mer is still the most helical one among four peptides. In 20% TFE / PBS, Both % helicities and 8223 / 8206 (1.25 ~ 1.75 for ideal canonical a-helix) indicates that bicyclo 12-mer are close to ideal a-helices.Table 4: Helicities of the synthesized 12-mers in 20% TFE / PBS buffer.C is L-Cys alkylated with TMB (1,3,5-trimethylenebenzene)NMR Studies

[0093] NMR studies gave further evidence supporting the proposed dominant C-capped helical conformation of bicyclo 12-mer. Four characteristic contacts anticipated for a C- carboxyamidated Schellman loop were all observed in the ROESY spectrum of the bicyclo 12-mer (FIGS. 8A-8B). Relatively low NOE intensities between C2aH and C-terminal amide / is probably due to quick H / D and H / H exchanges between the terminal amide and nearby solvent molecules. Simulations using those and other NMR constraints gave very similar low energy conformations (within 3 kcal / mol to the lowest one) except for the acetyl groups at the N-terminus (FIG. 9A). FIG. 9B shows the lowest-energy conformer with two / / -bonds characteristic of common Schellman loops. Five hydrogens (2 Cal and 3 N with smaller chemical shifts than their colleagues implies they are probably shielded by TMB, which is consistent in the simulated conformers: these five hydrogens are behind TMB and therefore affected by its shielding effect (FIG. 9C). This indicates that the TMB group forms a solid waterproof wall right above the backbone of the Schellman loop.

[0094] Ramachandran plot FIG. 10A features all bicyclo 12-mer residues of the 71 low energy conformers shown in FIG. 9A. Residue C2in the BSM corresponds to Ccap. That residue and others downstream of it towards the C’-terminus are expected to break from helicity, and the result matches well: C2, G and C3indeed do not have helical <p,v dihedral angles, whereas all the ones anticipated to be helical do. Even AcA1at the / V-terminus, where most deviation would beexpected, is mostly helical. Finally, dihedrals of G at C ’ accurately fall into the region of lefthanded oc-helices, as often observed in Schellman loops.

[0095] HID exchange experiments were conducted to measure protection of each carbonylamide / / -bond. Exchange rates of amide Hs were measured and then divided predicted rates of the same protons at random-coiled state to give protection factors. A convex curve is usually anticipated in peptides since / -bonds at the termini are destabilized. However, in the bicyclo 12- mer, there is no obvious fading at C-terminus. This again marks the good quality of the waterproof wall built by the TMB fragment. Incidentally, the NH of C2 is particularly protected (FIG. 10B); we think this is probably because of the cooperation of TMB and the proximal C4 Trp (FIG. 9B).

[0096] Many of the bicyclo 12-mer and the 17-mers tested in the aforementioned experiments are Ala rich peptides which are usually biased toward helices. To test generality, peptides 1 (Ac- RIARLEEKYK-NH2 (SEQ ID NO: 24) and 2 (Ac-QVARQLAEIY-NH2, SEQ ID NO: 25) were selected because they are not Ala rich, and have been used by others to test enforced helicity (see e.g, Wang, D. J. Am. Chem. Soc. 2006, 128 (28), 9248- 9256, which is incorporated herein by reference in its entirety). Sequences 1 and 2 correspond to helical fragments in proteins which are not helical as isolated peptides in aqueous solution; they are hydrophilic and hydrophobic, respectively, hence provide different types of comparison. Consequently, these and their BSM mimics BSM-1 (SEQ ID NO: 26) and -2 (SEQ ID NO: 27) (FIG. 11) were prepared. To make BSM mimics of any sequence, two changes are required. One is the residue at C3 will be mutated by Cys. The other is that three residues, Cys-Gly-Cys, will be added after the last expected helical residue in the sequence. These three residues correspond to Ccap, C’, and C”, and are not helical, so they cannot mutate expected helical residues unless a mimic of truncated helix is desired. Since all residues in 1 (SEQ ID NO: 24) and 2 (SEQ ID NO:25) were expected to be helical, three more residues were extended at their C-termini to form BSM mimics. Helicities of the peptides and peptidomimetics were compared on a per-residue basis. FIG. 11 shows neither of the control peptides have CD spectra resembling oc-helices, but BSM-1 (SEQ ID NO: 26) and -2 (SEQ ID NO: 27) do; the percent helicity and pertinent ellipticity ratios are given in Table 5. This proves the BSM capping method can be applied on more general situations.Table 5: Ellipticity Ratios 222 / 208 nm and Percent Helicity for Biological Peptides and BSMAnalogues at 25 °C in PBS Buffer

[0097] In conclusion, hydrophobic triangles comprise the majority of the data set of helical Schellman loops (FIG. 4B). This significant observation led us to the biomimetic approach in which could not have been conceived otherwise. BSMs are conveniently accessible via inherently efficient CLIPs reactions. Their unique bicyclic structure makes them more constrained and, presumably, more helix-inducing than the C-cap mimics reported to date which are monocycles. In fact, the cap motif of [C3-C”] 12-mer, one of our monocyclic controls which are less helical, is similar to a reported C-cap system (see e.g., Acharyya, A.; J. Phys. Chem. B 2019, 123, 1797- 1807, incorporated herein by reference in its entirety). BSMs support helical conformations well, even in short peptides and at elevated temperatures. Bioinformatic analysis also showed Schellman loops seldom contain hot spot residues. Consequently, substitutions of three Cys residues to form BSMs would not deter from their use in interface mimicry, because residues corresponding to hot-spots are usually incorporated outside the C-cap. Besides, BSM can also be applied on helical fragments without Schellman loops (FIG. 11). This requires the extension of three amino acids CGC at the end of the last helical residue, to form BSM mimics. Significant improvement of helicity on short biological peptides confirmed the feasibility of new C-capping method in peptides with various physiochemical properties.

[0098] As demonstrated above, C-cap impart a-helical conformations. The C-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 C-cap modules disclosed hereinare 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 serving as potential new PPI-disruption targets for novel treatment mechanisms.* * *

[0099] 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 C-capped peptide of formula I: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 isX1, X2, and X3are each independently S, O, Se, NH, or NV;Y1, Y2, and Y3are 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, unnatural amino acid, or a bond; n is 1, 2, or 3;Xaa is any natural or unnatural amino acid; andq is an integer from 1 to 40.

2. The C-capped peptide of claim 1, wherein Y1, Y2, and Y3are CH2.

3. The C-capped peptide of claim 1, wherein X1, X2, and X3are S.

4. The C-capped peptide of claim 1, wherein X1, X2, and X3are S and Y1, Y2, and Y3are CH2.

5. The C-capped peptide of claim 1, wherein AA1is glycine.

6. The C-capped peptide of claim 1, wherein the peptide contains at least one2H,3H,nC,13N, or1?O isotope.

7. The C-capped peptide of claim 1, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

8. The C-capped peptide of claim 7, wherein A1, A2, and A3are each H.

9. The C-capped peptide of claim 1, wherein R is H, alkyl, aryl or a carboxy protecting group.

10. The C-capped peptide of claim 1, comprising an amino acid sequence of C*XXC*GC* (SEQ ID NO: 1), wherein each C* is L-Cys alkylated with 1,3,5 trimethylenebenzene (TMB) and each X is any natural or unnatural amino acid, optionally wherein each X is independently glutamate, tyrosine, lysine, or isoleucine, and further optionally wherein the C-capped peptide comprises an amino acid sequence of C*EYC*GC* (SEQ ID NO: 2), C*YKC*GC* (SEQ ID NO: 3) or C*IYC*GC* (SEQ ID NO: 4), wherein each C* is L-Cys alkylated with 1,3,5 trimethylenebenzene (TMB).

11. A method of synthesizing a C-capped peptide, comprising reacting a peptide of formula IIwherein:B is O, S, NH, NR., or a bond; 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;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, unnatural amino acid, or a bond;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 of:wherein: n is 1, 2, or 3;A1, A2, and A3are each independently hydrogen, deuterium, halogen, or alkyl; andL1, L2, and L3are each independently selected from the group consisting of Cl, Br, I, mesylate, triflate, and tosylate.

12. The method of claim 11, comprising reacting the peptide of Formula II with 1,3,5- tris(bromomethyl)benzene.

13. A capping moiety for capping a C-terminal region of a peptide, comprising a compound of formula III: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 isX1, X2, and X3are each independently S, O, Se, NH, or NV;Y1, Y2, and Y3are each independently CH2, (CFF) , 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, unnatural amino acid, or a bond; and n is 1, 2, or 3.

14. The capping moiety of claim 13, X1, X2, and X3are each S and / or Y1, Y2, and Y3are each CH2.

15. The capping moiety of claim 13, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

16. A method of imparting helicity to at least a portion of a C-terminal region of a peptide, comprising 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;W isX1, X2, and X3are each independently S, O, Se, NH, or NV;Y1, Y2, and Y3are each independently CH2, (CH2)2, or CNfe;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, unnatural amino acid, or a bond; and n is 1, 2, or 3.

17. The method of claim 16, wherein X1, X2, and X3are each S and Y1, Y2, and Y3are each CH2,.

18. The method of claim 16, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

19. A method of reducing systemic degradation of a peptide, comprising 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;W isX1, X2, and X3are each independently S, O, Se, NH, or NV;Y1, Y2, and Y3are 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, unnatural amino acid, or a bond; and n is 1, 2, or 3.

20. The method of claim 19, X1, X2, and X3are each S and Y1, Y2, and Y3are each CH2.

21. The method of claim 19, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

22. A method of increasing cellular uptake of a peptide, comprising 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;W isX1, X2, and X3are each independently S, O, Se, NH, or NV;Y1, Y2, and Y3are each independently CH2, (CTH) , 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, unnatural amino acid, or a bond; and n is 1, 2, or 3.

23. The method of claim 22, X1, X2, and X3are each S and Y1, Y2, and Y3are each CH2.

24. The method of claim 22, whereinand A1, A2, and A3are each independently H, deuterium, halogen, or an unsubstituted Ci to C4 alkyl.

25. A compound comprising a structure of Formula 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 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;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.

26. The compound of claim 25, wherein Z is H, alkyl, or an amine protecting group and R is H, alkyl, aryl, or a carboxy protecting group.

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

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

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

30. The method of any one of claims 27 to 29, wherein the synthetic C-terminal cap is bicyclic, tricyclic, or has a higher order cyclic structure.

31. A C-capped peptide comprising a synthetic C-cap.

32. The C-capped peptide of claim 31, wherein the synthetic C-cap is bicyclic, tricyclic, or has a higher order cyclic structure.

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