Synthesis of membrane permeable macrocyclic peptides via imidazopyridinium grafting

Abstract

Macrocyclic peptides (MPs) are a class of compounds that have been shown to be particularly well suited for engaging difficult protein targets. However, their utility is limited by their generally poor cell permeability and bioavailability. The present disclosure reports an efficient solid-phase synthesis of novel MPs by trapping a reversible intramolecular imine linkage with a 2-carbonyl pyridine to create an imidazopyridinium (IP+)-linked ring. This chemistry is useful for the creation of macrocycles of different sizes and geometries, including head-to-side and side-to-side chain configurations. Many of the IP+-linked MPs exhibit far better passive membrane permeability than expected for "beyond Rule of 5" molecules, in some cases exceeding that of much lower molecular weight, traditional drug molecules. This chemistry has been demonstrated to be suitable for the creation of libraries of IP+-linked MPs and show that these libraries can be mined for protein ligands.

Description

SYNTHESIS OF MEMBRANE PERMEABLE MACROCYCLIC PEPTIDES VIA IMIDAZOPYRIDINIUM GRAFTINGGOVERNMENT SUPPORT

[0001] This invention was made with government support under CA273954 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0002] The present disclosure relates generally to an efficient solid-phase synthesis of novel macrocyclic peptides (MPs). In particular the disclosure relates to synthesis of novel macrocyclic peptides by trapping a reversible intramolecular imine linkage with a 2-carbonyl pyridine to create an imidazopyridinium (I P+)-linked ring.BACKGROUND

[0003] Macrocyclic peptides (MP) and related molecules have attracted considerable interest as probe molecules and drug leads, particularly for addressing difficult protein targets. Macrocyclization of peptides imparts many favorable properties, including increased resistance to proteases, decreased conformational flexibility and, in certain special cases, increased cell permeability by promoting intramolecular hydrogen bonds that mask otherwise highly hydrated amide N-H moieties. Moreover, powerful methods exist for the synthesis and screening of huge libraries of genetically-encoded MPs, such as phage display and mRNA display. Split and pool synthesis of DNA-encoded libraries (DELs) of macrocycles is another route to access large numbers of these molecules. For such applications, there continues to be a need for new macrocyclization reactions that proceed with high efficiency under gentle reaction conditions, allow the introduction of novel functional groups, and suffer from little or no competitive intermolecular coupling. The present disclosure describes a new method for the macrocyclization of peptides that meets all of these criteria and confers on the products favorable pharmacological properties.SUMMARY

[0004] Provided herein is a method of preparing a macrocyclic peptide (MP) having a stable imidazopyridinium, comprising: admixing under acidic conditions:(a) a peptide comprising a first terminus comprising a terminal amine group and a second terminus comprising a terminal aldehyde and at least one amino acid between the first terminus and the second terminus; and(b) about 2 to about 4 equivalents of a substituted or unsubstituted 2-carbonyl pyridine compound.

[0005] Further, provided herein is a process for preparing macrocyclic peptides (MP), having a stable imidazopyridinium (IP+) unit of Formula I:whereinC is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs- aryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, C^alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci^alkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulky;Rxat each instance, when present, is an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6’ position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs- aryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4, comprising: admixing a peptide of Formula IV containing a terminal amine group and a terminal aldehyde group:with about 2.0 to about 4.0 equivalents of a 2-carbonyl pyridine compound of Formula Bunder acidic conditions, wherein is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs- aryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, C^alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci^alkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulkyRxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6 position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4.

[0006] Additionally, provided herein is a process for preparing macrocyclic peptides of Formula l-a, comprising:whereinQ is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulkyRxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 2’ position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs- aryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4, comprising: a.) oxidizing a peptide of Formula II to yield a protected peptide of Formula IIIQ is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;PG is a protecting group; each AA is independently an amino acid;R1is H or Ci-ealkyl, andR8is CH2OH, CH2SH, or CH(OH)CH3b.) deprotecting the protected peptide of Formula III to yield a peptide of Formula IV;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;PG is a protecting group; each AA is independently an amino acid; andR1is H or Ci-ealkyl, c.) admixing a 2-carbony pyridine compound of Formula B with the peptide of Formula IV to yield the cyclic peptide of Formula I;whereinR7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulkyRxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6 position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs- aryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; andR1is H or Ci-ealkyl, and d.) releasing the cyclic peptides of Formula I from a solid support to yield macrocyclic peptides of Formula l-awherein is H, OH, Ci-ealkyl, Ci-ealkoxy, Cs- aryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; andR1is H or Ci-ealkyl.

[0007] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. While the compounds and methods disclosed herein are susceptible of cases in various forms, the description hereafter includes specific cases with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific cases described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 A shows the solid-phase synthesis of an IP+-linked MP A. The reaction was carried out on a 50 pmol scale based on loading. Mmt = Monomethoxytrityl, a) 25%HFIP / DCM, r.t., 2 x 30 min. b) AcOH / TFE (1 :1 , v / v), a 2-carbonyl pyridine compound (3.0 equiv.), rt., 10 hours. c TFA, r.t., 40 min.

[0009] Figure 1 B: Crude analytical HPLC trace of released cyclic peptide MP1 .

[0010] Figure 1C: High-resolution mass spectrum of the crude material released from beads (MP1).

[0011] Figure 2A shows various aldehydes containing P2A motif for Macrocyclization.Aldehydes and ketones containing a 2-carbonyl pyridine compound motif for cyclization. Nap (L-Ala(2-naphthyl)-OH) was used as UV indicator. All the reactions were carried out at 2 pmol and the purity of crude product was determined by LC-MS.

[0012] Figure 2B shows cyclization on TG beads followed by conjugation with FITC or Biotin.

[0013] Figure 3A shows side-to-head cyclization on TG Beads. Each linear peptide precursor was prepared in 2 pmol on TG beads.

[0014] Figure 3B shows side-to-head cyclization on TG Beads. All the reactions were carried out at 10 pmol scale based on loading. Purity of crude product determined by HPLC was given. Ahx: 6-aminohexanoic acid; Ipa: Isonipecotic acid.

[0015] Figure 4 shows the MP synthesized using side chain-to-side chain linkages. All the reactions were carried out at 10 pmol scale based on beads loading. Purity of crude product determined by HPLC was given. Dap: L-2,3-diaminopropionic acid; pAla: p-Alanine; Nle: L- norleucine.

[0016] Figure 5 shows the creation of stapled peptides via IP+chemistry. All the reactions were carried out at 10 pmol scale based on beads loading. Purity of crude product determined by HPLC was given.

[0017] Figure 6 shows the solid-phase synthesis of 100 IP+- linked MPs in individual wells of a microtiter plate. The numbers indicate % conversion to product.

[0018] Figure 7A shows the synthesis and screening of an IP+MP library. General structure of the library and the building blocks used in its construction.

[0019] Figure 7B. shows the synthesis and screening of an IP+MP library. Top: Schematic of the screening protocol used to identify ligands for fluorescently labeled Streptavidin (SA). Bottom: Increase in fluorescence polarization as a result titration of fluorescein-labeled MP29 (structure shown on right without the fluorescein) with unlabeled SA. See text for details of the library construction and screening.

[0020] Figure 8A shows the passive membrane permeability of certain IP+MPs. Rates of membrane passage (expressed in units of -logPe) for the compounds indicated as measured using PAMPA.

[0021] Figure 8B shows the structures of the most permeable MPs.

[0022] Figure 8C shows the structures of MP36 analogues lacking the IP+ unit. Their(poorer) passive membrane permeability is shown in A (yellow dots).

[0023] Figure 9A shows the passive membrane permeability of certain IP+MPs.

[0024] Figure 9B shows the structures of additional peptides that were tested for passive membrane permeability.

[0025] Figure 10 shows the circular dichroism spectra of MP24-28 and the linear precursor.

[0026] Figure 11 shows one-compound-one-well synthesis of 96 peptide sequences utilized in the synthesis of IP+MP libraries for screening of streptavidin ligands.

[0027] Figure 12 shows the fluorescence intensity of screening samples examined according to the present disclosure.

[0028] Figure 13 is a schematic of the chloroalkane penetration assay (CAPA).

[0029] Figure 14A is a plot of normalized fluorescence vs concentration for compounds showing improvement cell permeability of the IP+ macrocycles of the disclosure into living cells using the CAPA, relative to analogous compounds lacking the IP+ unit.

[0030] Figure 14B shows the compounds tested to prepare the plot in Figure 14A, LB9_2b (open circles) is an IP+ macrocycle of the disclosure and LB1 a (closed circles) is an analogous compound lacking the IP+ unit.DETAILED DESCRIPTION

[0031] There continues to be a need for new macrocyclization reactions that proceed with high efficiency under gentle reaction conditions, allow the introduction of novel functional groups, and suffer from little or no competitive intermolecular coupling. The present disclosure describes a new method for the macrocyclization of peptides that meets all of these criteria and confers on the products favorable pharmacological properties.

[0032] The disclosure provides methods of preparing MPs and other macrocyclic compound by trapping an intramolecular imine linkage with a substituted or unsubstituted 2- formyl- or 2-keto-pyridine to create a stable, imidazopyridinium (IP+) unit, a heterocycle that has not previously been incorporated into MPs. Given the broad availability of substitutedpyridines, the methods provide a simple route to introduce additional diversity into MP libraries over and above that of the amino acids.

[0033] The disclosed methods provide one or more advantages, including but not limited to (a) a remarkably efficient and flexible method for the solid-phase synthesis of MPs; (b) the creation of bead-displayed libraries of I P+-linked macrocycles, such as a novel streptavidin ligand from such a library; and / or (c) remarkably good passive membrane permeability of the resulting IP+MPs, such that even molecules far beyond Lipinski’s Rule of 5 (Ro5) permeate membranes at rates comparable to low molecular weight, drug-like compounds.Methods of the Disclosure

[0034] The methods of the present disclosure may be used to prepare MPs, including but not limited to, those provided in Table A.Table A

[0035] Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, cis-trans, conformational, and rotational) forms of the structure. For example, the R and Sconfigurations for each asymmetric center, (2) and (E) double bond isomers, and (2) and (E) conformational isomers are included in this disclosure, unless only one of the isomers is specifically indicated. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, cis / trans, conformational, and rotational mixtures of the present compounds are within the scope of the disclosure. In some cases, the compounds disclosed herein are stereoisomers. "Stereoisomers" refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein can exist as a single stereoisomer, or as a mixture of stereoisomers. Stereochemistry of the compounds shown herein indicate a relative stereochemistry, not absolute, unless discussed otherwise. As indicated herein, a single stereoisomer, diastereomer, or enantiomer refers to a compound that is at least more than 50% of the indicated stereoisomer, diastereomer, or enantiomer, and in some cases, at least 90% or 95% of the indicated stereoisomer, diastereomer, or enantiomer.

[0036] The compounds disclosed herein that have a double bond can exhibit Eor 2 (not shown) stereochemistry. In some cases, the compounds of Formula (I) exhibit E stereochemistry. In various cases, the compounds of Formula (I) exhibit 2stereochemistry at the double bond. The compounds of Formula (I) can have any stereochemical configuration at any sp3carbon atoms. In some cases, the compounds of the disclosure are optically pure. As used herein, “optically pure” refers to the predominant presence of one enantiomer of acompound if multiple stereochemical configurations can exist (e.g., at least 99% enantiomeric excess).

[0037] Unless otherwise indicated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure.

[0038] The compounds of the disclosure are defined herein by their chemical structures and / or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

[0039] As used herein, the term “alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms. The term Cnmeans the alkyl group has “n” carbon atoms. For example, Cealkyl refers to an alkyl group that has 6 carbon atoms. Ci- ?alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 6 carbon atoms), as well as all subgroups (e.g., 1-5, 2-5, 3-6, 1 , 2, 3, 4, 5, and 6 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and t-butyl (1 ,1 -dimethylethyl). Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0040] As used herein, the term “alkenyl” refers to straight chained and branched hydrocarbon groups, which contains at least one carbon-carbon double bond, containing two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms. The term Cnmeans the alkyl group has “n” carbon atoms. For example, Cealkenyl refers to an alkenyl group that has 6 carbon atoms. Cealkyl refers to an alkenyl group having a number of carbon atoms encompassing the entire range (i.e., 2 to 6 carbon atoms), as well as all subgroups (e.g., 2-5, 3-6, 2, 3, 4, 5, and 6 carbon atoms). Unless otherwise indicated, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

[0041] As used herein, the term “alkynyl” refers to straight chained and branched hydrocarbon groups, which contains at least one carbon-carbon triple bond, containing two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms. The term Cnmeans the alkyl group has “n” carbon atoms. For example, Cealkynyl refers to an alkynyl group that has 6 carbon atoms. Cealkyl refers to an alkynyl group having a number of carbon atoms encompassing the entire range (i.e., 2 to 6 carbon atoms), as well as all subgroups (e.g., 2-5, 3-6, 2, 3, 4, 5, and 6 carbon atoms). Unless otherwise indicated, an alkynyl group can be an unsubstituted alkynyl group or a substituted alkynyl group.

[0042] As used herein, the term “cycloalkyl” refers to a non-aromatic ring. Additionally, cycloalkyls of the disclosure can be monocyclic, bicyclic, bridged, fused or spirocyclic.

[0043] As used herein, the term “aryl” refers to a cyclic aromatic ring (e.g., a monocyclic aromatic ring with 5-6 total ring atoms, or a fused bicyclic ring with 10 total ring atoms). Unless otherwise indicated, an aryl group can be unsubstituted or substituted. Aryl groups can be isolated (e.g., phenyl) or fused to another aryl group (e.g., naphthyl), a cycloalkyl group (e.g., 1 ,4,5,6-tetrahydrocyclopenta[b]pyrrole), a heterocycle group (e.g., indoline), and / or a heteroaryl group (e.g., indole).

[0044] As used herein, the term "heterocycle" refers to a non-aromatic ring which contains one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur. Additionally, heterocycles of the disclosure can be monocyclic, bicyclic, bridged, fused or spirocyclic. For example, a heterocycle can be a monocyclic, bicyclic, bridged, fused, or spirocyclic 4-8 membered ring having 1 or 2 or 3 heteroatoms selected from N, O, and S. As another example, a heterocycle can be an 8-10 membered bicyclic, bridged, fused, or spirocyclic group having 1 or 2 or 3 ring heteroatoms selected from N, O, and S in the bicyclic ring. Nonlimiting examples of heterocycle groups include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, and oxazepane.

[0045] As used herein, the term "heteroaryl" refers to a cyclic aromatic ring having heteroatoms in the ring (e.g., a monocyclic aromatic ring with 5-6 total ring atoms, or a fused bicyclic ring with 10 total ring atoms), and containing one to three heteroatoms selected from nitrogen, oxygen, and sulfur atom in the aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted. Heteroaryl groups can be isolated (e.g., pyridyl, thiazyl, pyrryl) or fused to another heteroaryl group (e.g., purinyl), a cycloalkyl group (e.g., tetrahydroquinolinyl), a heterocycle group (e.g., dihydronaphthyridinyl), and / or an aryl group (e.g., benzothiazolyl, quinolyl, isoquinolinyl, or quinazolinyl).

[0046] As used herein, the term “alkoxy” refers to a “ — O-alkyl” group.

[0047] As used herein, the term “halo” refers to refers to a fluoro (F), chloro (Cl), bromo(Br), or iodo (I) group.

[0048] As used herein, the term “haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen. Such groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1 ,1 -difluoroethyl, 2-fluoroethyl, 1- chloro-2-fluoromethyl and 2-fluoroisobutyl.

[0049] As used herein, the term “hydroxyalkyl” refers to refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a hydroxyl group (OH). Such groups include but are not limited to, hydroxymethyl, hydroxyethyl, and the like.

[0050] As used herein, a “substituted” functional group is a functional, group having at least one hydrogen radical that is substituted with a non-hydrogen radical (i.e., a substituent). Examples of non-hydrogen radicals (or substituents) include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkyl, alkynyl, ether, aryl, heteroaryl, heterocycle, hydroxyl, oxy (or oxo), alkoxyl, ester, thioester, acyl, carboxyl, cyano, nitro, amino, sulfhydryl, and halo. When a substituted alkyl group includes more than one nonhydrogen radical, the substituents can be bound to the same carbon or different carbon atoms.

[0051] As used herein, the term “amino acid” and refers to organic compounds that contain both amino and carboxylic acid functional groups. Typically, amino acids may be subdivided into categories such as alpha (a), beta (P), gamma (y), etc., wherein the amino group is attached to a carbon atom that is attached to the carboxylic acid group, once removed from the carboxylic acid group, twice removed from the carboxylic acid group, etc., respectively. Also, many amino acids have a chiral center and thus can exist as either L or D isomers with only L isomers being able to form peptides. Additionally, amino acids may be subdivided based upon chemical properties such as polarity, ionization, etc. Further, amino acids may be subdivided based upon their side-chain groups such as aliphatic, acyclic, aromatic, polar, etc.

[0052] Over 500 amino acids exist in nature and these are further referred to as “natural” or “naturally occurring” amino acids. However, countless amino acids have been prepared or can be prepared containing different carbon chain lengths as well as different side chains. Of these amino acids there is a subgroup of 22 L-a-amino acids that may be used in peptide / protein synthesis which are known as “proteinogenic” or “canonical” amino acids. The proteinogenic amino acids are: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine. There exist hundreds of naturally occurring, non-proteinogenic amino acids such as: y-aminobutyric acid (GABA), levodopa (L-DOPA), triiodothyronine, hydroxyproline, ornithine, homoserine, cystine, canavanine, cystine, p-alanine, carnosine, N- formylmethionine, hypusine, 2-aminoisobutyric acid, citrulline, pantothenic acid, carnitine, 5- hydroxytrytophan, mimosine, 2,4-diaminobutyric acid, norleucine, methionine sulfoxine, methionine sulfone, a-aminobutyric acid, norleucine. There exist thousands of non-naturallyoccurring amino acids having varying length carbon chains and varying side chains such as: eflornithine, naphthylalanine, aspartylphenylalanine 1 -methyl ester, 3-nitrytyrosine, and nitroarginine.

[0053] As used herein, the term “sterically bulky” refers to a moiety that slows the rate of a chemical reaction due to steric bulk. Sterically bulky groups exhibit steric hindrance, which is usually manifested in intermolecular reactions. Steric hindrance is often exploited to control selectivity, such as slowing unwanted side-reactions. Steric hindrance can be observed in the relative rates of reaction. An example of such a measurement is that methyl bromide solvolyzes 107times faster than neopentyl bromide due to the inhibition of attack on the compound with the tertiary-butyl group. Sterically bulky generally encompasses any carbon or silicon atoms tri-substituted with atoms other than H. Examples of sterically bulky groups are tertiary-butyl, triphenylmethyl, p-methoxyphenyl-diphenylmethyl, and trimethylsilyl. A substituent is not sterically bulky if it does not include a group including a carbon or silicon atom tri-substituted with atoms other than H.

[0054] As used herein, the term “linker” refers to a compound having a chemical moiety that can be covalently bonded to a solid support (e.g., resin bead) and a protected moiety that can be covalently bonded to a reactant compound. Examples of this type of solid-phase synthesis can be used in the production of peptides, DNA, RNA, etc. Commonly used classes of linkers are acid-labile linkers, nucleophile-labile linkers, and photo-labile linkers.

[0055] As used herein, the term “protecting group” refers to a group used to temporarily mask the characteristic chemistry of a functional group because it interferes with another reaction. Suitable protecting groups should be easy to put on, easy to remove and in high yielding reactions, and inert to the conditions of the reaction required. Some common amino protecting groups are carbamates, amides, N-alkyls, amino acetals, N-benzyls, imines, enamines, N-phosphinyl, N-phosphoryl, N-silyl, N-sulfenyl, and N-sulfonyl. Examples of suitable protecting groups include, but are not limited to, monomethoxytrityl (MMT), carbobenzyloxy (Cbz), p-methoxybenzyloxycarbonyl (Moz or MeOZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), and allyloxycarbamate, acetyl (Ac), benzoyl (Bz), trichloroethyl chloroformate (Troc), p-toluenesulfonyl (Tosyl; Ts), nitrobenzenesulonyl (Nosyl), 2-nitrophenylsulfenyl (Nps), benzyl (Bn), p-methoxybenzyl (PMB), 3,4- dimethoxybenzyl (DMPM), and p-methoxyphenyl (PMP). A specific N-alkyl group is monomethoxytrityl which can be cleaved under neutral conditions.

[0056] As used herein, the term “electron donating group” refers to an atom or functional group that can donate electron density into neighboring atoms, usually a TT system via resonance or inductive effects thus making the TT system more nucleophilic. As a result ofthese electronic effects, for example, an aromatic ring to which such a group is attached is more likely to participate in electrophilic substitution reactions or a carbonyl group to which an electron donating group is attached becomes more nucleophilic, activating the carbonyl for further reactions. Examples of electron donating groups are: oxide, amino, hydroxy, alkoxy, acylamido, acyloxy, alkylphosphino, dialkylphosphino, alkylthio, sulfhydryl, alkyl, phenyl, and vinyl.

[0057] As used herein, the term “electron withdrawing group” refers to an atom or functional group that draws electron density from neighboring atoms towards itself, usually by resonance or inductive effects. Examples of electron withdrawing groups are: halogen, aldehyde, ketone, carboxylic ester, carboxylic acid, acyl halide, amide, cyano, and nitro.

[0058] Disclosed herein are methods of preparing a macrocyclic peptide (MP) having a stable imidazopyridinium. The methods can include admixing under acidic conditions (a) a peptide comprising a first terminus comprising a terminal amine group and a second terminus comprising a terminal aldehyde and at least one amino acid between the first terminus and the second terminus and (b) about 2 to about 4 equivalents of a substituted or unsubstituted 2-carbonyl pyridine compound.

[0059] The 2-carbonyl pyridine compounds are not particularly limiting. The 2-carbonyl pyridine can be a substituted or unsubstituted 2-formylpyridine. The 2-carbonyl pyridine can be a substituted or unsubstituted 2- ketopyridine. The 2-carbonyl pyridine compounds can generally have a structure. R7can generally be H or any functional group, provided the steric bulk is not so great as to inhibit the reaction. Surprisingly, as shown in Figure 2, macrocyclization proceeded smoothly even with several 2-ketopyridines (Py21-24). As the steric bulk of R7increases, the efficiency of the reaction generally decreases. R7can be an electron donating group. R7can be an electron withdrawing group. R7can be not sterically bulky. As shown in Figure 2, when R7is other than H, the reaction is less efficient when R6is bigger and does not activate the ketone by withdrawing electrons, thus making the carbonyl more electrophilic. This can be seen by comparing the yield of the macrocycle using Py21 and Py22. In neither case do the methyl or propyl substituents activate the ketone, and the larger propyl group hinders the reaction, relative to the smaller methyl group. However, in both cases the desired products were formed.

[0060] R7can be selected from H, OH, SH, CN, amino, halo, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. R7can be selected from H, OH, SH, CN,amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, Cs- cycloalkyl, a 5-10 membered heterocycloalkyl, Cs- aryl, and a 5-10 membered heteroaryl. R7can be selected from H, methyl, propyl, or pyridinyl.

[0061] Each Rxmcan generally be H or any functional group or two vicinal Rxmgroups, together with the carbon atoms to which they are attached can form a cyclic or bicyclic group, provided that the steric bulk of any Rxat the 6 position is not so great as to inhibit the reaction. An Rxat the 6 position can generally be any group that is not sterically bulky. As the combined electron donating ability of total Rxgroups increases, the efficiency of the reaction generally increases. As the combined electron withdrawing ability of total Rxincreases, the efficiency of the reaction generally decreases. The 2-carbonyl pyridine compound can be substituted with an electron donating group. As shown in Figure 2, a wide variety of substituted 2-carbonyl pyridine compounds can be used to prepare the I P+-linked macrocycles of the disclosure. 2-carbonyl pyridine compounds carrying electron donating or weakly electron withdrawing groups such as methyl, methoxy, chloro-, bromo, piperidyl and phenyl groups, provide the IP+-linked macrocycle in excellent yield and purity (>85%; Py1 -4, Py6-7, Py9-11 , Py14-15). 2-carbonyl pyridine compounds bearing moderate to strongly electron withdrawing groups such as -F, -CN or a carboxylic acid were less efficient substrates (Py8, Py12, Py13). Quinoline carboxaldehydes were also demonstrated to be excellent substrates (Py17, Py18, Py20). Thus, the scope of the pyridine moiety in the reaction is broad, though electron withdrawing substituents can result in somewhat lower yields. Two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group. Two vicinal Rxgroups together with the carbon atoms to which they are attached can form a substituted or unsubstituted piperdinyl, phenyl, or indonyl.

[0062] The pyridine moiety can be selected to provide a site of reactivity for further modification of the resulting MP. As shown in Figure 2, Py25, with a piperazinyl group bearing a free secondary amine, facilitated macrocyclization in highly efficient way and the free secondary amine on the piperazinyl group was readily modified with common labels such as biotin or carboxyfluorescein.

[0063] The 2-carbonyl pyridine can be selected from the group of:

[0064] The peptide of the disclosure can generally be any peptide comprising a first terminus comprising a terminal amine group and a second terminus comprising a terminal aldehyde and at least one amino acid between the first terminus and the second terminus. The terminal amine group of the first terminus can be the amine group of a terminal amino acid. The terminal aldehyde can be an activated aldehyde. The activated aldehyde can be a glyoxamide. The activated aldehyde can be a glyoxamide moiety modified with an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl between the two carbonyl groups of the glyoxamide.

[0065] The size of the peptide is not particularly limited. In general, the size of the peptide can be limited by the ability of the peptide to allow the two termini to approach each other in space so that the 2-carbonyl pyridine compound can react with both ends. The peptide can include at least one amino acid. The maximum number of amino acids in the peptide is not particularly limited. However, for some amino acids, e.g., alanine, as the number of amino acids increases, the amino acids self-assembly in a rigid helical structure which limits the flexibility of the molecule and hinders the ability of the two termini to approach in space andultimately the ability of the reaction to proceed. For amino acids that do not form helical structures and / or for mixtures of amino acids, the maximum number of amino acids in the peptide between the first terminus and the second terminus is not limited. As shown in Figure 3A, a series of linear precursors were synthesized in which 0-9 alanine residues separated the reactive Oxo (first terminus) and lysine (Lys) (second terminus) units. Beads displaying these molecules were then exposed to 2-formyl-quinoline to close the macrocycle. After release from the beads the products were analyzed by LC-MS. Compound MP2, in which no spacer alanine was present, was produced in only about 5% LC yield. A single alanine spacer resulted in about half of the starting material being converted to the desired product. The linear peptides containing 2-5 alanine units provided a high yield of clean product. The yield decreased again using peptides containing 6-9 alanine residues in view of helical structure formation. The peptide of the disclosure can comprise from 1 to 40 amino acids between the first terminus and second terminus, for example, about 1 to 40, about 1 to 35, about 1 to 30, about 1 to 25, about 2 to 20, about 3 to 15, or about 4 to 10 amino acids.

[0066] As shown in Figure 3B, various MPs that contain a variety of amino acids were prepared. Various protected amino acids were compatible with this reaction using the standard conditions with various 2-carbonyl pyridine compounds (for polar amino acids, the protecting groups were removed after cyclization). Cyclization proceeded efficiently with substrates containing Tyr (MP12), Trp (MP13), Arg (MP13), Lys (MP14), Cys (MP15), His (MP16), Glu (MP17). Methionine residues are oxidized quantitatively to the sulfoxide (see compound MP14) by NalCU. as expected. The structures of these macrocycles were confirmed by LCMS and NMR spectroscopy.

[0067] Mixed amino acid MPs with ring sizes greater than 24 atoms were constructed. As seen in Figure 3B, this resulted in efficient macrocyclization for compounds (MP18 and MP19) with 37 and 39 atoms, respectively, in the ring. It is believed that the IP+ chemistry will be useful for the efficient synthesis of macrocycles spanning a much wider range of ring sizes than might have been suggested from the data shown in Fig. 3A so long as there is sufficient flexibility in the linear precursor to allow formation of the intramolecular imine intermediate.

[0068] MPs can also be prepared from peptides in which the first terminus comprising the terminal amine group and / or the second terminus comprising the terminal aldehyde are on a side chain. As shown in Figure 4, side chain-to-side chain -connected MPs with different ring sizes were obtained in excellent purity using a variety of 2-carbony pyridine compounds. The peptides comprising the first terminus comprising the terminal amine group and / or the second terminus comprising the terminal aldehyde on a side chain can be used for “peptide stapling,” as shown in Figure 5. Peptide stapling is a technique in which appropriatelypositioned side chain residues are linked covalently to stabilize an alpha-helical conformation of the peptide. Stapled peptides are of broad interest as inhibitors of proteinprotein interactions. For example, a stapled peptide inhibitor of p53-Mdm2 / MdmX binding has recently entered clinical trials for the treatment of certain cancers with wild-type p53. As described in the Examples, all of the stapled MPs prepared in accordance with the methods of the disclosure displayed an enhanced degree of helical content. Further, MPs prepared in accordance with methods of the disclosure that contain the same ring but were formed using different 2-carbonyl pyridine compounds, display markedly different levels of helicity. Thus, the methods of the disclosure can be used to design and prepare finely tuned helical content of stapled peptides.

[0069] In the methods of the disclosure, the peptide can be attached to a solid support. The solid support can be a resin material. The solid support can be a swellable resin material. The swellable resin material can comprise a polystyrene bead, low cross-linked polystyrene bead, mixed block polystyrene-divinylbenzene bead, mixed block polystyrenepolyethylene glycol bead, polyacrylamide polyethylene glycol copolymer bead, or a combination thereof. The solid support can include a linker through which the peptide is attached to the solid support. The linker can be a rink-amide linker.

[0070] In the methods of the disclosure, the admixing under acidic conditions comprises admixing the peptide and the 2-carbonyl pyridine in the presence of an acid having a pKa of less than about 4.9. The acid having a pKa of less than about 4.9 can comprise acetic acid, adenine, arsenic acid, benzoic acid, butyric acid, chromic acid, formic acid, hydrofluoric acid, lactic acid, propionic acid, ascorbic acid, acetic acid, sulfuric acid, nitrous acid, oxalic acid, hydrochloric acid, phosphoric acid, sulfurous acid, or a combination thereof. The acid having a pKa of less than 4.9 can comprise acetic acid, benzoic acid, formic acid, lactic acid, propionic acid, ascorbic acid, or a combination thereof. The acid having a pKa of less than 4.9 can comprise acetic acid.

[0071] In the methods of the disclosure, the admixing of the peptide and the 2-carbonyl pyridine can occur in the presence of a solvent. The solvent can be any solvent that is miscible with the acid. The solvent can be a polar solvent. Suitable solvents can include, but are not limited to dimethylformamide, dimethylsulfoxide, dichloromethane, trichloromethane, trifluoroethanol, tetrahydrofuran, methylpyrollidone, water, and aqueous buffer.

[0072] The peptide and the 2-carbonyl pyridine can be admixed at a temperature in a range of about 10°C to about 50°C, for example, about 20°C to about 40°C, for example, about 25°C to about 35°C, about 25°C, about 30°C, or about 35°C.

[0073] The peptide and the 2-carbonyl pyridine can be admixed for about 2 hours to about 48 hours, for example, in a range of about 2 hours to about 48 hours, about 2 hours to about 36 hours, about 2 hours to about 24 hours, about 3 hours to about 18 hours, about 6 hours to about 12 hours, about 8 hours to about 10 hours, or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 1 1 hours, about 12 hours, about 18 hours, about 14 hours, about 36 hours, or about 48 hours.

[0074] The disclosure provides methods for preparing macrocyclic peptides having a stable imidazopyridinium unit, the macrocyclic peptide having a structure according to Formula I:wherein is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl; Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; R1is H or Ci-ealkyl; R7is H, OH, SH, CN, amino, halo, Ci- ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5- 10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulky; Rxat each instance, when present, is an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6’ position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs-ecycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4, comprising admixing a peptide of Formula IV containing a terminal amine group and a terminal aldehyde group:with about 2.0 to about 4.0 equivalents of a 2-carbonyl pyridine compound of Formula Bunder acidic conditions, wherein W' is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs- waryl, or a 5-10 membered heteroaryl; Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; R1is H or Ci-ealkyl; R7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5- 10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulky; Rxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6’ position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs-ecycloalkyl, Cs-waryl, a 5-7- membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4.

[0075] in Formula I and Formula IV can be the same or different. For example, inFormula IV, can be a solid support and in Formula Ican be a H, for example, if the product of admixing the compound of Formula IV and the compound of Formula B is further treated to remove the solid support.in Formula I and Formula B can be a solid support,H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl. V in Formula I andFormula B can be a solid support, H, OH, Ci-ealkyl, or Ci-ealkoxy.in Formula I andFormula B can be a solid support.in Formula I and Formula B can be H. Whenis a solid support, it can be any solid support disclosed herein. The solid support can be a resin bead. The resin bead can be a polyethylene glycol / polystyrene bead.

[0076] Q in Formula I and Formula IV can be a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl. Q in Formula I and Formula IV can be a bond. Q in Formula I and Formula IV can be an amino acid. Q in Formula I and Formula IV can be a C5-10 aryl. Q in Formula I and Formula IV can be a 5-10 membered heteroaryl. In general, Q in the peptide of Formula IV does not change throughout the method, such that Q in Formula IV and Q in Formula I are the same.

[0077] Each AA in Formula I and Formula IV are independently an amino acid. The amino acids can be any amino acids disclosed herein. In general, the methods of the disclosure do not affect the amino acids such that the amino acids in Formula IV are the same as the amino acids in Formula I. However, the amino acids in the peptide of Formula IV may include a protecting group that is removed in the macrocyclic peptide of Formula I.Additionally, the macrocyclic peptide of Formula I may be further modified post macrocyclic peptide formation. In some cases, the amino acids are selected such that the amino acid sequences do not form rigid helical structures. Each amino acid sequence in Formula I and Formula IV can include 1 -20 amino acids, for example, in a range of 1 -20, 2-18, 3-16, 4-14, 5-12, 6-10, 1 -7, 2-6, or 3-10, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20. The number of amino acids in each sequence do not change between the peptide of Formula IV and the macrocycle of Formula I. The number of amino acids in the two amino acid sequences in Formula IV and Formula I can be the same or different.

[0078] R1in Formula I and Formula IV can be H or Ci-ealkyl. R1in Formula I and Formula IV can be H. R1in Formula I and Formula IV can be Ci-ealkyl. In general, R1in the peptide of Formula IV does not change throughout the method, such that R1in Formula IV and R1in Formula I are the same.

[0079] The compound of Formula B can generally be any 2-carbonyl pyridine disclosed herein. R7can be any R7disclosed herein. Each Rxcan be any Rxdisclosed herein, with the proviso that when Rxis at the 6 position of the 2-carbonyl pyridine, the Rxis not sterically bulky.

[0080] The compound of Formula B can have a structure according to Formula B-a:wherein R7is any R7disclosed herein and R3, R4, R5, and R6are each independently selected from H, Ci-ealkyl, C^alkenyl, C^alkynyl, Ci-ehaloalkyl, Ci-ehydroxyalkyl, Ci-ealkoxy, halo, carboxyl, CN, Cs- cycloalkyl, Cs-waryl, a 3-10 membered heterocycle, and a 5-10 membered heteroaryl, wherein R3and R4, R4and R5, or R5and R6together with the carbon atoms to which they are attached may be taken together to form a Cs- cycloalkyl, Cs-waryl, a 3-10-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with Ciwalkyl, C^alkenyl, C^alkynyl, Ci- ehaloalkyl, Ciwalkoxy, halo, carboxyl, CN, Cs- cycloalkyl, Cs-waryl, a 3-10 membered heterocycle, or a 5-10 membered heteroaryl, provided that R6is not so sterically bulky as to hinder the reaction. R6can generally be any group that is not sterically bulky. As the combined electron donating ability of R3, R4, R5, and R6increases, the efficiency of the reaction generally increases. As the combined electron withdrawing ability of R3, R4, R5, and R6increases, the efficiency of the reaction generally decreases. R3, R4, R5, and R6can be electron donating groups. R3and R4, R4and R5, or R5and R6together with the carbon atoms to which they are attached can form a Cswcycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group. R3and R4, R4and R5, or R5and R6together with the carbon atoms to which they are attached can form a substituted or unsubstituted piperdinyl, phenyl, or indonyl.

[0081] The peptide of Formula IV and the 2-carbonyl pyridine of Formula B can be admixed with an acid having a pKa of less than about 4.9. The acid having a pKa of less than about 4.9 can comprise any acid having a pKa of less than about 4.9 disclosed herein.

[0082] The peptide of Formula IV and the 2-carbonyl pyridine of Formula B can be admixed in a solvent. The solvent can be any solvent that is miscible with the acid. The solvent can be a polar solvent. Suitable solvents can include, but are not limited to, dimethylformamide, dimethylsulfoxide, dichloromethane, trichloromethane, trifluoroethanol, tetrahydrofuran, methylpyrollidone, water, and aqueous buffer.

[0083] The peptide and the 2-carbonyl pyridine can be admixed at a temperature in a range of about 10°C to about 50°C, for example, about 20°C to about 40°C, about 25°C to about 35°C, about 25°C, about 30°C, or about 35°C.

[0084] The peptide and the 2-carbonyl pyridine can be admixed for about 2 hours to about 48 hours, for example, in a range of about 2 hours to about 48 hours, about 2 hours to about 36 hours, about 2 hours to about 24 hours, about 3 hours to about 18 hours, about 6 hours to about 12 hours, about 8 hours to about 10 hours, or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 1 1 hours, about 12 hours, about 18 hours, about 14 hours, about 36 hours, or about 48 hours.

[0085] In general, m can be an integer in a range of 0 to 4, for example, 0, 1 , 2, 3, or 4.

[0086] The methods of the disclosure can further comprise preparing the peptide of Formula IV by deprotecting a protected peptide of Formula III:wherein W is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs- aryl, or a 5-10 membered heteroaryl; Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; R1is H or Ci-ealkyl; and PG is a protecting group., , , Q, AA, and R1defined herein. The conversion of the protected peptide of Formula III to the peptide of Formula IV does not affectQ, AA, or R1. Accordingly, for a given reaction,Q, AA, or R1in Formula IV is the same asQ, AA, or R1in Formula III.

[0087] PG can be any protecting group disclosed herein. PG can be monomethoxytrityl.

[0088] Methods of removing protecting groups are well known in the art. When the protecting group is monomethoxytrityl, the protecting group can be removed by treating the peptide of Formula III with a mixture of hexafluoroisopropanol (HFIP) and dichloromethane (DCM) at room temperature (about 20-25°C) for about 15 minutes to about 1 hour. Theprotected peptide of Formula III can be treated once, twice, or three times with the mixture of hexafluoroisopropanol (HFIP) and dichloromethane (DCM). The hexafluoroisopropanol and dichloromethane can be provided in a ratio in a range of 1 :4 HFIP:DCM to 1 :2 HFIP:DCM, for example 1 :3 HFIP:DCM, by volume.

[0089] In the protected peptide of Formula III, Q can be a bond and the protected peptide of Formula III can have a structure of Formula lll-a:wherein AA, and R1can be anyAA, and R1defined herein.

[0090] The methods of the disclosure can further comprise preparing a protected peptide of Formula III, comprising oxidizing a peptide of Formula II:wherein Q, PG, AA, R1can be anyQ, PG, AA, and R1defined herein, and R8can be selected from CH2OH, CH2SH, and C(OH)CH3. R8can be CH2OH. R8can be CH2OH. R8can be CH(OH)CH3.

[0091] The oxidation of the peptide of Formula II to the peptide of Formula III does not generally affectQ, AA, or R1; however, the peptide of Formula II may have protecting groups on various amino acids that are susceptible to oxidation and the protecting groups can be removed to form a peptide of Formula III. Accordingly, for a given reaction,Q,AA, or R1in Formula IV is the same as Q, AA, or R1in Formula III, with various amino acids in protected or deprotected form.

[0092] Methods of oxidation are known in the art. The peptide of Formula II can be oxidized with sodium periodate (NaIC ) in aqueous solution at room temperature. The NaIC can be provided in a molar excess, for example, about 2 equivalents, about 3 equivalents, about 4 equivalents, about 5 equivalents, about 6 equivalents, or about 7 equivalents relative to the amount of peptide of Formula II.

[0093] Given the high level of efficiency and broad scope of the IP+-forming macrocyclization reaction, this chemistry was examined as to its suitability for the synthesis of MP libraries can also be prepared using the methods of the disclosure. The methods were carried out on a small amount of TentaGel beads in the wells of a microtiter filter plate, which is the most convenient format for the creation of combinatorial libraries, including DNA- encoded libraries (DELs). 20 different peptides, including eight tetrapeptides, four pentapeptides and four hexapeptides, were synthesized on 2 mg of 160 pm TentaGel RAM beads in individual wells of a 96 well microtiter filter plate open to the air. 18 of the 20 peptides had Mmt-protected Lys as the C-terminal residue, included a UV-active Nap, tyrosine (Tyr) or tryptophan (Trp) residue to facilitate subsequent LC-MS analysis, and terminated in a serine residue (subsequently oxidized to a glyoxamide). L15 and L16 contained the protected Lys as the third residue. L17 has a glyoxamide installed on a side chain. After completion of the peptide chain, the Lys and serine residues were deprotected and the peptide was oxidized with NalO4 to create the terminal aldehyde. Finally, three equivalents of a 2-carbonyl pyridine compound were added to each well to create the IP+- linked macrocycle. This protocol was carried out for five identical plates but in each case a different 2-carbonyl pyridine compound unit was added at the end. After incubation and washing, the material was released from the beads and completely deprotected using TFA. The products were analyzed by LC-MS.

[0094] As shown in Figure 6, the overwhelming majority of the products were produced in good to excellent purity. 83 of the IP+-containing MPs were >85% pure and 13 were 75-85% pure. Only 4 of the 100 MPs were produced in < 75% purity. These data strongly support that the IP+ grafting methods of the disclosure are suitable for the creation of libraries of MPs.

[0095] Model screening libraries can also be prepared using methods of the disclosure. The tripeptide sequence HP(Q / Y) has been shown to be a modest affinity ligand for Streptavidin (SA). A library of 480 compounds on 10 pm TentaGel beads was constructed by parallel solid-phase synthesis in microtiter filter plates and each set of beads was tested fortheir ability to capture fluorescently labeled SA. The general structure of the library is shown in Figure 7. A C-terminal Lys residue and an N-terminal Oxo (aldehyde) unit flanked four variable positions in which one of 14 different amino acids was employed Figure 7A. To bias the library towards peptides that resemble HP(Q / Y), amino acids aa1 , aa2, aa3, and aa11 were employed in the first position, amino acids aa1 -aa3 and aa8-aa10 in the second position, amino acids amino acids aa12-aa14 in the third position and amino acids aa4-aa7 in the fourth position. The linear precursors were then treated with one of five different 2- carbonyl pyridine compounds Figure 7A. After thorough washing, the beads were blocked with Starting Block to discourage non-specific protein binding, then incubated for one hour with Alexaflour 647-labeled SA (A647-SA, 150 nM) at room temperature. After thorough washing, the amount of fluorescence retained by the beads in each well was measured using a fluorescent plate reader. This protocol was conducted in triplicate. The five bead- displayed MPs that retained the highest levels of A647-SA in each run were synthesized as C-terminal fluorescein conjugates and their solution affinities for unlabeled SA were determined by titration followed by an increase in fluorescence polarization Figure 7B. These data showed I P+-linked MP29 to be the best SA ligand with a KDof approximately 7.0 μM Figure 7B. These data demonstrate that the methods of the disclosure are suitable for the construction of useful screening libraries of novel MPs.Cell Permeability Studies

[0096] A major limitation of most MPs is that they display poor passive membrane permeability, limiting their utility for engaging intracellular targets. The IP+motif is an interesting functional unit with respect to potentially influencing membrane permeability. It is relatively hydrophobic, especially in the case where quinoline or other more highly substituted 2-carbonyl pyridine compounds are employed to create the macrocycle, yet it carries a permanent positive charge (i.e., not due to a protonation event). Without intending to be bound by theory, it is believed that the positive charge can concentrate the compound on the cell surface through electrostatic interactions and that the hydrophobic character of the heterocycle can stimulate movement across the membrane, resulting in improved passive membrane permeability.

[0097] Twenty different I P+- linked MPs of various sizes and compositions were prepared according to the methods of the disclosure and their rates of membrane transit using PAMPA (parallel artificial membrane permeability assay) were measured. The PAMPA was performed in accordance with literature procedures, for example, as in Ottaviani et al., J. Med. Chem. 2006, 49, 3948-3954. Propranolol, a highly permeable low molecular weight (259 Da) drug and the much less permeable, charged small molecule Ranitidine, were employed as controls. In this assay compounds displaying a -log Pe below 6.0 areconsidered to be highly permeable, while those with a -log Pe between 6.0-7.0 are considered to be moderately permeable.

[0098] As shown in Figure 8A, a remarkable 45% of the MPs tested displayed a -logPe value below 6.0 (green dots), despite the fact that the masses of these compounds are all well above 500 Da. One of these macrocycles, MP40, with a molecular weight of 846 Da, actually traversed the membrane more rapidly than Propranolol. The particular 2-carbonyl pyridine compound used to create the macrocycle clearly had a significant effect on permeability. For example, as shown in Figure 8B, four MPs with the same peptide component, but formed using different 2-carbonyl pyridine compounds, displayed significantly different permeabilities, ranging from -logPe = 4.6 to 6.0.

[0099] The permeability of two very large compounds, MP18 and MP19 (shown in Figure 3B) were also tested, which have molecular weights of 1071 Da and 1234 Da, respectively. Remarkably, even these >1000 Da compounds displayed moderate membrane permeability in the PAMPA (-logPe of 6.4 and 6.3, respectively). Taken together, these data suggest that IP-i-containing MPs display much better membrane permeability than typical peptide macrocycles. The permeability of additional peptides is provided in Figure 9.

[0100] As shown in Figure 8C, the membrane permeability of MP36, which displays a - logPe value (5.94) close to the median of the 20 MPs analyzed, was compared with that of close analogues in which the ring had been closed using different chemistry. Specifically, MP47 and MP48 were created using intramolecular amination or acylation chemistry to close the ring. This is the equivalent of opening the pyridinium ring and deleting the positive charge. MP49 and MP50 were created by closing the ring through thioether bond formation and thus lack any trace of the IP+moiety. The passive permeabilities of these four molecules were assessed using PAMPA. As shown in Figure 8A (yellow dots), each of these molecules displayed a passive permeability between 10- and 100-fold poorer than the IP+-containing MP36. This comparative assessment shows that the IP+unit has a major positive effect on the passive membrane permeability of the MPs.EXAMPLES

[0101] The following examples are provided for illustration and are not intended to limit the scope of the invention.General Synthetic Route

[0102] Synthetic TentaGel beads were purchased from Rapp Polymere GmbH (Germany). Disposable reaction columns (Intavis AG) were used as reaction vessels for solid phase peptide synthesis. HPLC was carried out on a Waters systems equipped with a Waters 1525 binary HPLC pumps and a 2487 dual A absorbance detector, or a 2998 photodiode arraydetector. The mobile phase comprised of buffer A (H2O containing 0.1% trifluoroacetic acid (TFA)) and buffer B (CH3CN containing 0.1% TFA). Analytical HPLC was conducted using a Vydac C-18 column (5 pm, 250 x 4.6 mm, Alltech, Deerfield, IL) at a flow rate of 1 .0 mL / min with LIV detection at 220 and 254 nm. All steps involving water utilized distilled water filtered through a Barnstead Nanopure filtration system (Thermo Scientific). LC-MS analysis was carried out by Agilent 1100 Series equipped with SBC18 column, PDA detector and a linear gradient of 5% acetonitrile to 95% acetonitrile with 0.05% formic acid.

[0103] Deprotection of Fmoc group. Resin beads for solid stage synthesis included polystyrene / polyethylene glycol with rink-amide liners protected with an Fmoc functionality. The Fmoc group was deprotected by 20% piperidine in DMF (2 x 10 min). The resulting deprotected beads were extensively washed by DCM (2 x 1 min), DMF (2 x 1 min).

[0104] Amino acid to amino acid coupling. A peptide including a terminal Fmoc protected Serine (Ser) and an amino acid with an monomethoxytrityl (Mmt) protected amine(5.0 equiv to resin loading), Ethyl (hydroxyimino)cyanoacetate (Oxyma, 5.0 equiv) and N,N’- Diisopropylcarbodiimide (DIC, 5.5 equiv) were mixed in NMP for 5 min . Afterward, the resulting solution was added to the deprotected resin beads and the reaction was shaken for 1 h at 37°C to provide the peptide attached to the resin beads. Afterwards the resin beads with the peptide attached were washed with DMF 2 x.

[0105] On-bead oxidation of Serine (Ser) to give oxo-aldehyde. The Fmoc on the N- terminal Ser was removed as described above and the resulting beads were washed by DMF (2 x 1 min), 50% DMF-dH2O (2 x 1 min) and dH2O (2 x 1 min). Then a solution of dH2O containing NalO4 (5.0 equiv) was added to the beads and the reaction was shaken for 40 min at room temperature (the beads homogenized in the dH2O during the reaction). Once the reaction was complete, the resulting beads were successively washed by dH2O (5 x 1 min), 50% DMF-dH2O (2 x 1 min), DMF (2 x 1 min), DCM (2 x 1 min) to provide a resin bead having a peptide with a terminal glyoxamide group attached thereto.

[0106] Deprotection of monomethoxytrityl (Mmt) group. After the Ser was oxidized by NalO4, the resin beads having the peptide with the terminal glyoxamide group were treated with a solution of 25% HFIP / DCM (2 x 30 min) to deprotect the amino acid having an Mmtprotected amine. Then the resulting beads were extensively washed by DCM (3 x 1 min).

[0107] On-bead cyclization of peptide aldehydes. Once the Mmt group was totally removed, the pyridine-2-carboxaldehyde was dissolved in AcOH / TFE(1 :1 v / v) to give a clear solution, which was mixed with the beads. The reaction was shaken at 30 °C for 10 hours to provide a resin bead having a macrocyclic peptide attached thereto. Then the beads were washed by DMF (2 x 1 min), DCM (2 x 1 min) and Et20 (2 x 1 min).

[0108] Cleavage and purification. 100%TFA was added to the beads for 40 min to cleave the macrocyclic peptide from the resin beads. Then the resin was washed again with TFA to cleave any remaining macrocyclic peptide from the resin beads. The combined cleavage solutions were volatilized under a stream of Argon to give the resulting cyclized peptide, which was subsequently dissolved in water / acetonitrile, filtered, and purified by reversephase HPLC.Example 1 : Structure Elucidation of the Imidazopyridinium Structure

[0109] MP1 was prepared as shown in Figure 1A on 160 pm TentaGel beads with a Rink- amide (RAM) linker according to the above general procedures. In Figure 1 A, Oxo represents the glyoxamide formed by NalO4-mediated oxidation of an N-terminal serine. The Mmt protecting group was removed using a 1 :3 mixture of hexafluoropropanol (HFIP) / DCM, then the beads were exposed to 3 equivalents of pyridine-2-carboxaldehyde in a 1 :1 mixture of acetic acid (AcOH) / trifluoroethanol (TFE) for 10 hours at 30 °C. The products were released from the beads using trifluoroacetic acid (TFA) and analyzed by liquid chromatography / mass spectrometry (LC-MS). MP1 was formed in nearly quantitative yield.For large-scale synthesis, the reaction was carried out at 50 pmol using 125 mg 160 pm TentaGel beads (0.40 mmol / g). Cleavage of MP1 from beads by incubating in 100% TFA for 40 min, the crude product was obtained after drying TFA by Argon. Then cold Et20 was added to precipitate MP1 . After discarding the Et20, the MP1 was dried at room temperature to give the brown solid as TFA salt (22 mg, 36 pmol, 72%), nearly pure (some residual Et20) without further purification.

[0110] HRMS: Calculated for C24H34N7O5 [M+H]+500.2616, found 500.2615 Figure 1 C. The purity of the TFA salt was demonstrated by1H NMR spectroscopy and the ring structure was confirmed by Heteronuclear Multiple Bond Correlation (HMBC) spectroscopy.

[0111] Thus, Example 1 demonstrates an MP of the disclosure using methods of the disclosure.Example 2: Evaluation of Various 2-Carbonyl Pyridine CompoundsLys-Pro-Gly-Nap-O;;-? Cycfo-[Lys-Val-Gly-Nap]- yX

[0112] The ability of various 2-carbonyl pyridine compounds to form the intramolecular imine linkage was investigated using the reaction shown above. The resin bead including the peptide having a terminal glyoxamide and a terminal Mmt protected amine was prepared as described above. All the reactions were carried out at 2 pmol scale using 5 mg 160 pm Tenta Gel beads with Rink amide linker (0.4 mmol / g). Deprotection of Mmt was conducted at r.t. for 30 min twice using 25% HFIP / DCM (200 μL in total). 2-Pyridine carbonyl compounds were dissolved in a solution of AcOH / TFE (1 :1 , 200 μL in total) and were added to the beads at 30 °C and allowed to mix for 10 hours. After washing the resulting beads with DCM and Et20, 200 μL TFA was used to release the cyclized peptide from beads. The resulting TFA solution was dried by Argon to give the crude products which were then checked by LCMS to determine the purity. The 2-carbonyl pyridine compounds (P2CAs in the following reaction scheme) that were evaluated are listed in Figure 2.

[0113] Accordingly, Example 2 describes preparing macrocyclic peptides with various 2- carbonyl pyridine compounds using methods of the disclosure.Example 3: Preparation of Cvclo-fLvs-Pro-Glv-NaDl-Pv25-FITC

[0115] A 2-carbonyl pyridine compound having a piperazinyl group bearing a free secondary amine (Py25) was prepared according to the above scheme. Py25 was prepared according to the reported procedure: (HETEROARYL COMPOUNDS AS BTK INHIBITORS AND USES THEREOF, MERCK KGAA - US2016 / 96834, 2016, A1 ). 5-fluoropyridine-2- carbaldehyde (250 mg, 2 mmol, 1 .00 equiv) and Boc-piperazine (932 mg, 5 mmol, 2.5 equiv) were dissolved in DMF (15 mL), to which was added K2CO3 (690 mg, 5 mmol, 2.5 equiv). The resulting mixture was then stirred and heated at 100 °C for 3 hours. Then the reaction mixture was cooled to room temperature and diluted with 50 mL H2O. The resulting mixture was extracted with ethyl acetate (3 * 25 mL) and the organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified in a flash silica gel column eluting with MeOH in DCM (2% to 5% gradient) to afford Boc-Py25 as brown oil, which was then treated by pure TFA at room temperature for 30 min to remove Boc protecting group. Py25 was finally obtained by evaporating TFA using Argon without further purification. MS: m / z=192.2 [M+H]+.

[0116] Py25 was then used to form a to form the intramolecular imine linkage and the macrocyclic peptide, Cyclo-[Lys-Pro-Gly-Nap]-Py25, as described in Example 2, according to the above scheme.

[0118] Before conjugating with 5 / 6-FITC, the beads having the Cyclo-[Lys-Pro-Gly-Nap]- Py25 attached thereto were first washed by 1% DIPEA / DMF (3 * 1 min) and DMF (2 * 1 min). Then a solution of 5 / 6-FITC (5 pmol) in DMF was mixed with the beads at room temperature and protected from light for 2 hours. The resulting red beads were then washed by DMF (3 * 1 min) and DCM (3 * 1 min), after which TFA was added to release the FITC conjugated macrocycle at room temperature for 40 min. Finally, after removal of TFA, the product Cyclo-[Lys-Val-Gly-Nap]-Py25-FITC was obtained and analyzed by LCMS.

[0119] Thus, Example 3 demonstrates preparation and post-cyclization modification of a macrocyclic peptide of the disclosure according to methods of the disclosure.Example 4: Preparation of Cvclo-[Lvs-Pro-Glv-Nap]-Py25-Biotin

[0120] Beads having Cyclo-[Lys-Val-Gly-Nap]-Py25 (4 pmol, 10 mg TG beads) attached thereto were prepared according to Example 3. Before coupling with Biotin, the beads were first washed by 1% DIPEA / DMF (3 * 1 min) and DMF (2 * 1 min). Then a prepared solution of biotin (40 pmol, 10 equiv), Oxyma (40 pmol, 10 equiv) and DIC (44 pmol, 11 equiv) in NMP was mixed with the beads at 37 °C and shaken for 2 hours. The resulting red beads were then washed by DMF (3 * 1 min) and DCM (3 * 1 min), after which TFA was added to release the Biotin conjugated macrocycle at room temperature for 40 min. Finally, after removal of TFA, the product Cyclo-[Lys-Pro-Gly-Nap]-Py25-Biotin was obtained and analyzed by LCMS.

[0121] Thus, Example 4 demonstrates preparation and post-cyclization modification of a macrocyclic peptide of the disclosure according to methods of the disclosure.Example 5: Evaluation of the Impact of Spacing Distance Between Lys and Oxo-Aldehyde

[0122] Peptides MP2 to MP11 were prepared according to the above scheme to investigate the effect of alanine spacing between the terminal aldehyde and the terminal amine. All the reactions were carried out at 2 pmol scale using 5 mg 160 pm TG beads with rink-amide linker (0.4 mmol / g). All cyclized peptides were released from the beads using TFA, the purity was then analyzed by LC-MS. The corresponding number of alanine spacers for the MP compounds was as follows:MP2: 0 Ala; MP7: 5 Ala;MP3: 1 Ala; MP8: 6 Ala;MP4: 2 Ala; MP9: 7 Ala;MP5: 3 Ala; MP10: 8 AlaMP6: 4 Ala; MP11 : 9 Ala

[0123] When zero alanine spacers were used, a combination of MP2 and MP2-side were formed, as shown in the scheme below, with MP2-side forming as the major species (95%).

[0124] As shown in Table B, below, a single alanine spacer resulted in about half of the starting material being converted to the desired product. The linear peptides containing 2-5 alanine units provided a high yield of clean product. The yield decreased again using peptides containing 6-9 alanine residues. It is believed that the fall off in yield for substrates containing more than five consecutive alanine residues is due to a propensity to form helices that hinder cyclization. Therefore, linear peptides in which more flexible units (Ahx and Ipa) were placed between the terminal amine and terminal aldehyde reactive groups were prepared (MP12-MP19). MP12-MP19 are shown in Table A, above, and their amino acid sequence between the terminal amine and terminal aldehyde are provided below:MP12: Lys-Tyr; MP16: Lys-Leu-Pro-Ala-His-Leu;MP13: 1 Lys-Trp-Arg-Leu-Gly; MP17: Lys-Leu-Ala-Gly-Glu-Phe;MP14: Lys-Lys-Pro-Phe-Met(O); MP18: Lys-Lys-Thr-Ahx-Phe-Nle-lpa-pAla;MP15: Lys-Leu-Pro-Ala-Cys-Leu; MP19: Lys-Lys-Thr-Ahx-Phe-Nle-lpa-Leu-Gly.

[0125] As shown in Table B, this indeed resulted in efficient macrocyclization for compounds. Based on these data, it is believed that the IP+ chemistry is useful for the efficient synthesis of macrocycles spanning a much wider range of ring sizes than might have been suggested from the alanine data, so long as there is sufficient flexibility in the linear precursor to allow formation of the intramolecular imine intermediate.

[0126] MPs in which the linkage is between an oxo-modified side chain and an amine- containing side chain, such as a lysine (Lys) or Dap residue were also prepared (MP20, MP21 , MP22, and MP23). As shown in Figure 4, side chain-to-side chain-connected MPs with different ring sizes were obtained in excellent purity using a variety P2CAs (Table B).

[0127] Peptide stapling using side chain-to-side chain connections was also investigated using the methods of the disclosure. The twelve amino acid peptide, ITFEDLLDYYGP-NH2, which is a ligand for the HIV capsid protein that disrupts capsid assembly in vitro, but not in cultured cells due its cell impermeability was chosen as a model for the present synthetic methods.

[0128] The five linear substrates shown schematically in Figure 5, in which the amino- and aldehyde-containing residues had an i-i+4 or a i-i+7 spacing were constructed (MP24, MP25, MP26, MP27, and MP28). The beads displaying these compounds were treated with the indicated 2-carbonyl pyridine compound under the standard conditions. After cleavage, the products were analyzed by LC-MS. In each case, good to excellent conversion to the desired stapled peptides was observed (Table B). The degree of helicity was determined by circular dichroism spectroscopy. As shown in Figure 5, the linear peptide was a random coil. All of the stapled MPs displayed an enhanced degree of helical content. The best of these (» 74% helical) was MP24, in which the i and i+4 residues had been stapled with a 2-carbonyl pyridine compound. It is interesting that MP24, MP25 and MP26, which contain the same ring but were formed using a different 2-carbonyl pyridine compound, display markedly different levels of helicity. The two i-i+7-stapled peptides (MP27 and MP28), linked using a 2-carbonyl pyridine compound and Py17, also displayed measurably different levels of helicity. These data suggest that the use of different 2-carbonyl pyridine compound units in the creation of I P+- stapled MPs will provide a novel tool to fine tune the helical content of stapled peptides.

[0129] Table BCircular Dichroism

[0130] Tested peptides were dissolved in 30% TFE / PBS to give the final concentration of 100 μM. CD measurements were performed on a Jasco J-815 spectropolarimeter with a 1 mm Jasco quartz cell over the wavelength range of 190-250 nm. The cosolvents of 30% TFE / PBS was used as a blank and automatically subtracted from the samples during scanning. Data were recorded from 250 to 400 nm with a scan speed of 50 nm / min and data pitch of 0.5 nm, a bandwidth of 1 nm, a response of 1 s. The % a-helicity was calculated from BeStSel platform (https: / / bestsel.elte.hu / index.php). Figure 10.

[0131] Thus, Example 5 demonstrates the versatility of the methods of the disclosure for preparing macrocyclic peptides of the disclosure having various ring sizes.Example 6: Synthesis of IP+MP Libraries

[0132] A library of MPs was prepared as follows. 20 different peptides, including eight tetrapeptides, four pentapeptides, and four hexapeptides were synthesized on 2 mg of 90 pm TentaGel Rink Amide resin in individual wells of a 96 well microtiter filter plate open to the air. 17 of the 20 peptides had Mmt-protected lysine as the first residue, included a UV- active Nap, tyrosine (Tyr) or tryptophan (Trp) residue to facilitate subsequent LC-MS analysis, and terminated in a serine residue. Three substrates, L15, L16, and L17, contained the protected lysine as the third residue with a glyoxamide installed on a side chain. After completion of the peptide chain, the lysine and serine residues were deprotected and the peptide was oxidized with NaIC to create the oxo unit (terminal aldehyde). Finally, three equivalents of a P2CA were added to each well to create the I P+-linked macrocycle. This protocol was carried out in five identical plates but in each case a different P2CA unit was added at the end. After incubation and washing, the material was released from the resin and completely deprotected using TFA and the products analyzed by LC-MS. As shown in Figure 6, the overwhelming majority of the products were produced in good toexcellent purity. 83 of the IP+containing MPs were > 85% pure and 13 were 75-85% pure. Only four of the 100 MPs were produced in < 75% purity.

[0133] Thus, Example 6 demonstrates that macrocyclic peptides of the disclosure can be prepared as libraries using methods of the disclosure.Example 7: Synthesis of IP+MP Libraries for Screening of Streptavidin Ligands

[0134] A library of 480 I P+-linked MPs was constructed and tested against a target protein. 480 compounds were prepared on 10 pm Tenta Gel beads (0.3 mmol / g) by parallel solidphase synthesis in microtiter filer plates and tested for their ability to capture fluorescently labeled Streptavidin (SA). The general structure of the library is shown in Figure 7. A C- terminal lysine residue and an N-terminal Oxo unit (again derived from Ser oxidation) flanked four variable positions in which one of 14 different amino acids was employed (Figure 7). To bias the library towards peptides that resemble HP(Q / Y), a sequence that has been shown to be a modest affinity ligand for SA, amino acids aa1 , aa2, aa3, and aa11 were employed in the first position, amino acids aa1-aa3 and aa8-aa10 in the second position, amino acids amino acids aa12-aa14 in the third position and amino acids aa4-aa7 in the fourth position (Figure 7). The linear precursors were then treated with one of five different P2CAs. After thorough washing, the beads were blocked with Starting Block to discourage non-specific protein binding, then incubated for one hour with Alexaflour 647-labeled SA (A647-SA, 100 nM) at room temperature. After thorough washing, the amount of fluorescence retained by the beads in each well was measured using a fluorescent plate reader. This protocol was conducted in triplicate. The five bead-displayed MPs that retained the highest levels of A647- SA were synthesized as C-terminal fluorescein conjugates and their solution affinities for unlabeled SA were determined by titration followed by an increase in fluorescence polarization (Figure 7B). These data showed I P+- linked MP29 to be the best SA ligand with a KD of approximately 15 μM (Figure 7B). These data demonstrate that the IP+ grafting chemistry is suitable for the construction of useful screening libraries of novel MPs. Figure 11 shows an example of one-compound-one-well synthesis for 96 peptide sequences. In Figure 11 , First position (A1): aa1 : the wells of A1-A12 and B1-B12; aa2: C1 -C12 and DIDI 2; aa3: E1 -E12 and F1 -F12; aa11 : G1 -G12 and H1 -H12; Second position (A2): aa1 : A1 - H1 , A7-H7; aa2: A2-H2, A8-H8; aa3: A3-H3; A9-H9; aa8: A4-H4, A10-H10; aa9: A5-H5;A11 -H11 ; aa10: A6-H6, A12-H12; Third position (A3): aa12: A1 -A12, D1 -D12, G1 -G12; aa13: B1 -B12, E1-E12, H1-H12; aa14: 01-012, F1 -F12; Fourth position (A4): aa4: A1-A6, E1 -E6, D7-D12, H7-H12; aa5: B1 -B6, F1-F6, C7-C12, G7-G12; aa6: C1 -C6, G1-G6, B7- B12, F7-F12; aa7: D1 -D6; H1 -H6, A7-A12, E7-E12On-Bead Screening Procedure for Determining Fluorescence Intensity

[0135] An on-bead screening assay was used to measure binding activity based on fluorescence intensity (FLINT) changes. Reduction of FLINT corresponded to weaker binding and an increase of FLINT corresponded to strong binding and / or may be due to autofluorescence.Materials:• C-Slide Automated Cell Counting Chamber Slides (#63508-50 Electron Microscopy)• 500 μL filtered mobicols, gravity flow columns, luer-lock, screw cap (#M1002 with 10 urn filter)• 3. hydrophilic Multiscreen HTS BV filter plates, 1 .2 um, clear, nonsterile (#MSBVN1210) with foil stickers. Foil;. Multichannel pipette and tips;• Single channel pipettes (2, 10, 20, 200 μL) and tips• 1x PBST (Tween- 20 0.01%)• DMF• Starting block-PBS Blocking Buffer, Pierce (#PI37542)• 10 pm beads displaying compound / ligand.• Aiexaflour 647 conjugated Streptavidin. LoBind Eppendorf polypropylene snap cap (various sizes). Shaker or rotator. Mini-Centrifuge. per tubes, 0.1 mL, 8-strip, low profile with attached cap (#PC7065 ProCycle) and rack . 384-well plate, black, non-binding (NBS), low-volume, flat bottom, polystyrene (#3820 Corning). Microplate readerCount beads:Beads were counted using a Hemocytometer or cell counter on Tecan multimode plate reader, following the below procedure.1. 2 μL of vortexed beads (in DMF) were transferred into a new per tube and 9 μL of DMF and 9 μL methylene blue were added = 20 μL total2. The beads were centrifuged down and visually inspected to determine if the bead amounts are similar between the different compound conjugate beads being counted before counting (should be very little amount of beads)3. The beads were re-agitated to be evenly distributed in solution, then 10 μL of bead solution was transferred into the Hemocytometer and counted on microscope or counted using cell chip for Tecan4. An example of Hemocytometer math 4 squares is provided: 242 + 188 + 268 + 193 = 891 / 4 = 222.75 x 10000 (nL to mL) x 10 (dilution factor) = 22,275,000 / 1000 (mL to μL) = 22,275 beads per μL x 500 μL (total volume of stock) = 11 ,137,500 beads in total in the 500 pLsWhen using the cell chip, the number of beads per mL must be multiplied by the dilutionfactor:Ex: 3.68 * 105 / mL (final count on Tecan), needs to be multiplied by 10 (dilution factor), then divided by 1000 to get the number of beads per μL (3,680 / μL).Binding Assay (performed in triplicate):• About 300k beads for each compound are transferred to a filter plate and the beads were equilibrated in 1 x PBST overnight on a shaker, then drained;• The beads were incubated in 50 / 50 starting block (PBS) / 1 x PBST for 1 hour on the shaker, then drained.• 150 μL150 nM A647 SA was added to appropriate wells and incubated for 1 hour on shaker.• The beads were drained and washed with 50 / 50 SB / 1 xPBST for 20-45 mins, on the shaker 3 times.• All beads from each well were transferred to labeled per tubes.• The beads were gently agitated in the per tube and transferred to a 384-well plate for triplicates.• Any bubbles in 384-well plate were tapped down and the plate covered with foil sticker and centrifuged for 1 min.• Read on the TECAN under fluorophore used in A647 SA (Ex: 650 nm, Em:665 nm)Layout of 384-well Plate:Negative and positive controls were included to determine which of the ligands are possible hits. False hits may possibly be due to autofluorescence. Figure 12.

[0136] The possible hits were resynthesized. Hit 4 clearly showed no binding, which means it is likely a false hit due to autofluorescence. Hit 1 , Hit 2, and Hit 5 show weak binding (>100 μM). Only Hit 3 showed high binding affinity. Structure of fluorescein labeled Hit 3 for fluorescence polarization is shown below:

[0137] To determine the fluorescence polarization, the fluorescent ligands were dissolved in PBST, and molarity confirmed by absorbance (495 nm, E495 =78,000 M-1cm_1) using Nanodrop. FP binding saturation were performed in 384-well, medium bind black microtiter plates (Greiner Bio-One). Serial dilutions of streptavidin (high of 400 μM, 2-fold serial dilutions) were mixed 1 :1 with 100 nM ligand (20 pl final volume). Plates were incubated at r.t. for 20 min and read on an infinite M1000 Pro Plate Reader (Tecan) using 485 nm excitation and 535 nm emission filters. The KDvalues were obtained using Prism (GraphPad Software, Inc.) with a nonlinear regression with one site total.

[0138] Thus, Example 7 demonstrates the preparation of a library of IP+MP for screening of Streptavidin binding using methods of the disclosure.Example 8: Synthesis of MP47

[0139] MP47 was prepared according to the above scheme.

[0140] De-Mmt: the peptide was treated with 25%HFIP / DCM for 30 min two times.

[0141] Cyclization: After deprotection of Mmt, the beads were incubated in 5% DIPEA / DMF and shaken at 37 °C overnight.

[0142] Alkylation: 2-(bromomethyl)pyridine hydrobromide (10. 0 equiv) and DIPEA (20. 0 equiv) were dissolved in DMF, which was then mixed with beads at room temperature for 6 hours.

[0143] Cleavage: The beads were treated with pure TFA for 1 hour.

[0144] Thus, Example 8 demonstrates preparing macrocyclic peptides according to methods not of the disclosure.Example 9: Synthesis of MP48

[0145] MP48 was prepared according to the above scheme.

[0146] De-Mmt: the peptide was treated with 25%HFIP / DCM for 30 min two times.

[0147] Cyclization: Beads, after deprotection of Mmt, were incubated in 5% DIPEA / DMF and shaken at 37 °C overnight.

[0148] Acylation: 2-Picolinic acid (5.0 equiv), Oxyma (5.0 equiv) and DIC (5.5 equiv) were dissolved in DMF, and the resulting solution was mixed with beads at 37 °C for 6 hours.

[0149] Cleavage: The beads were treated with pure TFA for 1 hour.

[0150] Thus, Example 9 demonstrates preparation of macrocyclic peptides according to methods not of the disclosure.Example 10: Synthesis of MP49

[0151] MP49 was prepared according to the above scheme.

[0152] De-Mmt: peptides were treated with 1% TFA + 5% TIPS in DCM for 30 min three times.

[0153] Cyclization: Beads, after deprotection of Mmt, were incubated in 3% DIPEA / DMF and shaken at r.t. overnight.

[0154] Cleavage: The beads were treated with pure TFA for 1 hour.

[0155] Thus, Example 10 demonstrates preparation of macrocyclic peptides according to methods not of the disclosure.Example 11 : Synthesis of MP50

[0156] MP50 was prepared according to the above scheme.

[0157] De-Mmt: peptides were treated with 1% TFA + 5% TIPS in DCM for 30 min three times.

[0158] Cyclization: Beads, after deprotection of Mmt, were incubated in 3% DIPEA / DMF and shaken at r.t. overnight.

[0159] Cleavage: The beads were treated with pure TFA for 1 hour.

[0160] Thus, Example 11 demonstrates the preparation of a macrocyclic peptide according to methods not of the disclosure.Example 12: Attempted Preparation of Macrocyclic Peptides using 2-Carbonyl Pyrazine or 2- Carbonyl Pyrimidine

[0161] Preparation of an IP+MP using a 2-carbonyl pyrazine or 2-carbonyl pyrimidine to form the intramolecular imine linkage, instead of a 2-carbonyl pyridine, was attempted according to the following scheme:

[0162] Methods of the disclosure were followed, except the 2-carbonyl pyridine was replaced with pyrazine-2-carbaldehyde or pyramide-4-carbaldehyde. No macrocyclic peptides were found.

[0163] Thus, Example 12 demonstrates that the 2-carbonyl pyridine compound used in the methods of the disclosure cannot be replaced by other nitrogen containing heterocycle.

[0164] Example 13: Permeability in living cells

[0165] IP+MP were prepared and tested for permeability into living cells using the chloroalkane penetration assay (CAPA), as shown in Figure 13. The CAPA was performedin accordance with literature procedures, for example, as described in Peraro et at. J. Am. Chem. Soc. 2017, 130, 7792-7802.

[0166] A given concentration of a macrocycle of interest carrying a chloroalkane tag was incubated with cells that express a HaloTag protein. The HaloTag protein reacts covalently when exposed to a chloroalkane. Molecules that carried the chloroalkane tag that were highly cell permeable alkylated the HaloTag protein with high efficiency. Subsequent treatment of the cells with a cell permeable dye-chloroalkane conjugate would then provide little fluorescent labeling. Conversely, a less cell permeable molecule left unreacted HaloTag protein that could be labeled.

[0167] As shown in Figure 14A-B, the IP+macrocycles of the disclosure are about 50-100 times more permeable than analogues that lack the IP+unit, as evidenced by the lower fluorescence. This result is consistent with the results from the parallel artificial membrane permeability assay (PAMPA).

[0168] It should be understood that while this invention has been described in terms of specific embodiments set forth in detail, such embodiments are presented by way of illustration of the general principles of the invention, and the invention is not necessarily limited thereto. Certain modification and variations in any given material, process step or chemical formula will be readily apparent to those skill in the art without departing from the true spirit and scope of the present inventio, and all such modification and variations should be considered within the scope of the claims that follow.

Claims

What is claimed is:1 . A method of preparing a macrocyclic peptide (MP) having a stable imidazopyridinium comprising: admixing under acidic conditions:(a) a peptide comprising a first terminus comprising a terminal amine group and a second terminus comprising a terminal aldehyde and at least one amino acid between the first terminus and the second terminus; and(b) about 2 to about 4 equivalents of a substituted or unsubstituted 2-carbonyl pyridine compound.

2. The method of claim 1 , wherein the terminal aldehyde is an activated aldehyde.

3. The method of claim 1 or claim 2, wherein the activated aldehyde is a glyoxamide.

4. The method of any one of claims 1 to 3, wherein the peptide is attached to a solid support.

5. The method of any one of claims 1 to 4, wherein the admixing of the peptide and 2- carbonyl pyridine compound occurs in the presence of a solvent.

6. The method of any one of claims 1 to 5, wherein the admixing under acidic conditions comprises admixing in the presence of an acid having a pKa of less than about 4.9.

7. The method of any one of claims 4 to 6, wherein the solid support is a swellable resin material.

8. The method of claim 7, wherein the swellable resin material is polystyrene beads, low cross-linked polystyrene beads, mixed block polystyrene-divinylbenzene beads, mixed block polystyrene-polyethylene glycol bead, or polyacrylamide-polyethylene glycol copolymer beads.

9. The method of any one of claims 5 to 8, wherein the solvent is a 1 :1 acetic acid : trifluoroethanol solution.

10. The method of any one of claims 1 to 9, wherein the admixing of the peptide and 2- carbonyl pyridine compound occurs at a temperature in a range of about 10 °C to about 50 °C.11 . The method of any one of claims 1 to 10, wherein the admixing of the peptide and 2- carbonyl pyridine compound occurs at a temperature in a range of about 25 °C to about 35 °C.

12. The method of any one of claims 1 to 11 , wherein the 2-carbonyl pyridine is a substituted or unsubstituted 2-formyl-pyridine.

13. The method of any one of claims 1 to 11 , wherein the 2-carbonyl pyridine is a substituted or unsubstituted 2-keto-pyridine.

14. The method of any one of claims 1 to 13, wherein the 2-carbonyl pyridine compound is substituted with an electron donating group.

15. The method of any one of claims 1 to 14, wherein the 2-carbonyl pyridine compound comprises:

16. The method of any one of claims 1 to 15, wherein the peptide comprising a first terminus comprising a terminal amine group and a second terminus comprising a terminal aldehyde has a structure according to formula (IV):whereinGl is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently a natural amino acid or an unnatural amino acid; and R1is H or Ci-ealkyl.

17. The method of any one of claims 1 to 16, wherein the macrocyclic peptide has a structure according to Formula l-a:whereinQ is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulky;Rxat each instance, when present, is an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 2’ position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4.

18. A process for preparing macrocyclic peptides (MP), having a stable imidazopyridinium (IP+) unit of Formula I:wherein is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, C^alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulky;Rxat each instance, when present, is an electron donating or electron withdrawing group, with the provision that when Rxis present at the 6’ position, the Rxis not stericallybulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cswcycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4, comprising: admixing a peptide of Formula IV containing a terminal amine group and a terminal aldehyde group:with about 2.0 to about 4.0 equivalents of a 2-carbonyl pyridine compound of Formula Bunder acidic conditions, wherein is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulky;Rxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6 position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl mayeach be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4.

19. The process of claim 15, wherein the 2-carbonyl pyridine compound is a compound of Formula B-awhereinR3, R4, R5, and R6are each independently selected from H, Ci-ealkyl, C^alkenyl, C2- ealkynyl, Ci-ehaloalkyl, Ci-ehydroxyalkyl, Ci-ealkoxy, halo, carboxyl, CN, Cs-wcycloalkyl, Cs- waryl, a 3-10 membered heterocycle, and a 5-10 membered heteroaryl, wherein R3and R4, R4and R5, or R5and R6together with the carbon atoms to which they are attached may be taken together to form a Cs-wcycloalkyl, Cs-waryl, a 3-10-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with Ci-ealkyl, C^alkenyl, C^alkynyl, Ci-ehaloalkyl, Ci-ealkoxy, halo, carboxyl, CN, Cs-wcycloalkyl, Cs-waryl, a 3-10 membered heterocycle, or a 5-10 membered heteroaryl ; andR7is selected from H or Ci-ealkyl.

20. The process of claim 19, wherein the Cs-waryl is piperdinyl, phenyl, or indonyl.21 . The process of any one of claims 18 to 20 wherein the 2-carbonyl pyridine compound22. The process of any one of claims 18 to 21 , wherein the admixing of the peptide of Formula IV and the 2-carbonyl pyridine compound occurs in the presence of a solvent.

23. The process of claim 22, wherein the solvent comprises a 1 :1 mixture of acetic acid (AcOH):trifluoroethanol (TFE).

24. The process of any one of claims 18 to 23, wherein the admixing occurs for about 2 hours to about 48 hours.

25. The process of any one of claims 18 to 24, wherein the admixing occurs from about 10 °C to about 50 °C.

26. The process of any one of claims 18 to 25, further comprising preparing the peptide of Formula IV by deprotecting a protected peptide of Formula III:is a solid support, H, OH, C^alkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;PG is a protecting group;AA is an amino acid; andR1is H or Ci-6alkyl.

27. The process of any one of claims 18 to 26, wherein the PG comprises monomethoxytrityl.

28. The process of claim 27, wherein the PG is removed by treating the peptide of Formula III with a 1 :3 mixture of hexafluoroisopropanol (HFIP):dichloromethane (DCM).

29. The process of claim 28, wherein the treating occurs at r.t. (about 20 °C to about 25°C).

30. The process of any one of claims 26 to 29, wherein Q is a bond and the protected peptide of Formula III has a structure of Formula lll-a:whereinCl is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;PG is a protecting group;AA is an amino acid; and R1is H or Ci-ealkyl.31 . The process of any one of claims 26 to 30, further comprising preparing a peptide of Formula III comprising oxidizing a peptide of Formula II:wherein is a solid support, H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;PG is a protecting group;AA is an amino acid;R1is H or Ci-ealkyl; andR8is CH2OH, CH2SH, or CH(OH)CH3.

32. The process of claim 31 , wherein the oxidation is performed in solution in the presence of sodium periodate (NaICU).

33. The process of any one of claims 18 to 32, whereinCl comprises a solid support, the solid support comprises a rink-amide linker, and the peptides of Formulas I, II, III, lll-a, and IV, are attached to the solid support through the rink-amide linker.

34. The process of claim 33, wherein the rink-amide linker is an acid-labile linker.

35. The process of any one of claims 18 to 34, wherein the solid support is a resin bead.

36. The process of claim 35, wherein the resin bead is a 160 pm polyethylene glycol / polystyrene bead.

37. A process for preparing macrocyclic peptides of Formula l-a, comprising:whereinQ is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid;R1is H or Ci-ealkyl;R7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulkyRxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 2’ position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs-waryl, a 5-7-membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4, comprising: a.) oxidizing a peptide of Formula II to yield a protected peptide of Formula IIIQ is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;PG is a protecting group;each AA is independently an amino acid;R1is H or Ci-ealkyl; andR8is CH2OH, CH2SH, or CH(OH)CH3, b.) deprotecting the protected peptide of Formula III to yield a peptide of Formula IV;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl;PG is a protecting group; each AA is independently an amino acid; andR1is H or Ci-ealkyl, c.) admixing a 2-carbony pyridine compound of Formula B with the peptide of Formula IV to yield the cyclic peptide of Formula I;whereinR7is H, OH, SH, CN, amino, halo, Ci-ealkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C3-10 cycloalkyl, a 5-10 membered heterocycle, C5-10 aryl, or a 5-10 membered heteroaryl, wherein R7is not sterically bulkyRxat each instance, when present, is independently an electron donating or electron withdrawing group, with the proviso that when Rxis present at the 6 position, the Rxis not sterically bulky, or wherein two vicinal Rxgroups together with the carbon atoms to which they are attached may be taken together to form a Cs ecycloalkyl, Cs-waryl, a 5-7- membered heterocycle, or a 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl contain one or more of N, S, or O, and wherein the cycloalkyl, aryl, heterocycle, and heteroaryl may each be optionally substituted with an electron donating or electron withdrawing group; and m is an integer in a range of 0 to 4;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; andR1is H or Ci-ealkyl, and d.) releasing the cyclic peptides of Formula I from the solid support to yield macrocyclic peptides of Formula l-awherein is H, OH, Ci-ealkyl, Ci-ealkoxy, Cs-waryl, or a 5-10 membered heteroaryl;Q is a bond, an amino acid, a C5-10 aryl, or a 5-10 membered heteroaryl; each AA is independently an amino acid; andR1is H or Ci-ealkyl.

38. The method of any one of claims 1 to 15, wherein the peptide contains from one to forty amino acids between the first terminus and the second terminus.

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