WO2024263911A2

WO2024263911A2 - Methods for the synthesis and linearization of peptidomimetic macrocycles

Info

Publication number: WO2024263911A2
Application number: PCT/US2024/035013
Authority: WO
Inventors: Justin KIM; Elaine FOWLER
Original assignee: Dana Farber Cancer Institute Inc
Current assignee: Dana Farber Cancer Institute Inc
Priority date: 2023-06-22
Filing date: 2024-06-21
Publication date: 2024-12-26
Anticipated expiration: 2025-12-22
Also published as: WO2024263911A9; WO2024263911A3

Abstract

Disclosed are methodologies for the macrocyclization and linearization of peptides.

Description

Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO METHODS FOR THE SYNTHESIS AND LINEARIZATION OF PEPTIDOMIMETIC MACROCYCLES RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63/522,477, filed June 22, 2023, which is incorporated herein by reference in its entirety. SEQUENCE LISTING [0002] The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on June 20, 2024, is named 046094-781001WO-SL.xml and is 122,128 bytes in size. BACKGROUND OF THE DISCLOSURE [0003] Peptidic drugs occupy a privileged chemical space between small molecule and biological therapeutics, giving them a unique set of properties ideal for targeting challenging protein surfaces (Muttenthaler et al., Nat. Rev. Drug Discov., 2021, 20(4):309–325). This has been demonstrated by the clinical approval of over 80 peptidic drugs worldwide. Despite these successes, peptides can suffer from poor stability and low membrane permeability (Wang et al., Signal Transduct. Target. Ther., 2022, 7(1):48). These limitations can be mitigated by introducing structural modifications, such as non-canonical backbone elements (N-methylation (Chatterjee et al., Angew. Chemie Int. Ed., 2013, 52(1):254–269; Räder et al., Bioorganic Med. Chem., 2018, 26(10):2766–2773), D-amino acids (Garton et al., Proc. Natl. Acad. Sci., 2018, 115(7):1505–1510), aza-peptides (Altiti et al., Nat. Commun., 2022, 13(1):1–13)) to reduce proteolytic degradation. Backbone cyclization is widely used to constrain the peptide, rigidifying the structure into a bioactive conformation whilst often improving stability, cell membrane penetration, and pharmacokinetic properties (Dougherty et al., Chem. Rev., 2019, 119(17):10241–10287; Zhang et al., RSC Chem. Biol., 2022, 3(1):18–31; Vinogradov et al., J. Am. Chem. Soc., 2019, 141(10):4167–4181; Bechtler et al., RSC Med. Chem., 2021, 12(8):1325–1351). Reported macrocyclization technologies include ring-closing metathesis (Kim et al., Nat. Protoc., 2011, 6(6):761–771), click chemistry (Bock et al., Org. Lett., 2006, 8(5):919–922; Horne et al., Angew. Chemie Int. Ed., 2009, 48(26):4718–4724), Staudinger 1 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO ligation (Kleineweischede et al., Angew. Chemie Int. Ed., 2008, 47(32):5984–5988), Au(I)- mediated cyclization (Vanjari et al., J. Am. Chem. Soc., 2022, 144(11):4966–4976), Diels- Alder (Montgomery et al., J. Am. Chem. Soc., 2019, 141(41):16374–16381), and more (White et al., Nat. Chem., 2011, 3(7):509–524; Wills et al., ChemBioChem, 2021, 22(1):52–62; Keyes et al., J. Am. Chem. Soc.2023, 145(18):10071–10081). [0004] Most peptide cyclization strategies link amino acid sidechains. The alternative cyclization mode, head-to-tail, can be more difficult to access, particularly for unprotected peptides. Advantageously, head-to-tail macrocycles possess greater conformational rigidity over alternative cyclization modes and can exhibit greater protease stability as the vulnerable C-/N-termini are removed (Shinbara et al., Front. Chem., 2020, 8:447). Head-to-tail cyclization is prevalent in natural products and identified bioactive macrocycles such as cyclosporine A have achieved great therapeutic success (Tedesco et al., J. Transplant. 2012, 2012:1–7; Limbach et al., J. Am. Chem. Soc., 2022, 144(28):12602–12607). Small ring sizes in particular can be exceptionally challenging to generate due to reduced entropy following cyclization, as well as competing epimerization and oligomerization reactions (Bechtler et al., RSC Med. Chem., 2021, 12(8):1325–1351; Wills et al., Tetrahedron, 2022, 126:133071). Cyclization strategies often employ protected linear peptides (Thombare et al., Angew. Chemie Int. Ed., 2019, 58(15):4998–5002; Yamagami et al., European J. Org. Chem., 2021, 2021(22):3133– 3138; Bouayad‐Gervais et al., Pept. Sci., 2022, 114(3):e24254), or enzymatic technologies (Kobayashi et al., J. Am. Chem. Soc., 2023, 145(6):3270–3275). Accessing these macrocycles is not trivial and new strategies are necessary to explore this lucrative chemical space. [0005] Few methods have been reported for the synthesis of small cyclic peptides that can be linearized in response to stimuli. Current methods harness disulfides (Qian et al., Angew. Chemie Int. Ed., 2015, 54(20):5874–5878), iminoboronates (Bandyopadhyay et al., J. Am. Chem. Soc., 2016, 138(7):2098–2101), tetrazines (Borges et al., ChemBioChem, 2023, 24:4), carbamates (Zeng et al., J. Am. Chem. Soc., 2022, 144(51):23614–23621), or triazenes (Nwajiobi et al., J. Am. Chem. Soc., 2022, 144(10):4633–4641). None of these technologies is directly applicable to head-to-tail cyclized peptides. In fact, most strategies rely on canonical amino acid side chain reactivity, fundamentally limiting the range of amino acids that can be incorporated into a peptide, and the electrophiles involved are often large and cumbersome. Additionally, the generated cyclic peptides often suffer from poor stability. Technologies to cyclize and linearize head-to-tail linked, stable peptides are greatly needed. 2 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [0006] Linearization is a useful tool for applications across chemical biology such as targeted drug delivery and the sequencing of macrocyclic peptide hits following a library screening. This macrocycle screening strategy would be a valuable addition to the current toolbox that contains one-bead-one-compound, phage-display, mRNA display and DNA-encoded library technologies, all of which require each library member to be labelled for hit identification (Li et al., J. Med. Chem., 2022, 65(18):11913–11926; Prudent et al., Nat. Rev. Chem., 2021, 5(1):62-71). However, this label can influence target binding affinity and the physical properties of library members, impacting hit quality. Affinity selection (AS) with unlabelled library members is the ideal screening scenario and offers unique opportunities for the curation of library properties. Tandem mass spectrometry (MS/MS) has been used to sequence linear peptides following AS screening with >10⁸ library diversities (Gates et al., Proc. Natl. Acad. Sci., 2018, 115(23):E5298–E5306; Touti et al., Nat. Chem. Biol., 2019, 15(4):410–418; Quartararo et al., Nat. Commun., 2020, 11(1):1–11; Koh et al., Int. J. Pept. Res. Ther., 2022, 28(2):62). However, MS sequencing of macrocyclic peptides is extremely challenging due to degeneracy (Townsend et al., Bioorganic Med. Chem., 2018, 26(6):1232–1238). SUMMARY OF DISCLOSURE [0007] The current disclosure solves the problems outlined above by providing linear peptides with an electrophile (e.g., an acetamide (a) with the -NH- group on the N-terminal amino acid not shown) that can be converted into cyclic peptides (b) under specific conditions (e.g., at specific temperatures), then linearized to linear peptides (c) in response to stimuli with SmI₂:

. [0008] A first aspect of the present disclosure is directed to a compound represented by formula I:

pharmaceutically acceptable salt or stereoisomer thereof, wherein: X is an electron withdrawing group; 3 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. [0009] A second aspect of the present disclosure is directed to a compound (macrocyclic peptide) represented by formula II:

pharmaceutically acceptable salt or stereoisomer thereof, wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. [0010] A third aspect of the present disclosure is directed to a compound represented by formula III:

pharmaceutically acceptable salt or stereoisomer thereof, wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. [0011] Further aspects of the present disclosure are directed to processes of preparing compounds of formula I-III and their pharmaceutically acceptable salts or stereoisomers. [0012] Presently disclosed compounds may offer several benefits in connection with peptide cyclization strategies and uses of libraries of the macrocyclic peptides in affinity screening. The hydroxamate linkage that is formed upon cyclization is a stable, low molecular weight unit that offers unique benefits in comparison to other peptide cyclization methodologies as the cyclic peptidomimetics generated closely resemble native cyclic peptides. The hydroxamate linkages are chemoselectively cleavable under mild conditions, to generate linear peptides (compounds of formula (III)) prior to tandem mass spectrometry-based sequencing, that may mitigate aforementioned difficulties associated with degeneracy. The present methodsmay generate a range of macrocycle ring sizes under mild, aqueous conditions. The cyclization and linearization processes are compatible with split-and-pool solid phase peptide synthesis, non- 4 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO canonical amino acids (including non-proteinaceous, but natural, amino acids such as Ornithine (Orn)), and post-cyclization functionalization with useful tags such as biotin, and are amenable to combinatorial chemistry to efficiently generate large pools of compounds. BRIEF DESCRIPTION OF THE DRAWINGS [0013] FIG.1 shows the macrocyclization and linearization scope. FIG.1A shows a general scheme at a specific temperature. FIG.1B shows different 13- to 25-membered macrocycles, their production yields and linearization yields, including 8a, 9a, 10a, 11a, 12a, and 13a, (SEQ ID NOs: 11, 14, 17, 20, 23, and 26, respectively). FIG.1C shows macrocycles featuring non- canonical (non-natural) amino acids and those 40- or more membered macrocyclic rings, their production yields and linearization yields. Structures of linear, N-terminal iodoacetamide capped peptides are displayed, including 14a, 15a, 16a, 17a, 18a, and 19a (SEQ ID NOs: 29, 32, 35, 38, and 41, respectively). Unless otherwise stated, the cyclization conditions were 1 mM peptide concentration in 25 mM MOPS buffer, pH 8 at 38°C for 48 h. The linearization reactions were all carried out by titrating SmI2 into the reaction mixture until the blue color was maintained, after 1 h, samples were quenched in air and analyzed by HPLC. ^aReaction was performed at 0.5 mM concentration of 7a. ^bReaction was performed for 24 h. f: D- phenylalanine, mV: N-methylvaline. [0014] FIG. 2 shows split-and-pool SPPS, macrocyclization, and linearization of an eight- peptide mixture including HPLC traces. The eight peptides include L-Orn-QDGA, L-Orn- ADGA, LEQDGA, LEADGA, L-Orna-QYGA, L-Orn-AYGA, LEQYGA, and LEAYGA (SEQ ID NOs: 75-82). [0015] FIG. 3 shows the synthesis of RGD containing macrocycles using hydroxamic acid macrocyclization chemistry. [0016] FIG. 4A shows macrocycle 10b (SEQ ID NO: 18) structural validation through alternative synthetic route involving HATU mediated cyclization of S4 (the protected form of S3 (SEQ ID NO: 92), shown in the Figure). Hydroxamate linkage was incorporated through SPPS and the linear peptide S4 (SEQ ID NO: 47) was cleaved from the resin with protecting groups intact to enable HATU mediated cyclization to form macrocycle 10b2 (SEQ ID NO: 48). FIG. 4B shows ¹H NMR comparison of the macrocycles generated by hydroxamic acid and HATU mediated macrocyclizations indicated that the product was identical. SPPS: solid 5 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO phase peptide synthesis, DIC: N,N'-diisopropylcarbodiimide, DMF: dimethylformamide, TFA: trifluoroacetic acid, TIPS: triisopropylsilane. [0017] FIG. 5A shows the structural validation of linearized peptide 6 through alternative synthetic route using SPPS, including SEQ ID NOs: 5 and 10. Utilization of Rink amide resin enabled access to the C-terminal amide linear peptide. N-terminal capping with glycolic acid installed the glycolamide motif. FIG. 5B shows ¹H NMR and ¹³C NMR comparison of the linear peptide 6b (SEQ ID NO: 10) generated by SPPS and SmI₂ linearization of hydroxamate linked macrocycle 5 (SEQ ID NO: 5) indicated that the product was identical. SPPS: solid phase peptide synthesis, TFA: trifluoroacetic acid, TIPS: triisopropylsilane. [0018] FIG.6 shows the generation of head-to-tail macrocyclization incompetent C-terminal amide, N-terminal haloacetamide functionalized linear peptides containing nucleophilic amino acids: lysine (S5a–c (SEQ ID NOs: 49-51)), arginine (S6 (SEQ ID NO: 52)), methionine (S7a– c (SEQ ID NOs: 54-56)), and histidine (S8a–c (SEQ ID NOs: 57-59)). Linear peptides were generated through SPPS, haloacetamide capping, and cleavage. The N-terminal haloacetamide functionalized linear peptides were subjected to macrocyclization conditions for the specified times and analyzed by HPLC at 220 nm wavelength comparing AUC of peptidic peaks. MOPS: 3-(N-morpholino)propanesulfonic acid, SPPS: solid phase peptide synthesis. [0019] FIG. 7 is a series of graphs showing ELISA data for Integrin αVβ3, including 29b-f (SEQ ID NOs: 84, 1, 2, 88, and 90, respectively). [0020] FIG. 8 is a series of graphs showing ELISA data for Integrin α_Vβ₆, including 29b-f (SEQ ID NOs: 84, 1, 2, 88, and 90, respectively). [0021] FIG.9 is a series of graphs showing ELISA data for Integrin α_IIbβ₃, including 29b-f (SEQ ID NOs: 84, 1, 2, 88, and 90, respectively). [0022] FIG.10 shows HMBC highlighting interactions between C25H and Histidine ring of histidine-linked LTGFHA 21 (SEQ ID NO: 62) with the structure shown below. [0023] FIG. 11 shows stability of cyclized peptide 5 (SEQ ID NO: 5) in PBS pH 2.5 over time. [0024] FIG. 12 shows stability of cyclized peptide 5 (SEQ ID NO: 5) in PBS pH 10 over time. [0025] FIG. 13 shows stability of cyclized peptide 5 (SEQ ID NO: 5) in PBS pH 7.4 over time. [0026] FIG.14 shows the proteolytic and oxidative stability of cyclized peptide 5 (SEQ ID NO: 5) over time. 6 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO DETAILED DESCRIPTION [0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure. [0028] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like. [0029] Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2%, or 1%) of the particular value modified by the term “about.” [0030] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group that that minimum number of heteroatoms. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure. [0031] With respect to compounds of the present disclosure, and to the extent the following terms are used herein to further describe them, the following definitions apply. [0032] As used herein, the term “halogen” (or “halo” or “halide”) refers to fluorine, chlorine, bromine, or iodine. [0033] As used herein, the term “electron withdrawing group” refers to an atom or functional group containing an electronegative atom that attracts more electron density from the atoms to which they are attached (e.g., an electron withdrawing group), such as a halogen atom (e.g., I, Br, Cl), a tosyl group (-OTs), or an alkoxy group. [0034] Unless stated otherwise, and to the extent not further defined for any particular group(s) in the compounds of formula (I-III) disclosed herein may be substituted or unsubstituted. To the extent not disclosed otherwise for any particular group(s), representative 7 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO examples of substituents may include alkyl (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), substituted alkyl (e.g., substituted C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), alkoxy (e.g., C1-C6, C₁-C₅, C₁-C₄, C₁-C₃, C₁-C₂, C₁), substituted alkoxy (e.g., substituted C₁-C₆, C₁-C₅, C₁-C₄, C₁- C3, C1-C2, C1), haloalkyl (e.g., CF3), alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkenyl (e.g., substituted C2-C6, C2-C5, C2-C4, C2-C3, C2), alkynyl (e.g., C2-C6, C2-C5, C2-C4, C₂-C₃, C₂), substituted alkynyl (e.g., substituted C₂-C₆, C₂-C₅, C₂-C₄, C₂-C₃, C₂), cyclic (e.g., C₃-C₁₂, C₅-C₆), substituted cyclic (e.g., substituted C₃-C₁₂, C₅-C₆), carbocyclic (e.g., C₃-C₁₂, C5-C6), substituted carbocyclic (e.g., substituted C3-C12, C5-C6), heterocyclic (e.g., 3- to 12- membered, 5-to 6-membered), substituted heterocyclic (e.g., substituted 3- to 12-membered, 5-to 6-membered), aryl (e.g., benzyl and phenyl), substituted aryl (e.g., substituted benzyl or substituted phenyl), heteroaryl (e.g., pyridyl or pyrimidyl), substituted heteroaryl (e.g., substituted pyridyl or substituted pyrimidyl), aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl), halo, hydroxyl, aryloxy (e.g., C₆-C₁₂, C₆), substituted aryloxy (e.g., substituted C6-C12, C6), alkylthio (e.g., C1-C6), substituted alkylthio (e.g., substituted C1-C6), arylthio (e.g., C6-C12, C6), substituted arylthio (e.g., substituted C6-C12, C6), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfinamide, substituted sulfinamide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid, and peptide groups. Terminal substituents can be selected from the group consisting of C₁-C₆ alkyl, halo, hydroxy or cyano. [0035] In one aspect, compounds of the disclosure are represented by formula I:

pharmaceutically acceptable salt or stereoisomer thereof, wherein: X is an electron withdrawing group; each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3 to about 50. [0036] In a second aspect, compounds of the disclosure are represented by formula II:

pharmaceutically acceptable salt or stereoisomer thereof, 8 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3 to about 50. [0037] In a third aspect, compounds of the disclosure are represented by formula III:

pharmaceutically acceptable salt or stereoisomer thereof, wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3 to about 50. [0038] In some embodiments, n is an integer from 13-46. In some embodiments, n is an integer from 13-42. In some embodiments, n is an integer from 13-36. In some embodiments, n is an integer from 13-30. In some embodiments, n is an integer from 13-25. In some embodiments, n is an integer from 16-25. [0039] In some embodiments, X is halo or optionally substituted tosyl. In some embodiments, X is halo. In some embodiments, X is iodo or chloro. [0040] In some embodiments, the non-naturally occurring amino acid contains N- methylation, N-acylation, an aromatic backbone, or a tag. Tags typically contain a detectable moiety such as a label. Representative examples of tags include dyes and chromogenic agents. The term “label” includes any moiety that allows the compound to which it is attached to be captured, detected, or visualized. A label may be directly detectable (i.e., it does not require any further reaction or manipulation to be detectable, e.g., a fluorophore or chromophore is directly detectable) or it may be indirectly detectable (i.e., it is made detectable through reaction with or binding to another entity that is detectable, e.g., a hapten is detectable by immunostaining after reaction with an appropriate antibody comprising a reporter such as a fluorophore). Representative examples of labels include affinity tags, radiometric labels (e.g., radionuclides (such as, for example, ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I, and the like)), fluorescent dyes, phosphorescent dyes, chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like), spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, and platinum) or nanoclusters, enzymes (such as, for example, those used in an ELISA, i.e., horseradish 9 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), magnetic labels (such as, for example, Dynabeads™), and haptens. [0041] In certain embodiments, the label comprises a fluorescent dye. Representative examples of fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine (FITC), naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6- carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., 5-carboxytetramethylrhodamine (TAMRA), carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, or tetramethylrhodamine (TMR)), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g. Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800), and the like. For more examples of suitable fluorescent dyes and methods for coupling fluorescent dyes to other chemical entities see, for example, The Handbook of Fluorescent Probes and Research Products, 9th Ed., Molecular Probes, Inc., Eugene, Oregon and Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11^th Ed., Life Technologies. [0042] In some embodiments, the tag or label is attached via a reactive group present in a naturally occurring amino acid such as the sidechain of lysine or cysteine. For example, methods of biotinylating the lysine sidechain amine are described herein. [0043] In some embodiments, the tag is an affinity tag, which as known in the art refers to agents that take part in an interaction (e.g., antigen and antibody, enzyme and substrate, receptor and ligand) that facilitates capture and/or purification of the molecule. Representative examples include small chemical compounds (such as biotin,

, and derivatives thereof), short amino acid sequences (e.g., 2 to 20 amino acids in length, 4 to 12 amino acids in length, such as the (His)₆ tag, (His)₄ tag, (His)₃ tag, (His)₂ tag, (Leu)₄ tag, (Leu)₃ 10 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No. 046094-781001WO tag, (Leu)2 tag, HA tag, FLAG tag, VSV-G tag, HSV tag, and V5 tag), chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, and glutathione-S-transferase (GST). [0044] In some embodiments, the tag is a chromogenic agent, which as known in the art refers to a chemical compound that induces a color reaction. Representative examples include azo reagents such as methyl orange and methyl red, nitrophenols, phthaleins such as phenolphthalein or thymolphthalein, sulfonephthaleins such as bromophenol blue or bromocresol green, indophenols such as 2,6-dichlorophenolindophenol, azine reagents such as thiazine dye methylene blue, indigo carmine, derivatives of diphenylamine such as diphenylamine-4-sulfonic acid and variamine blue, arsenazo III, catechol violet, dithizone, 1- (2′-pyridylazo)-2-naphthol, 4-(2′-pyridylazo)resorcinol, chrome azurol S, eriochrome black T, eriochrome blue-black B, pyrogallol red, alizarin complexone, methylthymol blue, and xylenol orange. [0045] In some embodiments, the tag is a biotin tag/NeutrAvidin bead, biotin tag/streptavidin bead, His6-tag/NTA bead, Fc tag/protein G beads, His10-tag/NTA bead, biotinylated Avi- tag/streptavidin bead, or an EDC/NHS coupling/carboxylic acid bead. Other tags which may be suitable for use in the present dislcosure are disclosed in Huang et al., Chem. Rev., 2019, 119:10360-10391. [0046] In some embodiments, the non-naturally occurring amino acid is an α-hydroxy acid, α-mercapto acid, a thioacid, a D-amino acid, or a β-amino acid. In some embodiments, the non- naturally occurring amino acid is a side-chain chloroacetyl amino acid, a side-chain azide amino acid, a side-chain alkyne amino acid, or fluorescent amino acid. Representative examples of non-naturally occurring amino acids include:

11 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No. 046094-781001WO

. Other non-naturally occurring amino acids which may be suitable for use in the present disclosure are disclosed in Huang et al., Chem. Rev., 2019, 119:10360-10391. [0047] In some embodiments, the optional substituent for the compound of formula I-III is independently alkyl, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkyenyloxy, alkynyloxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, aralkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-aralkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, N-alkyl-N- 12 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocycloalkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocycloalkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N- heteroarylaminocarbonyl, cyano, nitro, and azido. [0048] Compounds of the present disclosure may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. A pharmaceutically acceptable salt of the compounds of this disclosure can be formed, for example, by reaction of an appropriate free base of a compound of the disclosure and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, P. L., "Salt selection for basic drugs," International Journal of Pharmaceutics, 33:201-217 (1986); Bastin, R. J., et al., "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4:427-435 (2000); and Berge, S. M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66:1-19 (1977). [0049] Compounds of the present disclosure may have at least one chiral center and thus may be in the form of a stereoisomer, which as used herein, embraces all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers which include the (R-) or (S-) configurations of the compounds), mixtures of mirror image isomers (physical mixtures of the enantiomers, and racemates or racemic mixtures) of compounds, geometric (cis/trans or E/Z, R/S) isomers of compounds and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The chiral centers of the compounds may undergo epimerization in vivo; thus, for these compounds, administration of the compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Accordingly, the compounds of the present disclosure may be made and used in the form of individual isomers and substantially free of other isomers, or in the form of a mixture of various isomers, e.g., racemic mixtures of stereoisomers. The stereochemistry of standard L- and D-amino acids is known to the skilled person. 13 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [0050] In some embodiments, the compounds of the present disclosure are isotopic derivatives in that it has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound includes deuterium or multiple deuterium atoms. As used herein, the term “compound” embraces isotopic derivatives. Methods of Synthesis [0051] In another aspect, the present disclosure is directed to a method for making a compound of formula I-III, or a pharmaceutically acceptable salt or stereoisomer thereof. The compounds of the present disclosure will be better understood in connection with the synthetic schemes that are described in various working examples and which illustrate non-limiting methods by which the compounds may be prepared, e.g., compounds of formulas I-III. [0052] In another aspect, the present disclosure is directed to methods for preparing a compound of formula I:

stereoisomer thereof, comprising: (i) functionalizing a resin with an O-alkylhydroxylamine, wherein the amine is protected with a protecting group; (ii) coupling the functionalized resin with an optionally substituted naturally or non- naturally occurring amino acid having a terminal amine group, wherein the amine group is protected with a protecting group; (iii) deprotecting the amine group of the optionally substituted naturally or non-naturally occurring amino acid from step (ii); (iv) repeating steps (ii) and (iii) a number of times until the desired length of n is reached; (v) capping the resin; and (vi) cleaving the compound of formula I off the resin. [0053] In some embodiments, the amine protecting group is 9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), benzyl carbamate (Cbz), benzyl (Bn), triphenylmethylamine (Tr), or p-toluenesulfonamide (Ts). [0054] Reaction parameters which may be suitable for preparing a compound of formula I are disclosed in Stawikowski et al., Curr. Protoc. Protein Sci., 2002, chapter: unit 18-1; Coin et al., Nature Protocols, 2007, 2:3247-3256; Mueller et al., Front. Bioeng. Biotechnol., 2020, 14 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 8:162; Merrifield, J. Am. Chem. Soc., 1963, 85(14):2149-2154; Isidro-Llobet et al., J. Org. Chem., 2019, 84(8):4615-4628. [0055] In some embodiments, the resin is a polystyrene resin. In some embodiments, the resin is a polyacrylate, a polyacrylamide, or a polyethylene glycol resin. [0056] In some embodiments, the resin is capped with a haloacetamide or a pseudo- haloacetamide, e.g., O-tosyl. [0057] In another aspect, the present disclosure is directed to methods for preparing a compound of formula II:

[0058] In some embodiments, the solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DCM, CHCl₃, CCl₄, DCE, toluene, MeCN, or THF. [0059] In some embodiments, the solvent is a protic solvent. In some embodiments, the protic solvent is MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP. [0060] In some embodiments, the solvent is a buffered aqueous solvent. In some embodiments, the buffered aqueous solvent is phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane (TRIS), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), or 3-(N-morpholino)propanesulfonic acid (MOPS). [0061] In some embodiments, the mixing is carried out at a temperature of about 0-100°C. In some embodiments, the mixing is carried out at a temperature of about 30-60°C. In some embodiments, the mixing is carried out at a temperature of about 30-55°C. In some embodiments, the mixing is carried out at a temperature of about 35-50°C. [0062] In some embodiments, the pH is about 7.5 to about 13. In some embodiments, the pH is about 7.5 to about 12. In some embodiments, the pH is about 7.5 to about 11. In some embodiments, the pH is about 7.5 to about 10. [0063] In some embodiments, the mixing is carried out for about 0.25 to about 240 hours. In some embodiments, the mixing is carried out for about 24 to about 96 hours. In some embodiments, the mixing is carried out for about 24 to about 48 hours. In some embodiments, the mixing is carried out for about 1 to about 24 hours. In some embodiments, the mixing is 15 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO carried out for about 120 hours. In some embodiments, the mixing is carried out for about 96 hours. In some embodiments, the mixing is carried out for about 72 hours. In some embodiments, the mixing is carried out for about 48 hours. In some embodiments, the mixing is carried out for about 24 hours. In some embodiments, the mixing is carried out for about 12 hours. In some embodiments, the mixing is carried out for about 6 hours. In some embodiments, the mixing is carried out for about 3 hours. In some embodiments, the mixing is carried out for about 2 hours. In some embodiments, the mixing is carried out for about 1 hour. In some embodiments, the mixing is carried out for about 0.5 hours. [0064] In some embodiments, the method for preparing a compound of formula II optionally further comprises reacting with a tag. [0065] In another aspect, the present disclosure is directed to methods for preparing a compound of formula III:

stereoisomer thereof, comprising reacting a compound of formula II:

the presence of a solvent. [0066] In some embodiments, the solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DCM, CHCl₃, CCl₄, DCE, toluene, MeCN, or THF. [0067] In some embodiments, the solvent is a protic solvent. In some embodiments, the protic solvent is water, MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP. [0068] In some embodiments, the solvent is a solvent mixture. In some embodiments, the solvent mixture is THF/MeOH. In some embodiments, the THF/MeOH mixture is 1:1 THF/MeOH. In some embodiments, the THF/MeOH mixture is 3:1 THF/MeOH. [0069] In some embodiments, SmI₂ is in excess. In some embodiments, SmI₂ is in excess of about 2 to about 300 equivalents. In some embodiments, SmI2 is in excess of about 25 to about 200 equivalents. In some embodiments, SmI2 is in excess of about 50 to about 150 equivalents. In some embodiments, SmI₂ is in excess of about 250 equivalents. In some embodiments, SmI₂ is in excess of about 200 equivalents. In some embodiments, SmI₂ is in excess of about 150 equivalents. In some embodiments, SmI2 is in excess of about 75 equivalents. In some embodiments, the molarity of SmI2 is about 4 mM. 16 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [0070] In some embodiments, the reacting is carried out at a temperature of about 0-100°C. In some embodiments, the reacting is carried out at a temperature of about 20-50°C. In some embodiments, the reacting is carried out at about 38°C. In some embodiments, the reacting is carried out at about room temperature (20-25°C). [0071] In some embodiments, the mixing is carried out for about 1 to about 96 hours. In some embodiments, the mixing is carried out for about 24 to about 96 hours. In some embodiments, the mixing is carried out for about 24 to about 72 hours. In some embodiments, the mixing is carried out for about 48 to about 96 hours. In some embodiments, the mixing is carried out for about 24 to about 48 hours. In some embodiments, the reacting is carried out for about 24 hours. In some embodiments, the reacting is carried out for about 12 hours. In some embodiments, the reacting is carried out for about 6 hours. In some embodiments, the reacting is carried out for about 3 hours. In some embodiments, the reacting is carried out for about 2 hours. In some embodiments, the reacting is carried out for about 1 hour. In some embodiments, the reacting is carried out for about 0.5 hours. In some embodiments, the reacting is carried out for about 0.25 hours. Methods of Use [0072] Compounds of formulas (II) and (III) may be used in affinity screening to identify putative therapeutic macrocyclic peptides that bind targets, e.g., therapeutically validated targets, and then sequence the linearized compound that binds the target, respectively. In some aspects, the present disclosure is directed to methods of identifying a macrocyclic peptide that binds a putative therapeutic target, comprising: (i) conducting affinity selection that comprises incubating a library of macrocyclic peptides of the formula (II) with a putative therapeutic target immobilized on a solid support, under conditions to allow any one or more members of the library to bind the target; (ii) washing to remove unbound macrocyclic peptides; (iii) eluting macrocyclic peptides that bind the target; (iv) linearizing the thus- eluted one or more macrocyclic peptides by reacting with SmI2; and (v) analyzing the one or more linearized macrocyclic peptides via tandem liquid chromatography/mass spectrometry (LC-MS/MS), thereby determining the amino acid sequence of the one or more macrocyclic peptides in the library that bind the target. [0073] Split-and-pool solid phase peptide synthesis (SPPS) can be used to make macrocyclic peptide libraries of various sizes. A mixture of solid supports is randomly split into subpools. 17 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO An optionally substituted naturally or non-naturally occurring amino acid is coupled to the supports in each subpool. The supports are repooled, mixed, and another optionally substituted naturally or non-naturally occurring amino acid is coupled to the supports in each subpool. The process of splitting, chemistry, and pooling is iterated until the desired length of the amino acid chain is reached, at which point the peptide is cyclized. [0074] Affinity screening can be used to identify linear peptides of particular interest. See, Prudent et al., Nat. Rev. Chem., 2021, 5:62-71; Lu et al., Anal. Chem., 2019, 91(13):8162- 8169; Annis et al., Curr. Opin. Chem. Biol., 2007, 11(5):618-526. [0075] Tandem mass spectrometry (MS/MS) can be used to sequence linear peptides. See, Coon et al., Biotechniques, 2018, 38(4): 519-523; Hunt et al., Proc. Natl. Acad. Sci. USA, 1986, 83(17):6233-6237. [0076] In some embodiments, the putative therapeutic target comprises a protein or a transcription factor. Putative therapeutic targets which may be suitable for use in the present disclosure are disclosed in Huang et al., Chem. Rev., 2019, 119:10360-10391. [0077] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims. EXAMPLES [0078] Example 1: General Information [0079] Solid phase peptide synthesis (SPPS) was conducted manually in either 3- or 10-mL fritted syringes that were attached to an end-over-end rotator for mixing during reaction steps. Filtration was carried out under vacuum. Room temperature (rt) is defined as 22.5 ± 2.5°C. Gas-tight syringes with stainless steel needles or cannulae were used to transfer air- and moisture-sensitive liquids. Organic solutions were concentrated at 27°C on rotary evaporators capable of achieving a minimum pressure of ~2 torr unless otherwise stated. Reaction heating was performed using aluminum metal heating blocks. Macrocyclization reactions were carried out under an air atmosphere. Linearization reactions were conducted in glass reaction vials under a positive pressure of nitrogen using solvents degassed under a stream of N₂. [0080] Materials [0081] All solvents were purchased from Fisher Scientific or Sigma–Aldrich. Unless otherwise stated, chemical reagents were purchased from Fisher Scientific, Sigma–Aldrich, 18 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO Alfa Aesar, Oakwood Chemical, Acros Organics, Combi-Blocks, or TCI America. Solid phase peptide synthesis (SPPS) resins used: 2-chlorotrityl chloride resin (Chem-Impex, 100–200 mesh, 1.43 mmol/g loading) for general peptide synthesis, 2-chlorotrityl chloride resin (Chem- Impex, 200–400 mesh, 1.47 mmol/g loading) for combinatorial library synthesis. [0082] General Instrumentation [0083] All solvents were purchased from Fisher Scientific or Sigma–Aldrich. Unless otherwise stated, chemical reagents were purchased from Fisher Scientific, Sigma–Aldrich, Alfa Aesar, Oakwood Chemical, Acros Organics, Combi-Blocks, or TCI America. Solid phase peptide synthesis (SPPS) resins used: 2-chlorotrityl chloride resin (Chem-Impex, 100–200 mesh, 1.43 mmol/g loading) for general peptide synthesis, 2-chlorotrityl chloride resin (Chem- Impex, 200–400 mesh, 1.47 mmol/g loading) for combinatorial library synthesis. [0084] UV-vis data were obtained with an Agilent Cary 60 UV-vis spectrophotometer. Infrared data (IR) were obtained with a Cary 630 Fourier transform infrared spectrometer equipped with a diamond Attenuated total reflectance (ATR) objective and are reported as follows: frequency of absorption (cm^–1), intensity of absorption (s = strong, m = medium, w = weak, br = broad). High resolution mass spectra (HRMS) were recorded on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer using an electrospray ionization (ESI), atmospheric pressure ionization (API), or electron ionization (EI) source. Automated C18 reverse phase chromatography was performed using an Isolera One (Biotage) purification system. High performance liquid chromatography (HPLC) purification was performed using an Agilent 1260 Infinity system. High performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 Infinity II system. [0085] Example 2: Synthesis and macrocyclization of C-terminal hydroxamic acid, N- terminal electrophilic peptides [0086] SPPS was used to access C-terminal hydroxamic acid functionalized peptides containing different N-terminal electrophiles. The C-terminal hydroxamic acid was installed by functionalizing 2-chlorotrityl chloride resin with N-hydroxyphthalimide using reported procedures (Sinatra et al., Angew. Chemie Int. Ed., 2020, 59(50):22494–22499). With the resin in hand, a peptide was synthesized containing six amino acids including a lysine and aspartic acid residue to test the chemoselectivity of the desired cyclization in the presence of common reactive motifs. The N-terminus was capped with a series of electrophiles before the peptide was cleaved and fully deprotected under acidic conditions. To achieve chemo- and 19 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO regioselective cyclization in the presence of unprotected amino acids, mild aqueous conditions were used for the electrophile screen. Product yield was estimated using high performance liquid chromatography (HPLC) UV analysis at 220 nm (Table 1, entries 1–4). Table 1. Macrocyclization of linear peptide 4a.

20 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 14 25 mM HEPES 8 1 50 24 92 1 7 15 25 mM MOPS 8 1 50 24 94 0 6 16 25 mM MOPS 8 2 38 48 >95 0 2 17 25 mM MOPS 8 4 38 48 65 21 14 18 25 mM MOPS 8 6 38 48 25 65 10 19 50 mM MOPS 8 4 38 24 84 3 13 Conditions described in General macrocyclization procedure A with product yield estimated by HPLC based on AUC peaks at 220 nm. Abbreviations: Temp – temperature, PBS – phosphate buffered saline, HEPES – N-2-hydroxyethylpiperazine-N'- 2-ethanesulfonic acid, MOPS – 3-(N-morpholino)propanesulfonic acid. [0087] Macrocycle 5 was generated from linear peptide 4 featuring an N-terminal (pseudo)haloacetamide electrophile (X = I, Br, Cl, OTs). A single major product was formed in each case, indicating exquisite chemoselectivity of the hydroxamic acid alkylation in the presence of amines and carboxylic acids. The bromo- and iodoacetamide capped peptides exhibited full starting material conversion after 24 h at 38°C, with similar purity profiles while the chloroacetamide capped derivative cyclized significantly slower. Only 25% conversion was observed under the reaction conditions. The tosylated variant showed significant cyclization; however, the purity profile was poorer than for the halogenated derivatives. The iodide electrophile was carried forward for its superior reactivity and reaction purity. [0088] Next, reaction conditions were screened to optimize macrocyclization and minimize side product formation (Table 1, entries 5–15, full screen: S13). The reaction proceeded most efficiently under basic conditions. Little product was formed in water (Table 1, entry 9) or under acidic conditions (Table 1, entries 5–6). Minimal side products were generated across almost all conditions, especially at high pH. At low pH, in addition to poor reactivity, slow hydrolysis of the hydroxamic acid proved problematic. Complete reaction and >95% product formation was observed in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH 8 in 24 h (Table 1, entry 12). Next, while reactions were run at 50°C for a shorter period, reaction yields were not markedly improved (Table 1, entries 13–15). Finally, peptide concentrations were varied from 1 to 6 mM (Table 1, entries 16–19). [0089] After validating the macrocyclization, we turned to the linearization of cyclo- DQLGKA (5). Treatment of macrocycle 5 with SmI₂ led to the formation of linearized peptide 6 in degassed tetrahydrofuran with methanol as cosolvent (Table 2). After screening conditions using a fixed amount of SmI2, we found that a titration approach maximized conversion. Table 2. Peptide Linearization conditions of peptide 5 to 6. 21 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

Conditions: Macrocycle 5 (4 mM), SmI₂, degassed solvent, 1h. Product yield estimated by HPLC based on AUC of peptidic peaks at 220 nm. [0090] Example 3: Substrate scope of hydroxamic acid macrocyclization with iodoacetamide electrophile [0091] The macrocyclization and linearization scope was established in the presence of different amino acids across a range of ring sizes (FIG.1). After a small condition screen (Table 3), 13-membered macrocycle 7b was formed at 0.5 mM reaction concentration in high yield despite the small ring size. The superiority of MOPS buffer over PBS was clear from this macrocyclization screen as the PBS conditions led to significant hydroxamic acid hydrolysis, resulting in reduced macrocycle yield. Peptides containing serine, tyrosine, tryptophan, and threonine all cyclized and linearized with high yields and minimal side products. Non- canonical amino acids such as N-methylamino acids, D-amino acids, β- amino acids, and aromatic backbone amino acids could all be easily incorporated into macrocyclic peptides using our strategy. Both cyclization and linearization proceeded cleanly and in high yield in each case. Larger ring sizes (40–46 atoms) could also be synthesized efficiently in 50 mM MOPS buffer, pH 8. [0092] The structures of the cyclized and linearized peptides were further validated by synthesis using alternative methods. To confirm that macrocyclization occurs through 22 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO hydroxamic acid O-alkylation, macrocycle 10b was prepared by HATU-mediated cyclization of an SPPS-derived linear peptide containing an internal hydroxamate linkage (FIG.4A). The ¹H and ¹³C NMR spectra of the macrocyclic peptide obtained through both methods were identical (FIG. 4B). To verify the product of SmI2-mediated linearization, NMR spectra of peptide 6 was compared to that of a C-terminal amide, N-terminal glycolic acid containing peptide synthesized on Rink amide resin (FIG.5A). [0093] Example 4: Chemoselectivity of hydroxamic acid macrocyclization with chloroacetamide electrophile [0094] The compatibility of the macrocyclization protocol was evaluated with the complete set of nucleophilic amino acid side chains. N-terminal haloacetamide peptides featuring a C- terminal amide in lieu of the hydroxamic acid were subjected to the cyclization conditions to determine whether the side chains of any residue would interfere (FIG.6). Lysine and arginine residues did not react with the iodoacetamide at pH 8, but the more nucleophilic methionine and histidine residues did. Methionine cyclization was completely mitigated by replacing the iodoacetamide electrophile with the less reactive chloroacetamide. This strategy also reduced histidine cyclization significantly. [0095] To optimize hydroxamic acid reactivity with the less reactive chloroacetamide electrophile, N-terminal chloroacetamide, C-terminal hydroxamic acid LTGFHA (SEQ ID NO: 1) peptide was prepared and subjected to several cyclization conditions (Table 4). The previously optimized conditions, MOPS, pH 8, generated significant levels of the histidine cyclized product 21 alongside desired product 20b with little selectivity (Table 4, entry 1). The hydroxamic acid reacted considerably more slowly with chloroacetamide than with the more reactive iodoacetamide electrophile, with reaction times ranging between 48–96 h for complete starting material conversion. At pH 7, the major product formed was histidine-cyclized macrocycle 21 (Table 4, entry 4); however, as the pH was increased to 10, the desired hydroxamate linked product 20b was obtained as the major product. Under these conditions, the reaction neared completion at 48 h (Table 4, entry 6). A greater population of the hydroxamate anion at higher pH is consistent with the enhanced nucleophilicity of the hydroxamic acid (pKa 8–9) and increased formation of the head-to-tail cyclized product (Bauer et al., Angew. Chemie Int. Ed. Eng., 1974, 13(6):376–384). [0096] Additional methionine and histidine containing peptides were cyclized via the chloroacetamide electrophile in good yields: 23 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No. 046094-781001WO

[0097] Therefore, whilst chloroacetamide resulted in slower macrocyclization, it ensured good product yields for methionine and histidine containing peptides. Using PBS, pH 9 at 50°C enabled the hydroxamate-linked macrocycles to be accessed more rapidly and with higher yields than at lower temperature, even for the 37-membered, non-canonical macrocycle 23b (78%). [0098] Example 5: Cysteine containing macrocycles can be accessed using a simple protecting group strategy

[0099] To render the macrocyclization and linearization technology compatible with cysteine-containing peptides and enable complete coverage of canonical amino acid space, we developed a cyclization protocol that complements our optimized route. Cysteine is a versatile amino acid with demonstrated utility as a nucleophilic handle for creating small molecule- protein conjugates, bivalent drug assemblies, or bicyclic peptide ligands (Erlanson et al., Proc. Natl. Acad. Sci., 2000, 97(17):9367–9372; Rafie et al., Bioconjug. Chem., 2018, 29(6):1834- 1840; Chen et al., J. Am. Chem. Soc., 2013, 135(17):6562–6569). The ability to modify and diversify macrocyclic peptide scaffolds rapidly has proven advantageous in early-stage drug discovery (Habeshian et al., Nat. Commun., 2022, 13(1):3823; Guo et al., Nat. Chem., 2019, 11(3):254–263). 24 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00100] The cysteine-compatible method exploits the facile disassembly of protected peptides from the acid sensitive 2-chlorototrityl chloride resin on which they are synthesized. An iodoacetamide capped cysteine-containing peptide was first synthesized by SPPS then cleaved from the resin under mildly acidic conditions (1% trifluoroacetic acid in dichloromethane), producing trityl-protected cysteine peptide 24 with ca. 90% purity by HPLC. The crude peptide was cyclized and fully deprotected to provide pure peptide 25 in ca. 75% purity without the need for chromatographic purification. HPLC purification yielded pure peptide in 47% isolated yield over two steps. The cysteine containing macrocycle was linearized to generate peptide 26 in 71% yield. [00101] The peptidomimetic macrocycles generated by this method can be functionalized in situ to easily append biologically useful tags, such as biotin, in high yield. Due to the high chemoselectivity and efficiency of the macrocyclization reaction, amino acid residues can be effortlessly labeled in a one-pot process. [00102] The stability of the peptidomimetic macrocycle was assessed under different conditions. High stability of the hydroxamate linked cyclic peptides is necessary for potential therapeutics and for library screening applications. Macrocycle 5 was highly stable in different pH buffers at 37°C. Less than 3% degradation was observed after 48 h in pH 2.5, 7.4, and 10 buffer, showcasing the robustness of the hydroxamate linkage. Even prolonged exposure to these conditions led to minimal decomposition; less than 15% decomposition was observed after 28 days at pH 2.5 and 10. At pH 7.4, ca.12% degradation occurred after 14 days; however, the major impurities observed were unrelated to the hydroxamate linkage, primarily Gln deamination and epimerization. Only minor impurities were observed at basic pH. Under acidic conditions, dehydration products were observed, likely due to acid-catalyzed condensation between lysine and aspartic acid residues, also unrelated to the hydroxamate linkage. [00103] Macrocycle stability was also high under harsh oxidative conditions. Only ca.20% degradation was observed after 48 h at 37°C in the presence of hydrogen peroxide. In contrast, macrocycles generated through cysteine cyclization are often unstable under oxidative conditions. The stability of macrocycle 5 was also assessed in the presence of common proteases proteinase K and pepsin. Less than 5% degradation was observed following incubation with either protease for 48 h at 37°C, highlighting the high stability of our hydroxamate molecular clasp. 25 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00104] Example 6: Macrocyclization and linearization are highly effective with multiple peptidic species present [00105] To demonstrate the potential of the technology for combinatorial library synthesis, a mixture of 8 peptides was synthesized by split-and-pool SPPS (FIG.2). HPLC-MS analysis of the crude peptide mixture distinctly showed each peptide with low impurity levels. Cyclization and linearization of the peptide mixture proceeded cleanly with minimal side products. These reactions were highly effective on the crude peptides with no HPLC purification necessary for product formation. This is an important factor for tag-free combinatorial library synthesis and highlights the high selectivity and efficiency of the developed reactions even in the presence of multiple species. [00106] Example 7: Hydroxamate-linked peptidomimetic macrocycles exhibit high integrin α_Vβ₃ binding affinity [00107] Biologically active macrocycles were generated using this hydroxamate linkage. Integrin proteins are transmembrane glycoprotein receptors vital for cell adhesion and signaling (Slack et al., Nat. Rev. Drug Discov., 2022, 21(1):60–78). These proteins recognize the Arginine-Glycine-Aspartic acid (RGD) binding motif present in their endogenous ligands and as such can be inhibited by therapeutics that exploit this motif (Hatley, R. J. D.; Macdonald, S. J. F.; Slack, R. J.; Le, J.; Ludbrook, S. B.; Lukey, P. T. An αv-RGD Integrin Inhibitor Toolbox: Drug Discovery Insight, Challenges and Opportunities. Angew. Chemie Int. Ed.2018, 57 (13), 3298–3321; Dechantsreiter et al., J. Med. Chem., 1999, 42(16):3033–3040; Mas-Moruno et al., Anticancer. Agents Med. Chem., 2010, 10(10):753–768). To this end, a series of simple alanine containing RGD macrocyclic peptides were synthesized with 13–28 atom ring sizes and tested their activity against integrin proteins using ELISA (Table 5) (Kapp et al., Sci. Rep., 2017, 7(1):39805). This method can generate a diverse range of macrocycle sizes that can be challenging to access using alternative cyclization strategies due to linker size or cyclization efficiencies. The low molecular weight and small size of the hydroxamate linkage facilitates unique access to a minimal 13-membered cyclo-RGD macrocycle 29a alongside larger variants with high cyclization efficiency, enabling comprehensive exploration of how macrocycle size impacts integrin binding affinity. [00108] The RGD containing peptides were generated in high yields despite the wide range of macrocycle ring sizes. Macrocycles 29b–f were all accessed via macrocyclization of the unprotected linear peptides in MOPS, pH 8 buffer at 38°C with yields ranging from 86–98%. 26 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO The 13-membered cyclo-RGD macrocycle (29a) was synthesized using the protecting group strategy (Example 5) to avoid cyclization occurring between the aspartic acid carboxylic acid and the hydroxamic acid. Macrocyclization facilitated by cesium carbonate rapidly yielded 57% cyclized product that was treated with trifluoroacetic acid for 1 h to generate macrocycle 29a. This highlights the ease with which challenging macrocycles may be accessed using the protecting group strategy. [00109] With the range of RGD containing macrocycles in hand (FIG. 3 and Table 5), integrin αVβ3 binding affinity was evaluated using ELISA (FIG. 7). The intermediate macrocycles cyclo-RGDAA (29c) (SEQ ID NO: 2) and cyclo-ARGDAA (29d) (SEQ ID NO: 3) with ring sizes of 19 and 22 atoms, exhibited optimum potency towards integrin α_Vβ₃ with IC₅₀ values of 5.6 and 5.8 nM, respectively. Binding affinity is reduced with increased ring size, with IC50 values of 18 and 37 nM for the 25 and 28-membered macrocycles, respectively. Notably, the small macrocycles cyclo-RGD (29a) and cyclo-RGDA (29b) (SEQ ID NO: 4) exhibited a more significant drop in IC₅₀, with affinities of 4.1 µM and 163 nM, respectively. The reduced activity is likely due to the distorted RGD conformation in the smaller, more rigid macrocycles, particularly prominent for the 13-membered cyclo-RGD macrocycle (29a). [00110] Integrin proteins are linked to numerous diseases, including cancer, fibrosis, and cardiovascular indications (Ley et al., Nat. Rev. Drug Discov., 2016, 15(3):173–183). These proteins consist of an α and β subunit, and the resulting heterodimers share some characteristics, such as binding affinity, toward the RGD pharmacophore. As such, it is extremely useful to explore the selectivity of RGD containing peptides towards other RGD binding integrin family proteins. To this end, ELISA was used to measure the binding affinity of RGD containing macrocycles 29a–f to integrins αVβ6 and αIIbβ3 (FIG.8 and FIG.9). Integrin α_Vβ₆ is involved in activation of the pro-fibrotic mediator transforming growth factor-β1 and is a therapeutic target for fibrosis (John et al., Nat. Commun., 2020, 11(1):4659). The integrin αIIbβ3 is a key modulator of platelet aggregation and is vital for hemostasis (van den Kerkhof et al., Int. J. Mol. Sci., 2021, 22(7):3366). Inhibitors for both proteins usually contain either the RGD motif itself or pharmacophore mimics. Hence, the α_Vβ₃ inhibitors were explored for selectivity for the αVβ3 protein over other family members. Potent αVβ3 binding macrocycles 29c–f all exhibited IC50 values 2–4 orders of magnitude higher for both proteins, indicating good selectivity toward the α_Vβ₃ protein. Despite their near identical binding affinities toward integrin αVβ3, cyclo-RGDAA (29c) (SED ID NO: 2) and cyclo-ARGDAA (29d) (SEQ ID NO: 3) differed in αVβ6 and αIIbβ3 affinity, with the larger macrocycle 29d exhibiting greater 27 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO selectivity for αVβ3. Small macrocycles cyclo-RGD (29a) and cyclo-RGDA (29b) (SEQ ID NO: 4) showed little to no binding to αVβ6 and αIIbβ3 integrins. Specific and defined targeting of integrin proteins is advantageous as it enables more comprehensive understanding of the biological effects of an inhibitor. [00111] Example 8: General Synthetic Procedures [00112] General procedure for the synthesis of hydroxamic acid substituted resin

[00113] Following the literature procedure (Sinatra et al., Angew. Chemie Int. Ed., 2020, 59(50):22494–22499).2-chlorotrityl chloride resin (1.43 mmol/g, 1 equiv) was swelled in N,N- dimethylformamide (10 mL/g resin) for 1 h. Then, a solution of N-hydroxyphthalimide (3.50 equiv) and triethylamine (3.50 equiv) in N,N-dimethylformamide (1.5 mL/g resin) was added and the resin suspension was rotated end over end on a rotator for 48 h at room temperature. The resin was filtered then washed sequentially with N,N-dimethylformamide (5 × 5 mL/g resin) and dichloromethane (5 × 5 mL/g resin). A solution of dichloromethane/methanol/N,N- diisopropylethylamine (80/15/5 v/v/v, 2 × 5 mL/g resin) was then added to the resin and the suspension was rotated for 1 h at room temperature twice. [00114] The phthalimide was deprotected by adding a solution of hydrazine monohydrate in methanol (5% v/v, 2 × 3 mL/g resin) to the resin and rotating the resulting suspension for 30 min at room temperature twice before the resin was filtered and washed sequentially with methanol (5 × 10 mL/g resin), N,N-dimethylformamide (5 × 10 mL/g resin), and dichloromethane (5 × 10 mL/g resin). [00115] General procedure for the synthesis of hydroxamic acid functionalized peptides 28 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00116] Using the hydroxamic acid functionalized resin prepared above, the full-length peptides were synthesized through repeated amino acid coupling reactions and Fmoc deprotection reactions as described. The first amino acid coupling was carried out for 24 h at room temperature. Subsequent amino acid couplings were carried out for 1.5 h for canonical amino acids and 5 h for non-canonical amino acids. [00117] Amino acid couplings: A vial was sequentially charged with Fmoc protected amino acid (4.1 equiv) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N- methylmethanaminium hexafluorophosphate N-oxide (HATU, 4 equiv). N,N- dimethylformamide (0.45 M) was then added followed by N,N-diisopropylethylamine (8 equiv). This solution was added to the fritted syringe containing resin (1 equiv). The resin suspension was rotated end over end on a rotator at room temperature. After 1.5–24 h, the resin was filtered then washed sequentially with

dimethylformamide (5 × 10 mL/g resin) and dichloromethane (5 × 10 mL/g resin) [00118] Fmoc deprotection reactions: A solution of piperidine/N,N-dimethylformamide (20% v/v, 2 × 10 mL/g resin) was added to the resin and the resulting suspension was rotated for 5 min at room temperature before filtering and repeating. The resin was filtered then washed sequentially with N,N-dimethylformamide (5 × 10 mL/g resin) and dichloromethane (5 × 10 mL/g resin). [00119] After coupling the final amino acid and performing the final deprotection, the resin was capped with an electrophile and cleaved from the resin as described below. [00120] General procedure for the determination of resin loading by UV-Vis spectroscopy [00121] Following the initial amino acid coupling, the resin loading was determined using UV-vis spectroscopic analysis of the piperidine/N,N-dimethylformamide (20% v/v) deprotection solution to quantify the amount of released dibenzofulvene. The absorbance of 29 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO the solution at 301 nm wavelength was measured and the concentration determined using the Beer-Lambert law: A = ε × c × l Where A = absorbance, ε = molar extinction coefficient (for dibenzofulvene ε301 nm = 8021 M cm^-1)², c = concentration and l = path length (1 cm). [00122] The spectroscopically determined resin loading ranged between 0.9–1.1 mmol/g following the hydroxamic acid functionalization protocol. [00123] General procedure for peptide N-terminal capping with different electrophiles

[00124] For iodoacetamide capped peptides: A vial was sequentially charged with iodoacetic acid (5.1 equiv) and tetramethylfluoroformamidinium hexafluorophosphate (TFFH, 5 equiv). Dichloromethane (0.2 M) was then added followed by 2,6-lutidine (10 equiv). This solution was added to the fritted syringe containing resin (1 equiv). The resin suspension was rotated end over end on a rotator at room temperature in the dark. After 1 h, the resin was filtered and washed sequentially with N,N-dimethylformamide (5 × 10 mL/g resin) then dichloromethane (5 × 10 mL/g resin) and dried under a N₂ stream for cleavage. [00125] For bromoacetamide capped peptides: A vial was sequentially charged with bromoacetic acid (5.1 equiv) and tetramethylfluoroformamidinium hexafluorophosphate (TFFH, 5 equiv). Dichloromethane (0.2 M) was then added followed by 2,6-lutidine (10 equiv). This solution was added to the fritted syringe containing resin (1 equiv). The resin suspension was rotated end over end on a rotator for 1 h at room temperature in the dark. After 1 h, the resin was filtered and washed sequentially with N,N-dimethylformamide (5 × 10 mL/g resin) then dichloromethane (5 × 10 mL/g resin) and dried under a N2 stream for cleavage. [00126] For chloroacetamide capped peptides: A vial was sequentially charged with chloroacetyl chloride (10 equiv) and dichloromethane (0.2 M), followed by 2,6-lutidine (10 equiv). This solution was added to the fritted syringe containing resin (1 equiv). The resin suspension was rotated end over end on a rotator for 1 h at room temperature. After 1 h, the resin was filtered and washed sequentially with N,N-dimethylformamide (5 × 10 mL/g resin) then dichloromethane (5 × 10 mL/g resin) and dried under a N₂ stream for cleavage. 30 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00127] For O-tosylacetamide capped peptides:

[00128] Diethylaminosulfur trifluoride (DAST, 50.5 µL, 0.383 mmol, 1.05 equiv) was added to a solution of S9 (83.8 mg, 0.364 mmol, 1 equiv) in dry dichloromethane (3.6 mL) cooled to 0°C. The reaction mixture was stirred for 1 h at room temperature, then diluted with dichloromethane (10 mL), washed twice with ice water (2 × 5 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure to give S10, which was used without purification in the subsequent reaction. [00129] A vial was sequentially charged with crude S10 (5.1 equiv), dichloromethane (0.2 M) and 2,6-lutidine (10 equiv). This solution was added to the fritted syringe containing resin (1 equiv). The resin suspension was rotated end over end on a rotator for 1.5 h at room temperature. After 1 h, the resin was filtered and washed sequentially with N,N- dimethylformamide (5 × 10 mL/g resin) then dichloromethane (5 × 10 mL/g resin) and dried under a N2 stream for cleavage. [00130] General procedure for peptide resin cleavage [00131] A solution of 92.5/2.5/2.5/2.5 v/v/v/v trifluoroacetic acid/triisopropylsilane/H2O/dichloromethane cleavage mixture (7.5 mL/g resin) was added to a fritted syringe containing dry resin and the resin suspension was rotated end over end on a rotator for 1 h at room temperature. After 1 h, the filtrate was collected, and the resin washed with dichloromethane (3 × 10 mL/g resin). The combined filtrate and washings were partially concentrated to ca. 20% volume before ice-cold diethyl ether was added to precipitate the peptides, which were then filtered, triturated with diethyl ether and dried under reduced pressure. [00132] General peptide purification procedures [00133] Automated flash column chromatography: All peptides were purified using a Biotage® Sfär C18 D - Duo 100 Å 30 µm 6 g column. The column was equilibrated with H2O+0.1% TFA (3 CV (column volumes)) prior to wet loading. Peptidic peaks were identified using UV 220 nm analysis and fractions were combined and concentrated under reduced pressure to isolate pure products. Specific conditions are noted for each substrate. [00134] Automated flash column chromatography: Procedure A 31 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00135] Eluent: Isocratic 100% H2O+0.1% TFA (3 CV), gradient 0→35% MeCN/H2O+0.1% TFA (10 CV), isocratic 100% MeCN+0.1% TFA (3 CV). [00136] Automated flash column chromatography: Procedure B (desalt) [00137] Eluent: Isocratic 100% H2O+0.1% TFA (4 CV), isocratic 70% MeCN/H2O+0.1% TFA (5 CV), isocratic 100% MeCN+0.1% TFA (3 CV). [00138] Preparative High Performance Liquid Chromatography (HPLC) procedures [00139] All peptides were purified using an Agilent InfinityLab Pursuit XRs C18, 21.2 x 250 mm, 5 µm. Preparative LC column on an Agilent Infinity system. Flow rate of 20 mL/min. General method: gradient, 0→5% MeCN/H₂O+0.1% TFA (2 min), then 5→X% MeCN/H₂O+0.1% TFA (22 min), then gradient X→100% MeCN+0.1% TFA (1 min), then isocratic 100% MeCN+0.1% TFA (1 min). X is specified for each peptide. [00140] General HPLC analytical methods [00141] Carried out using an Agilent 1260 Infinity II system equipped with a InfinityLab Poroshell 120 EC-C18 analytical LC column (4.6 × 50 mm, 2.7 μm particle size, 1 mL/min flow rate) and an InfinityLab Poroshell 120 (EC-C18, 4.6 x 5 mm, 2.7 µm) guard column. [00142] HPLC Analytical Method A: 0→60% MeCN, 20 min [00143] Eluent: 100% H₂O+0.1% TFA (2.5 min), gradient 0→60% MeCN/H₂O+0.1% TFA (20 min), gradient 60→100% MeCN/H2O+0.1% TFA (0.5 min), 100% MeCN+0.1% TFA (1 min). [00144] HPLC Analytical Method B: 0→40% MeCN, 20 min [00145] Eluent: 100% H2O+0.1% TFA (2.5 min), gradient 0→40% MeCN/H2O+0.1% TFA (20 min), gradient 40→100% MeCN/H₂O+0.1% TFA (0.5 min), 100% MeCN+0.1% TFA (1 min). [00146] HPLC Analytical Method C: 0→60% MeCN, 10 min [00147] Eluent: 100% H2O+0.1% TFA (2.5 min), gradient 0→60% MeCN/H2O+0.1% TFA (10 min), gradient 60→100% MeCN/H₂O+0.1% TFA (0.5 min), 100% MeCN+0.1% TFA (1.5 min). [00148] HPLC Analytical Method D: 0→95% MeCN, 20 min [00149] Eluent: 100% H₂O+0.1% TFA (2.5 min), gradient 0→95% MeCN/H₂O+0.1% TFA (20 min), gradient 95→100% MeCN/H₂O+0.1% TFA (0.5 min), 100% MeCN+0.1% TFA (1 min). [00150] Example 9: Peptide Macrocyclizations 32 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00151] General macrocyclization procedure A - Purified peptide was dissolved in phosphate buffered saline (PBS) buffer, pH 8 at 1 mM concentration with sonication. The reaction mixture was then heated to 38°C and stirred for the specified amount of time. Samples were taken at t = 0 h and at the end of the reaction for analysis by HPLC. Approximate yields were determined as a percentage of the total area under the curve of peptidic peaks. Products were confirmed by a combination of mass spectrometry and NMR analysis. [00152] General macrocyclization procedure B - Purified peptide was dissolved in 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS buffer), pH 8 at 1 mM concentration with sonication. The reaction mixture was then heated to 38°C and stirred for the specified amount of time. Samples were taken at t = 0 h and at the end of the reaction for analysis by HPLC. Approximate yields were determined as a percentage of the total area under the curve of peptidic peaks. Products were confirmed by a combination of mass spectrometry and NMR analysis. [00153] Example 10: Electrophile screen for cyclo-DQLGKA (5) (SEQ ID NO: 5) (Table 1, entries 1–4) [00154] Peptides were synthesized by SPPS using methods detailed above. The generated crude peptides were purified by prep HPLC prior to screening. [00155] HRMS analysis of linear peptides: [00156] Iodoacetamide capped DQLGKA (4a) (SEQ ID NO: 6) HRMS (ESI) (m/z): calc’d for C28H49IN9O11 [M+H]⁺: 814.2591, found: 814.2595. [00157] Bromoacetamide capped DQLGKA (4b) (SEQ ID NO: 7) HRMS (ESI) (m/z): calc’d for C₂₈H₄₉BrN₉O₁₁ [M+H]⁺: 766.2729, found: 766.2733. [00158] Chloroacetamide capped DQLGKA (4c) (SEQ ID NO: 8) HRMS (ESI) (m/z): calc’d for C28H49ClN9O11 [M+H]⁺: 722.3235, found: 722.3237. [00159] O-Tosylacetamide capped DQLGKA (4d) (SEQ ID NO: 9) HRMS (ESI) (m/z): calc’d for C35H56N9O14S [M+H]⁺: 858.3662, found: 858.3668 33 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00160] Iodoacetamide electrophile (4a)

[00161] Macrocyclization of peptide 4a (0.6 mg, 0.74 µmol) (SEQ ID NO: 6) was carried out in PBS, pH 8 (0.74 mL) at 38°C using General Macrocyclization Procedure A. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. t = 0 h

t = 24 h

[00163] Macrocyclization of peptide 4b (0.9 mg, 1.18 µmol) (SEQ ID NO: 7) was carried out in PBS, pH 8 (1.18 mL) at 38°C using General Macrocyclization Procedure A. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. t = 0 h

34 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 2 (4b) 9.34 1135.5 252.2 0.0705 91.4 t = 24 h

[00165] Macrocyclization of peptide 4c (0.9 mg, 1.25 µmol) (SEQ ID NO: 8) was carried out in PBS, pH 8 (1.25 mL) at 38°C using General Macrocyclization Procedure A. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. t = 0 h

t = 24 h

35 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00167] Macrocyclization of peptide 4d (0.8 mg, 0.93 µmol) (SEQ ID NO: 9) was carried out in PBS, pH 8 (0.93 mL) at 38°C using General Macrocyclization Procedure A. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. t = 0 h

t = 24 h

[00168] Example 11: Optimization of DQLGKA cyclization with iodoacetamide electrophile (Table 1) [00169] Purified peptide 4a (0.5–1 mg, 0.62–1.23 µmol) (SEQ ID NO: 6) was dissolved in the specified buffer at the specified concentration with sonication. The reaction mixture was then heated and stirred for the specified amount of time. Aliquots of the reaction mixture were taken at t = 0 h and at the end of the reaction, diluted two-fold in H₂O/MeCN (4/1 v/v) then analyzed using HPLC with HPLC Analytical Method A: 0→60% MeCN. HPLC showing optimized conditions: Entry 11

[00170] Example 12: NMR characterization of DQLGKA (SEQ ID NO: 5) cyclization with iodoacetamide electrophile

36 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00171] General procedure for the synthesis of hydroxamic acid functionalized peptides was used as described above on a 0.172 mmol scale. The peptide was purified by automated reverse phase chromatography (gradient 0→30% MeCN/H₂O+0.1% TFA, 8 CV) to give iodoacetamide capped peptide 4a (58 mg, 83%) (SEQ ID NO: 6).¹H NMR (500 MHz, CD3OD, 25°C): δ 8.84 (d, J = 6.7 Hz, 0.1H, NH), 8.64 (d, J = 6.8 Hz, 0.2H, NH), 8.32 (t, J = 6.0 Hz, 0.2H, NH), 8.10 (d, J = 7.0 Hz, 0.2H, NH), 8.05 (d, J = 6.2 Hz, 0.4H, NH), 7.81 (d, J = 7.8 Hz, 0.2H, NH), 4.64 (t, J = 6.7 Hz, 1H), 4.38 (dd, J = 8.9, 5.3 Hz, 1H), 4.33–4.25 (m, 2H), 4.25– 4.17 (m, 1H), 3.94 (d, J = 16.8 Hz, 1H), 3.83–3.73 (m, 3H), 2.98–2.86 (m, 3H), 2.77 (dd, J = 17.0, 6.5 Hz, 1H), 2.41–2.31 (m, 2H), 2.19–2.13 (m, 1H), 1.99–1.91 (m, 1H), 1.90–1.84 (m, 1H), 1.81–1.61 (m, 6H), 1.49–1.39 (m, 2H), 1.37 (d, J = 7.1 Hz, 3H), 0.99 (d, J = 6.2 Hz, 3H), 0.93 (d, J = 6.2 Hz, 3H).¹³C NMR (126 MHz, CD3OD, 25°C): δ: 177.8, 175.6, 174.3, 174.1, 173.6, 173.5, 171.9, 171.8, 171.7, 161.0 (app d, J = 39 Hz, TFA salt), 55.1, 54.4, 54.3, 52.2, 49.8, 44.0, 40.7, 40.6, 36.0, 32.3, 32.2, 28.1, 27.9, 25.9, 23.5, 23.3, 21.9, 18.1, –2.1. FTIR (thin film) cm^-1: 3265 (br), 3064 (br), 2937 (br), 1640 (s), 1521 (s), 1420 (m), 1167 (s), 1137 (s), 1029 (m), 954 (m), 906 (m), 954 (m), 797 (m). HRMS (ESI) (m/z): calc’d for C28H49IN9O11 [M+H]⁺: 814.2591, found: 814.2595.

[00172] Macrocyclization was carried out on peptide 4a (10 mg, 12 μmol) (SEQ ID NO: 6) using General Macrocyclization Procedure B. The macrocycle was purified by automated 37 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO reverse phase chromatography (isocratic 100% H2O+0.1% TFA (8 CV), gradient 0→35% MeCN/H2O+0.1% TFA (12 CV)) to give macrocycle 5 (8.3 mg, 99%). ¹H NMR (500 MHz, CD₃OD, 25°C): δ 9.04 (d, J = 8.0 Hz, 0.2H, NH), 8.49 (d, J = 4.3 Hz, 0.5H, NH), 8.09 (br s, 0.7H, NH), 7.98 (d, J = 9.0 Hz,, 0.2H, NH), 4.74 (t, J = 5.9 Hz, 1H, 3), 4.57 (d, J = 16.3 Hz, 1H, 1), 4.49 (dd, J = 10.2, 4.9 Hz, 1H, 19), 4.45–4.41 (m, 1H, 7), 4.26 (d, J = 16.3 Hz, 1H, 1), 4.17 (q, J = 6.8 Hz, 1H, 25), 4.10 (d, J = 17.6 Hz, 1H, 17), 4.07–4.04 (m, 1H, 12), 3.63 (d, J = 17.3 Hz, 1H, 17), 3.00–2.86 (m, 4H, 5, 24), 2.39–2.26 (m, 2H, 10), 2.07–1.99 (m, 2H, 9), 1.91– 1.82 (m, 1H, 21), 1.78–1.57 (m, 6H, 21, 14, 15, 23), 1.48–1.33 (m, 5H, 22, 27), 0.98 (d, J = 5.8 Hz, 3H, 16), 0.94 (d, J = 5.8 Hz, 3H, 16).¹³C NMR (126 MHz, CD₃OD, 25°C): δ 177.8 (11), 175.5 (13), 174.2 (6), 173.7 (20), 173.1 (8), 173.0 (4), 172.3 (26), 171.6 (2), 171.4 (18), 160.8 (q, J = 38.3 Hz, TFA salt), 117.2 (q, J = 285 Hz, TFA salt), 77.3 (1), 54.9 (12), 54.2 (7), 54.0 (19), 51.6 (3), 48.5 (25, d-MeOD overlapping*), 43.7 (17), 40.6 (24), 40.4 (14), 36.7 (5), 33.1 (21), 32.5 (10), 29.4 (9), 27.8 (23), 25.8 (15), 23.7 (22), 23.0 (16’), 22.4 (16), 17.6 (27). FTIR (thin film) cm^-1: 3314 (br), 2933 (w), 1655 (br s), 1539 (m), 1454 (w), 1170 (s), 1070 (w). HRMS (ESI) (m/z): calc’d for C28H48N9O11 [M+H]⁺: 686.3468, found: 686.3470. [00173] HPLC analysis t = 0 h – Pure peptide 4a

t = 24 h

[00174] Example 13: Peptide Linearizations (Table 2) [00175] General Linearization Procedure [00176] Samarium(II) iodide (0.1 M in tetrahydrofuran (THF)) was added to a 4 mM solution of peptide in degassed tetrahydrofuran/methanol (3/1 v/v) under a N₂ atmosphere until the deep blue color was maintained. The reaction mixture was then stirred under N₂ for 1 h at room temperature before an aliquot was taken, quenched in air, concentrated under reduced pressure, and redissolved in H2O+1% formic acid (FA) for HPLC analysis. The product peptides were isolated following automated flash column chromatography (procedure B desalt) and the fractions concentrated to yield pure peptide. [00177] Linearization of macrocycle cyclo-DQLGKA (5) (SEQ ID NO: 5) 38 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00178] Samarium(II) iodide (0.1 M in tetrahydrofuran, 0.5 mL) was added dropwise to a solution of macrocycle 5 (4 mg, 4.91 µmol) in a degassed mixture of tetrahydrofuran/methanol (3/1 v/v, 0.3 mL) until a deep blue solution was formed. The reaction mixture was then stirred under N2 at room temperature for 1 h before analysis by HPLC (HPLC Analytical Method B: 0→40% MeCN). The reaction was then quenched by stirring in air for 1 h and concentrated under a N₂ stream. The crude residue was initially purified by automated flash column chromatography (isocratic 100% H2O+0.1% TFA (5 CV), then gradient 0→40% MeCN/H2O+0.1% TFA (8 CV)). The concentrated product was then further purified by prep HPLC (gradient 5→75% MeCN/H₂O+0.1%TFA, 22 min) to give linear peptide 6 (2 mg, 50% yield) (SEQ ID NO: 10). ¹H NMR (500 MHz, CD3OD, 25°C): δ 8.66 (d, J = 6.6 Hz, 0.4H, NH), 8.32 (t, J = 5.8 Hz, 0.4H, NH), 8.26 (d, J = 7.7 Hz, 0.3H, NH), 8.09 (d, J = 6.0 Hz, 0.7H, NH), 8.03 (d, J = 6.9 Hz, 0.5H, NH), 7.86 (d, J = 7.5 Hz, 0.4H, NH), 4.77 (t, J = 6.2 Hz, 1H), 4.40–4.25 (m, 3H), 4.23–4.19 (m, 1H), 4.11–4.00 (m, 2H), 3.93 (dd, J = 16.6, 4.0 Hz, 1H), 3.77 (dd, J = 16.8, 4.7 Hz, 1H), 2.97–2.82 (m, 4H), 2.36 (t, J = 7.1 Hz, 2H), 2.18–2.11 (m, 1H), 2.01–1.84 (m, 2H), 1.84–1.60 (m, 6H), 1.53–1.43 (m, 2H), 1.40 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 6.0 Hz, 3H), 0.93 (d, J = 6.1 Hz, 3H).¹³C NMR (126 MHz, CD₃OD, 25°C): δ 177.9, 177.6, 175.7, 175.5, 174.5, 174.4, 173.7, 173.6, 172.1, 62.7, 55.2, 54.5, 54.5, 50.9, 50.3, 44.1, 40.7, 40.6, 36.4, 32.3, 32.0, 28.0, 27.9, 25.9, 23.6, 23.3, 21.9, 18.1. FTIR (thin film) cm^-1: 3299 (br), 39 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 2922 (w), 1644 (s), 1536 (m), 1431 (m), 1182 (s), 1133 (s), 835 (m), 798 (m), 723 (m). HRMS (ESI) (m/z): calc’d for C28H50N9O11 [M+H]⁺: 688.3624, found: 688.3626. HPLC analysis: Method B: 0→40% MeCN

[00179] Example 14: Macrocyclization and linearization of N-terminal iodoacetamide containing peptides (FIG.1) [00180] Concentration study for PGA 13-membered macrocycle 7b (FIG.1A) [00181] Iodoacetamide capped PGA 7a: HRMS (ESI) (m/z): calc’d for C12H20IN4O5 [M+H]⁺: 427.0473, found: 427.0473. [00182] PGA macrocyclization Table 3. Macrocyclization screen for iodoacetamide N-terminal PGA 7a forming 13- membered macrocycle 7b with peptide concentration reduced from 2 mM to 0.5 mM to achieve optimum monomer formation (Product yield estimated by HPLC analysis at 220 nm wavelength comparing AUC of peptidic peaks. PBS: phosphate buffered saline, MOPS: 3-(N- morpholino)propanesulfonic acid).

40 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00183] Macrocyclization was carried out using General Macrocyclization Procedure B with varying peptide concentration of A – 2 mM, B – 1 mM, C – 0.5 mM, D – 1 mM in PBS pH 8. The peak present at t_R = 7.67 has a mass corresponding to the dimer (proposed structure S1). Formation of this peak was avoided by decreasing the reaction concentration. The peak present at tR = 6.24 (condition D) had a mass corresponding to the completely hydrolyzed peptide (proposed structure S2). [00184] Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. The reaction was analyzed every 24 h until complete consumption of starting material peptide. [00185] HPLC Analysis t = 0 h – Pure 7a

Entry 4: t = 72 h, 1 mM peptide 7a in PBS, pH 8, 38°C 41 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00186] 13-membered macrocycle (FIG.1B) [00187] PGA (7a–c): Macrocyclization

[00188] Macrocyclization of peptide 7a (0.5 mg, 1.41 µmol) was carried out in 25 mM MOPS buffer (2.8 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00189] Macrocycle 7b HRMS (ESI) (m/z): calc’d for C12H19N4O5 [M+H]⁺: 299.1350, found: 299.1349. [00190] HPLC Analysis t = 0 h

t = 48 h

[00191] PGA (7a–c): Linearization

[00192] Linearization of macrocycle 7b (1.1 mg, 3.7 µmol) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in 42 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO tetrahydrofuran, 0.20 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00193] Linear peptide 7c HRMS (ESI) (m/z): calc’d for C₁₂H₂₀N₄O₅ [M+H]⁺: 301.1506, found: 301.1505. HPLC Analysis

[00194] 16–25-membered macrocycles (FIG.1B) [00195] QYKA (8a–c): Macrocyclization

[00196] Macrocyclization of peptide 8a (0.6 mg, 0.87 µmol) (SEQ ID NO: 11) was carried out in 25 mM MOPS buffer (0.87 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00197] Iodoacetamide capped peptide 8a HRMS (ESI) (m/z) (SEQ ID NO: 11): calc’d for C₂₂H₃₁IN₅O₉ [M+H]⁺: 636.1161, found: 636.1163. [00198] Macrocycle 8b (SEQ ID NO: 12) HRMS (ESI) (m/z): calc’d for C25H38N7O8 [M+H]⁺: 564.2776, found: 564.2778. [00199] HPLC Analysis t = 0 h – Pure 8a

t = 48 h

43 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00200] QYKA (8a–c): Linearization

[00201] Linearization of macrocycle 8b (1.2 mg, 2.1 µmol) (SEQ ID NO: 12) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.30 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00202] Linear peptide 8c (SEQ ID NO: 13) HRMS (ESI) (m/z): calc’d for C₂₅H₄₀N₇O₈ [M+H]⁺: 566.2933, found: 566.2934. HPLC Analysis

[00203] TDFA (9a–c): SPPS

[00204] SPPS was performed using procedures reported above on 15 μmol scale. Crude peptide was purified using automated flash column chromatography (gradient 0→30% MeCN/H2O+0.1% TFA, 8 CV) to give iodoacetamide capped peptide 9a (8 mg, 87%) (SEQ ID NO: 14).¹H NMR (500 MHz, CD3OD, 25°C): δ 8.39 (d, J = 7.8 Hz, 0.4H, NH), 8.29 (d, J = 7.4 Hz, 0.6H, NH), 8.06 (d, J = 7.6 Hz, 0.8H, NH), 7.88 (d, J = 7.3 Hz, 0.5H, NH), 7.32– 44 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 7.26 (m, 2H), 7.25–7.17 (m, 3H), 4.69–4.61 (m, 1H), 4.57–4.49 (m, 1H), 4.38–4.24 (m, 2H), 4.20–4.16 (m, 1H), 3.91 (d, J = 10.1 Hz, 1H), 3.77 (d, J = 10.1 Hz, 1H), 3.19 (dd, J = 14.1, 5.0 Hz, 1H), 2.95 (dd, J = 14.1, 9.1 Hz, 1H), 2.81 (dd, J = 17.1, 6.1 Hz, 1H), 2.67 (dd, J = 17.1, 6.9 Hz, 1H), 1.35 (d, J = 7.2 Hz, 3H), 1.16 (d, J = 6.4 Hz, 3H).¹³C NMR (126 MHz, CD3OD, 25°C): δ: 174.1, 173.0, 172.8, 172.3, 171.7, 171.4, 138.3, 130.3, 129.6, 127.8, 68.5, 60.3, 56.5, 51.6, 49.0 (CD₃OD overlapping with 1C), 38.1, 36.1, 19.8, 18.0, -2.2. FTIR (thin film) cm^-1: 3299 (br), 2926 (m), 2855 (w), 1655 (s), 1536 (m), 1454 (w), 1402 (w), 1204 (m), 1137 (w). HRMS (ESI) (m/z): calc’d for C22H31IN5O9 [M+H]⁺: 636.1161, found: 636.1163. [00205] TDFA (9a–c): Macrocyclization

[00206] Macrocyclization of peptide 9a (6 mg, 9.45 µmol) (SEQ ID NO: 14) was carried out in 25 mM MOPS buffer (9.5 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. After complete reaction, the product was purified using automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 8 CV, gradient 0→35% MeCN/H₂O+0.1% TFA, 12 CV) to give macrocycle 9b (4 mg, 83%) (SEQ ID NO: 15).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.72 (d, J = 6.9 Hz, 0.5H, NH), 8.43 (t, J = 9.9 Hz, 0.3H, NH), 8.20 (d, J = 7.2 Hz, 0.3H, NH), 7.94 (d, J = 7.7 Hz, 0.6H, NH), 7.31–7.25 (m, 2H, 16), 7.25–7.19 (m, 3H, 15, 17), 4.55 (d, J = 15.7 Hz, 1H, 1), 4.37–4.33 (m, 2H, 3, 18), 4.24 (d, J = 15.7 Hz, 1H, 1), 4.18–4.13 (m, 2H, 7, 11), 4.12– 4.04 (m, 1H, 5), 3.29–3.21 (m, 2H, 13), 2.87 (d, J = 6.9 Hz, 2H, 9), 1.35 (d, J = 6.9 Hz, 3H, 20), 1.20 (d, J = 6.3 Hz, 3H, 6). ¹³C NMR (126 MHz, CD3OD, 25°C): δ: 174.0 (10), 173.0, 173.0, 172.9, 172.9 (overlapping 2, 4, 8, 12), 171.3 (19), 139.1 (14), 130.2 (15), 129.6 (16), 127.8 (17), 75.4 (1), 68.1 (5), 60.1 (3), 59.4 (11), 53.4 (7), 48.7 (CD3OD overlapping with 18*), 36.7 (13), 35.3 (9), 20.2 (6), 17.5 (20). FTIR (thin film) cm^-1: 3288 (br), 2926 (w), 2855 (w), 1681 (s), 1603 (m), 1549 (w), 1443 (w), 1208 (m), 1141 (m), 842 (w), 805 (w), 723 (w). HRMS (ESI) (m/z): calc’d for C₂₂H₃₀N₅O₉ [M+H]⁺: 508.2038, found: 508.2040. [00207] HPLC analysis 45 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO t = 0 h – Pure 9a

t = 48 h

[00208] TDFA (9a–c): Linearization

[00209] Linearization of macrocycle 9b (0.5 mg, 0.99 µmol) (SEQ ID NO: 15) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.7 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00210] Linear peptide 9c (SEQ ID NO: 16) HRMS (ESI) (m/z): calc’d for C₂₂H₃₂N₅O₉ [M+H]⁺: 510.2195, found: 510.2197. HPLC Analysis

[00211] YSAWGF (10a–c): Macrocyclization 46 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00212] Macrocyclization of peptide 10a (4.6 mg, 5.04 µmol) (SEQ ID NO: 17) was carried out in 25 mM MOPS buffer (5.0 mL) using General Macrocyclization Procedure B. Reaction analysis at 0 h was carried out using Method D: 0→95% MeCN, t = 48 h reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. Purification was carried out by prep HPLC (gradient, 5→40% MeCN/H₂O+0.1% TFA) to give macrocycle 10b (3.2 mg, 81%) (SEQ ID NO: 18).¹H NMR (500 MHz, CD3OD, 25°C): δ 9.09 (d, J = 8.6 Hz, 0.5H, NH), 8.40 (d, J = 4.3 Hz, 0.4H, NH), 8.02 (d, J = 6.6 Hz, 0.6H, NH), 7.78 (d, J = 8.1 Hz, 0.6H, NH), 7.74 (t, J = 5.9 Hz, 0.7H, NH), 7.70 (d, J = 7.7 Hz, 0.6H, NH), 7.61 (d, J = 7.9 Hz, 1H, 21), 7.34– 7.31 (m, 3H, 24, 33), 7.28–7.20 (m, 3H, 34, 35), 7.15–6.95 (m, 5H, 7, 22, 23, 26), 6.69 (d, J = 8.4 Hz, 2H, 8), 4.72–4.59 (m, 2H, 3, 16), 4.48–4.45 (m, 1H, 10), 4.43–4.34 (m, 2H, 1, 29), 4.16–4.01 (m, 3H, 1, 13, 27), 3.83–3.76 (m, 2H, 12), 3.67 (dd, J = 16.8, 5.1 Hz, 1H, 1), 3.49 (dd, J = 15.1, 4.5 Hz, 1H, 18), 3.22 (dd, J = 15.0, 10.4 Hz, 1H, 18), 3.09–3.04 (m, 2H, 31), 3.03–2.84 (m, 2H, 5), 1.13 (d, J = 7.3 Hz, 3H, 15). ¹³C NMR (126 MHz, CD3OD, 25°C): δ 175.1 (14), 174.4 (17), 173.8 (4), 173.5 (11), 171.0 (2), 170.9 (28), 170.6 (30), 160.1 (q, J = 40 Hz, TFA), 157.5 (9), 138.1 (25), 137.5 (32), 131.4 (7), 130.5 (34), 129.7 (33), 128.7 (6), 128.6 (20), 128.1 (35), 123.9 (26), 122.6 (23), 120.0 (22), 119.3 (21), 116.3 (8), 112.3 (24), 111.4 (19), 77.2 (1), 63.4 (12), 57.0 (10), 56.0 (3), 55.6 (16), 53.8 (29), 52.1 (13), 43.7 (27), 38.6 (31), 37.7 (5), 28.2 (18), 16.9 (15). FTIR (thin film) cm^-1: 3332 (br), 2930 (w), 1670 (s), 1517 (m), 1454 (m), 1208 (m), 1141 (m), 842 (w), 805 (w). Macrocycle 10b (SEQ ID NO: 18) HRMS (ESI) (m/z): calc’d for C39H45N8O10 [M+H]⁺: 785.3253, found: 785.3257. Iodoacetamide capped peptide 10a (SEQ ID NO: 17) HRMS (ESI) (m/z): calc’d for C39H46IN8O10 [M+H]⁺: 913.2376, found: 913.2380 [00213] HPLC analysis t = 0 h – Pure 10a 47 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

t = 48 h

[00214] YSAWGF (10a–c): Linearization

[00215] Linearization of macrocycle 10b (1.0 mg, 12.8 µmol) (SEQ ID NO: 18) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.20 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. [00216] Linear peptide 10c (SEQ ID NO: 19) HRMS (ESI) (m/z): calc’d for C39H47N8O10 [M+H]⁺: 787.3410, found: 787.3415. HPLC Analysis

[00217] VQYKA (11a-c): Macrocyclization 48 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00218] Macrocyclization of peptide 11a (0.5 mg, 0.63 µmol) (SEQ ID NO: 20) was carried out in 25 mM MOPS (0.63 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00219] Iodoacetamide capped peptide 11a (SEQ ID NO: 20) HRMS (ESI) (m/z): calc’d for C30H48IN8O9 [M+H]⁺: 791.2583, found: 791.2585. Macrocycle 11b (SEQ ID NO: 21) HRMS (ESI) (m/z): calc’d for C₃₀H₄₇N₈O₉ [M+H]⁺: 663.3461, found: 663.3463. [00220] HPLC Analysis t = 0 h – Pure 11a

t = 48 h

[00221] VQYKA (11a-c): Linearization

[00222] Linearization of macrocycle 11b (1.0 mg, 1.5 µmol) (SEQ ID NO: 21) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 49 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO M in tetrahydrofuran, 0.25 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00223] Linear peptide 11c (SEQ ID NO: 22) HRMS (ESI) (m/z): calc’d for C₃₀H₄₉N₈O₉ [M+H]⁺: 665.3617, found: 665.3619. HPLC Analysis

[00224] AKLGFA (12a-c): Macrocyclization

[00225] Macrocyclization of peptide 12a (0.6 mg, 0.76 µmol) (SEQ ID NO: 23) was carried out in 25 mM MOPS buffer (0.76 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00226] Iodoacetamide capped peptide 12a (SEQ ID NO: 23) HRMS (ESI) (m/z): calc’d for C31H50IN8O8 [M+H]⁺: 789.2791, found: 789.2794. Macrocycle 12b (SEQ ID NO: 24) HRMS (ESI) (m/z): calc’d for C₃₁H₄₉N₈O₈ [M+H]⁺: 661.3668, found: 661.3671. [00227] HPLC Analysis t = 0 h – Pure 12a

[00228] AKLGFA (12a-c): Linearization 50 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00229] Linearization of macrocycle 12b (1.0 mg, 1.52 µmol) (SEQ ID NO: 24) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.25 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00230] Linear peptide 12c (SEQ ID NO: 25) HRMS (ESI) (m/z): calc’d for C31H51N8O8 [M+H]⁺: 663.3824, found: 663.3827. HPLC Analysis

[00231] GYSAWGF (13a-c): Macrocyclization

[00232] Macrocyclization of peptide 13a (5.0 mg, 5.16 µmol) (SEQ ID NO: 26) was carried out in 25 mM MOPS buffer (5.16 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00233] Iodoacetamide capped peptide 13a (SEQ ID NO: 26) HRMS (ESI) (m/z): calc’d for C41H49IN9O11 [M+H]⁺: 970.2591, found: 970.2594. Macrocycle 13b (SEQ ID NO: 27) HRMS (ESI) (m/z): calc’d for C₄₁H₄₈N₉O₁₁ [M+H]⁺: 842.3468, found: 842.3472. [00234] HPLC Analysis t = 0 h – Pure 13a 51 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

t = 24 h

[00235] GYSAWGF (13a-c): Linearization

[00236] Samarium(II) iodide (0.1 M in tetrahydrofuran, 0.6 mL) was added dropwise to a solution of macrocycle 13b (4 mg, 4.76 µmol) (SEQ ID NO: 27) in a degassed mixture of tetrahydrofuran/methanol (3/1 v/v, 0.3 mL) until a deep blue solution was formed. The reaction mixture was then stirred under N₂ at room temperature for 1 h before analyzing with HPLC (HPLC Analytical Method B: 0→40% MeCN). The reaction was then quenched by stirring in air for 1 h and concentrated under a N₂ stream. The crude residue was purified by automated flash column chromatography (isocratic 100% H₂O+0.1% TFA (5 CV), then gradient 0→40% MeCN/H2O+0.1% TFA (8 CV)) to give the linear peptide 13c (2 mg, 50% yield) (SEQ ID NO: 28).¹H NMR (500 MHz, CD3OD, 25°C): δ 8.27 (d, J = 5.7 Hz, 0.2H, NH), 8.12 (t, J = 8.0 Hz, 0.2H, NH), 8.06 (d, J = 7.1 Hz, 0.1H, NH), 7.99–7.91 (m, 0.3H, NH), 7.88 (d, J = 7.1 Hz, 0.1H, NH), 7.57 (d, J = 7.9 Hz, 1H, 27), 7.33 (d, J = 8.1 Hz, 1H, 24), 7.27–7.22 (m, 4H, 35, 36), 7.19–7.16 (m, 1H, 37), 7.12 (s, 1H, 22), 7.08 (t, J = 7.9 Hz, 1H, 25), 7.05–6.96 (m, 3H, 9, 26), 52 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 6.67 (d, J = 8.5 Hz, 2H, 10), 4.60 (dd, J = 9.4, 5.1 Hz, 1H, 31), 4.56–4.51 (m, 2H, 5, 18), 4.40 (t, J = 5.3 Hz, 1H, 12), 4.18 (q, J = 7.2 Hz, 1H, 15), 4.00 (s, 2H, 1), 3.95–3.80 (m, 4H, 3, 14, 29), 3.76 (dd, J = 11.2, 5.4 Hz, 1H, 14), 3.61 (d, J = 16.6 Hz, 1H, 29), 3.40–3.35 (m, 1H, 20), 3.20 (dd, J = 13.9, 5.1 Hz, 1H, 33), 3.12 (dd, J = 14.8, 9.5 Hz, 1H, 20), 3.04 (dd, J = 14.1, 5.6 Hz, 1H, 7), 2.94 (dd, J = 14.0, 9.4 Hz, 1H, 33), 2.84 (dd, J = 14.0, 8.6 Hz, 1H, 7), 1.20 (d, J = 7.2 Hz, 3H, 17). ¹³C NMR (126 MHz, CD₃OD, 25°C): δ 176.2 (32), 176.0 (2), 175.4 (16), 174.8 (19), 173.9 (6), 173.1 (13), 171.7 (4), 171.5 (30), 157.4 (11), 138.6 (34), 138.1 (23), 131.3 (9), 130.3 (35), 129.5 (36), 128.7 (8), 128.6 (28), 127.7 (37), 124.5 (22), 122.5 (25), 119.9 (26), 119.2 (27), 116.3 (10), 112.3 (24), 111.1 (21), 63.2 (14), 62.6 (1), 56.9 (12), 56.8 (5), 56.4 (18), 56.1 (31), 51.6 (15), 43.8 (29), 43.1 (3), 38.7 (33), 37.7 (7), 28.1 (20), 17.0 (17). FTIR (thin film) cm^-1: 3314 (br), 2926 (w), 2855 (w), 1655 (s), 1517 (m), 1457 (m), 1338 (w), 1204 (m), 1137 (w), 1081 (w), 835 (w). HRMS (ESI) (m/z): calc’d for C41H50N9O11 [M+H]⁺: 844.3624, found: 844.3629. HPLC analysis

[00237] Macrocycles featuring non-canonical amino acids (FIG.1C) [00238] D-f-mV-R (14a-c): SPPS

[00239] SPPS was performed using procedures reported above on 36 μmol scale. Crude peptide was purified using automated flash column chromatography (gradient 0→30% MeCN/H₂O+0.1% TFA, 8 CV) to give iodoacetamide capped peptide 14a (14 mg, 53%) (SEQ ID NO: 29).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.24 (d, J = 7.5 Hz, 0.7H, NH), 7.79 (d, J = 8.1 Hz, 0.6H, NH), 7.37 (t, J = 5.7 Hz, 0.4H, NH), 7.32–7.19 (m, 5H), 5.09 (q, J = 7.5 Hz, 1H), 4.66 (dd, J = 7.8, 5.7 Hz, 1H), 4.54 (d, J = 10.8 Hz, 1H), 4.27 (q, J = 7.4 Hz, 1H), 3.82– 3.69 (m, 2H), 3.24–3.17 (m, 2H), 3.08 (dd, J = 13.6, 7.6 Hz, 1H), 3.05–2.92 (m, 4H), 2.66 (dd, J = 16.9, 5.8 Hz, 1H), 2.57 (dd, J = 16.9, 7.8 Hz, 1H), 2.18–2.07 (m, 1H), 1.82–1.69 (m, 2H), 1.66–1.50 (m, 2H), 0.90 (d, J = 6.4 Hz, 3H), 0.56 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD, 25°C): δ 174.4, 173.6, 172.3, 171.6, 171.3, 170.3, 161.4 (d, J = 37 Hz, TFA), 158.5, 53 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 137.6, 130.5, 129.7, 128.1, 64.0, 52.7, 51.6, 51.5, 42.0, 39.0, 36.7, 31.7, 30.4, 27.6, 26.3, 20.1, 19.2, –2.2. FTIR (thin film) cm^-1: 3280 (br), 2926 (m), 2855 (m), 1670 (s), 1536 (m), 1379 (m), 1204 (s), 1137 (m), 1081 (w), 839 (w), 801 (w), 723 (w). HRMS (ESI) (m/z): calc’d for C27H41IN8O8 [M+H]⁺: 733.2165, found: 733.2168. [00240] D-f-mV-R (14a-c): Macrocyclization

[00241] Macrocyclization of peptide 14a (9.1 mg, 12.4 µmol) (SEQ ID NO: 29) was carried out in 25 mM MOPS buffer (12.4 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. After complete reaction, the product was purified using automated flash column chromatography (isocratic 100% H2O+0.1% TFA, 8 CV, gradient 0→40% MeCN/H2O+0.1% TFA, 12 CV) to give macrocycle 14b (6.5 mg, 87%) (SEQ ID NO: 30).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.69 (d, J = 6.4 Hz, 0.2H, NH), 7.62 (d, J = 7.7 Hz, 0.1H, NH), 7.43 (t, J = 5.5 Hz, 0.2H, NH), 7.28– 7.25 (m, 4H, 11, 12), 7.23–7.18 (m, 1H, 13), 4.98 (dd, J = 10.3, 5.4 Hz, 1H, 7), 4.86–4.85 (m, 1H, 3, overlapping with H₂O), 4.58 (d, J = 9.6 Hz, 1H, 15), 4.45 (d, J = 15.9 Hz, 1H, 1), 4.25 (d, J = 15.9 Hz, 1H, 1), 4.15 (dd, J = 8.3, 6.0 Hz, 1H, 20), 4.07 (dd, J = 13.2, 3.5 Hz, 2H, MOPS), 3.75 (t, J = 12.5 Hz, 2H, MOPS), 3.55 (d, J = 12.4 Hz, 2H, MOPS), 3.35 (t, J = 7.2 Hz, 2H, MOPS), 3.25–2.99 (m, 6H, 9, 24, MOPS), 2.97 (t, J = 6.5 Hz, 2H, MOPS), 2.89–2.84 (m, 4H, 5, 14), 2.69 (dd, J = 16.5, 7.2 Hz, 1H, 5), 2.24–2.19 (m, 2H, MOPS), 2.09–1.89 (m, 2H, 17, 22), 1.89–1.81 (m, 1H, 22), 1.67–1.49 (m, 2H, 23), 0.92 (d, J = 6.5 Hz, 3H, 18/19), 0.45 (d, J = 6.7 Hz, 3H, 18/19).¹³C NMR (126 MHz, CD3OD, 25°C): δ 174.8 (8), 173.5 (6), 172.7 (4), 172.3 (16), 170.8 (overlapping 2C, 2, 21), 161.2 (q, J = 38 Hz, TFA), 158.6 (25), 137.6 (9), 130.5 (11), 129.7 (12), 128.1 (13), 75.5 (1), 65.2 (MOPS), 64.7 (15), 58.0 (MOPS), 53.3 (MOPS), 52.9 (7), 52.7 (20), 50.3 (3), 49.8 (MOPS), 41.9 (24), 38.4 (9), 36.5 (5), 32.3 (14), 29.1 (22), 28.0 (17), 26.5 (23), 20.8 (18/19), 20.5 (MOPS), 20.0 (18/19). FTIR (thin film) 54 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO cm^-1: 3403 (br), 3001 (w), 2915 (w), 1662 (m), 1435 (m), 1409 (m), 1312 (m), 1018 (s), 954 (s), 701 (m), 671 (m). HRMS (ESI) (m/z): calc’d for C27H41IN8O8 [M+H]⁺: 733.2165, found: 733.2168. [00242] HPLC analysis t = 0 h – Pure 14a

t = 24 h

[00243] D-f-mV-R (14a-c): Linearization

[00244] Linearization of macrocycle 14b (0.5 mg, 0.83 µmol) (SEQ ID NO: 30) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.20 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00245] Linear peptide 14c (SEQ ID NO: 31) HRMS (ESI) (m/z): calc’d for C₂₇H₄₃N₈O₈ [M+H]⁺: 607.3198, found: 607.3202. HPLC Analysis

[00246] Pip-G-ArCH2-GIE (15a-c): Macrocyclization 55 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00247] Macrocyclization of peptide 15a (2.1 mg, 2.36 µmol) (SEQ ID NO: 32) was carried out in 25 mM MOPS buffer (2.36 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. Analysis by HPLC, 254 nm wavelength. [00248] Iodoacetamide capped peptide 15a (SEQ ID NO: 32) HRMS (ESI) (m/z): calc’d for C₃₈H₅₁IN₇O₁₀ [M+H]⁺: 892.2737, found: 892.2740. Macrocycle 15b (SEQ ID NO: 33) HRMS (ESI) (m/z): calc’d for C38H50N7O10 [M+H]⁺: 764.3614, found: 764.3616. [00249] HPLC Analysis t = 0 h – Pure 15a

t = 48 h

[00251] Linearization of macrocycle 15b (0.8 mg, 1.05 µmol) (SEQ ID NO: 33) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 56 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO M in tetrahydrofuran, 0.30 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. [00252] Linear peptide 15c (SEQ ID NO: 34) HRMS (ESI) (m/z): calc’d for C₃₈H₅₂N₇O₁₀ [M+H]⁺: 766.3770, found: 766.3773. HPLC Analysis

[00253] DAA-f-mV-R (16a-c): Macrocyclization

[00254] Macrocyclization of peptide 16a (5.6 mg, 6.4 µmol) (SEQ ID NO: 35) was carried out in 25 mM MOPS buffer (6.4 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00255] Iodoacetamide capped peptide 16a HRMS (ESI) (m/z): calc’d for C33H52IN10O10 [M+H]⁺: 875.2907, found: 875.2911. Macrocycle 16b (SEQ ID NO: 36) HRMS (ESI) (m/z): calc’d for C33H51N10O10 [M+H]⁺: 747.3784, found: 747.3787. [00256] HPLC Analysis t = 0 h – Pure 16a

t = 48 h

[00257] DAA-f-mV-R (16a-c): Linearization 57 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00258] Linearization of macrocycle 16b (1.0 mg, 1.34 µmol) (SEQ ID NO: 36) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.60 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. [00259] Linear peptide 16c (SEQ ID NO: 37) HRMS (ESI) (m/z): calc’d for C₃₃H₅₃N₁₀O₁₀ [M+H]⁺: 749.3941, found: 749.3944. HPLC Analysis

[00260] SSAGSLF-Asp* (17a-c): Macrocyclization (* the Asp is coupled via its sidechain)

[00261] Macrocyclization of peptide 17a (5.6 mg, 5.80 µmol) (SEQ ID NO: 38) was carried out in 25 mM MOPS buffer (5.8 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00262] Iodoacetamide capped peptide 17a (SEQ ID NO: 38) HRMS (ESI) (m/z): calc’d for C35H53IN9O15 [M+H]⁺: 966.2700, found: 966.2704. Macrocycle 17b (SEQ ID NO: 39) HRMS (ESI) (m/z): calc’d for C₃₅H₅₂N₉O₁₅ [M+H]⁺: 838.3577, found: 838.3582. [00263] HPLC Analysis t = 0 h – Pure 17a

58 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO t = 45 h

[00264] SSAGSLF-Asp* (17a-c): Linearization (* the Asp is coupled via its sidechain)

[00265] Linearization of macrocycle 17b (1.0 mg, 1.19 µmol) (SEQ ID NO: 39) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.20 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. [00266] Linear peptide 17c (SEQ ID NO: 40) HRMS (ESI) (m/z): calc’d for C₃₅H₅₄N₉O₁₅ [M+H]⁺: 840.3734, found: 840.3737. HPLC Analysis

[00267] 40+-membered macrocycles (FIG.1C) [00268] EFPSAGDQLGKA (18a-c): Macrocyclization

59 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00269] Macrocyclization of peptide 18a (3.8 mg, 2.71 µmol) (SEQ ID NO: 41) was carried out in 25 mM MOPS buffer (2.71 mL) using General Macrocyclization Procedure B. Macrocyclization of peptide 18a (0.4 mg, 0.29 µmol) (SEQ ID NO: 41) was also carried out in 50 mM MOPS buffer (0.29 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00270] Iodoacetamide capped peptide 18a (SEQ ID NO: 41) HRMS (ESI) (m/z): calc’d for C₅₅H₈₅IN₁₅O₂₀ [M+H]⁺: 1402.5134, found: 1402.5140. Macrocycle 18b (SEQ ID NO: 42) HRMS (ESI) (m/z): calc’d for C55H84N15O20 [M+H]⁺: 1274.6012, found: 1274.6019. [00271] HPLC Analysis t = 0 h – Pure 18a

t = 50 h; 25 mM MOPS, pH 8, 38°C

t = 48 h, 50 mM MOPS, pH 8, 38°C

60 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00272] EFPSAGDQLGKA (18a-c): Linearization

[00273] Linearization of macrocycle 18b (1.0 mg, 0.79 µmol) (SEQ ID NO: 42) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 1.0 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. [00274] Linear peptide 18c (SEQ ID NO: 43) HRMS (ESI) (m/z): calc’d for C55H86N15O20 [M+H]⁺: 1276.6168, found: 1276.6178. HPLC Analysis

[00275] ADLAGKQFRGEPSV (19a-c): Macrocyclization

61 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00276] Macrocyclization of peptide 19a (3.8 mg, 2.29 µmol) (SEQ ID NO: 44) was carried out in 25 mM MOPS buffer (2.29 mL) using General Macrocyclization Procedure B. Macrocyclization of peptide 19a (0.4 mg, 0.24 µmol) (SEQ ID NO: 44) was also carried out in 50 mM MOPS buffer (0.24 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00277] Iodoacetamide capped peptide 19a (SEQ ID NO: 44) HRMS (ESI) (m/z): calc’d for C₆₆H₁₀₇IN₂₀O₂₂ [M+2H]²⁺: 829.3451, found: 829.3455. Macrocycle 19b (SEQ ID NO: 45) HRMS (ESI) (m/z): calc’d for C66H105N20O22 [M+H]⁺: 1529.7707, found: 1529.7712. [00278] HPLC Analysis t = 0 h – Pure 19a

t = 5 days; 25 mM MOPS, pH 8, 38°C

62 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00279] ADLAGKQFRGEPSV (19a-c): Linearization

[00280] Linearization of macrocycle 19b (1.0 mg, 0.65 µmol) (SEQ ID NO: 45) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.35 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. [00281] Linear peptide 19c (SEQ ID NO: 46) HRMS (ESI) (m/z): calc’d for C66H107N20O22 [M+H]⁺: 1531.7863, found: 1531.7875. HPLC Analysis

[00282] Example 15: Peptide structural validation [00283] Cyclization structural validation through HATU cyclization of S4 (FIG.4) 63 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00284] Fmoc-glycine-2-chlorotrityl resin (0.493 mmol/g) was swelled in N,N- dimethylformamide for 1 h before use. SPPS conditions used as described above. The hydroxamate moiety was incorporated using alternative amide coupling conditions: (Boc- aminooxy)-acetic acid (5.1 equiv), N,N’-diisopropylcarbodiimide (DIC, 5 equiv) in N,N- dimethylformamide (0.25 M) for 1 h at room temperature. Complete coupling was confirmed through cleavage of ca. 5 mg resin and analysis by HPLC-MS. The final Fmoc-Phe-OH coupling was repeated twice for 10 h at room temperature to ensure complete coupling. Finally, the resin was cleaved using 5% TFA/dichloromethane (5 mL/g resin) for 1 h at room temperature before the cleavage solution was collected, the resin was washed thrice with dichloromethane and the combined solutions were concentrated under a N₂ stream. The crude residue was purified by automated flash column chromatography (gradient 0→80% MeCN/H2O+0.1% TFA, 10 CV) to give protected linear peptide S4 (9.0 mg, 12%) (SEQ ID NO: 47).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.07 (d, J = 8.2 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.52 (s, 1H), 7.33–7.16 (m, 9H), 6.91 (d, J = 8.1 Hz, 2H), 4.74 (dd, J = 8.1, 5.7 Hz, 1H), 4.61 (dd, J = 9.3, 5.1 Hz, 1H), 4.35–4.26 (m, 2H), 4.19–4.07 (m, 2H), 3.98–3.86 (m, 3H), 3.62– 3.60 (m, 1H), 3.54–3.51 (m, 1H), 3.35 (m, 1H), 3.18–3.09 (m, 4H), 2.93 (dd, J = 14.1, 9.2 Hz, 1H), 1.65 (s, 9H), 1.29 (br s, 12H), 1.12 (s, 9H).¹³C NMR (126 MHz, CD₃OD, 25°C): δ: 174.5, 173.9, 173.6, 172.5, 172.1, 170.7, 167.3, 155.6, 151.1, 136.8, 135.2, 133.0, 131.8, 131.0, 130.5, 130.2, 129.0, 125.5, 125.3, 125.2, 123.7, 120.1, 117.4, 116.1, 84.8, 79.5, 76.2, 75.1, 62.3, 56.3, 64 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 55.7, 54.5, 53.7, 50.8, 41.9, 38.3, 37.9, 29.2, 28.4, 28.4, 27.6, 18.2. FTIR (thin film) cm^-1: 3284 (br), 2978 (w), 2930 (w), 1677 (s), 1633 (s), 1525 (m), 1454 (m), 1372 (m), 1256 (w), 1208 (m), 1159 (m), 1088 (w), 842 (w). HRMS (ESI) (m/z): calc’d for C₅₂H₇₁N₈O₁₃ [M+H]⁺: 1015.5135, found: 1015.5138.

[00285] HATU (4.6 mg, 1.5 equiv, 12.1 mol) and N,N-diisopropylethylamine (4.2 μL, 3 equiv, 24.2 μmol) were sequentially added to the linear peptide S4 (9 mg, 1.0 equiv, 8.1 μmol) (SEQ ID NO: 47) in anhydrous N,N-dimethylformamide (8.0 mL) under a N2 atmosphere. The reaction mixture was stirred at room temperature for 5 h before concentrating under vacuum. The crude residue was purified by automated flash column chromatography (gradient 5→50% MeCN/H2O+0.1% TFA (2 CV), gradient 50→100% MeCN/H2O+0.1% TFA (8 CV)). The residue was then dissolved in a cleavage solution of TFA/TIPS/H2O/dichloromethane (0.5 mL, 92.5/2.5/2.5/2.5, v/v/v/v) and warmed to 35°C for 2 h before being concentrated under a N₂ stream and purified by automated flash column chromatography (gradient 0→55% MeCN/H2O+0.1% TFA, 10 CV) to give macrocycle 10b2 (3.0 mg, 48%) (SEQ ID NO: 48). ¹H NMR (500 MHz, CD₃OD, 25°C): δ 9.09 (d, J = 8.7 Hz, 0.8H, NH), 8.39 (d, J = 4.3 Hz, 0.8H, NH), 8.03 (d, J = 6.8 Hz, 0.9H, NH), 7.78 (d, J = 8.1 Hz, 1H, NH), 7.74 (t, J = 5.9 Hz, 0.9H, NH), 7.70 (d, J = 7.6 Hz, 0.6H, NH), 7.61 (d, J = 7.8 Hz, 1H), 7.34–7.31 (m, 3H), 7.27– 7.20 (m, 3H), 7.12–7.98 (m, 5H), 6.69 (d, J = 8.5 Hz, 2H), 4.71–4.64 (m, 1H), 4.64–4.61 (m, 1H), 4.46 (dd, J = 11.8, 5.2 Hz, 1H), 4.43–4.34 (m, 2H), 4.15–4.02 (m, 3H), 3.83–3.76 (m, 65 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 2H), 3.67 (dd, J = 16.9, 5.3 Hz, 1H), 3.49 (dd, J = 15.0, 4.4 Hz, 1H), 3.22 (dd, J = 15.0, 10.5 Hz, 1H), 3.10–3.03 (m, 2H), 3.01–2.97 (m, 1H), 2.95–2.89 (m, 1H), 1.13 (d, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD, 25°C): δ 175.1, 174.4, 173.8, 173.5, 171.0, 170.9, 170.6, 157.5, 138.2, 137.5, 131.4, 130.5, 129.7, 128.7, 128.6, 128.1, 123.9, 122.6, 120.0, 119.3, 116.3, 112.3, 111.4, 77.2, 63.5, 57.0, 56.0, 55.6, 53.8, 52.1, 43.7, 38.6, 37.7, 28.2, 16.9. FTIR (thin film) cm- ¹: 3347 (br), 2926 (w), 1662 (s), 1517 (m), 1457 (m), 1208 (m), 1141 (m), 1070 (w), 842 (w), 805 (w). HRMS (ESI) (m/z): calc’d for C₃₉H₄₅N₈O₁₀ [M+H]⁺: 785.3253, found: 785.3256. ¹HNMR and ¹³CNMR comparison between this synthesized peptide 10b2 (SEQ ID NO: 48), and macrocyclic peptide 10b (SEQ ID NO: 18) generated through cyclization via the hydroxamic acid confirmed that the structure of the macrocyclic peptide is linked via the OH moiety of the hydroxamic acid. [00286] Example 16: Linearization structural validation through glycolic acid capping (FIG.

[00287] Rink amide resin (0.51 mmol/g) was swelled in N,N-dimethylformamide (10 mL/g resin) for 1 h before use. SPPS conditions were used as described above with coupling times of 1 h. [00288] N-terminal capping: a vial was sequentially charged with glycolic acid (5.0 equiv) and HATU (5.0 equiv). N,N-Dimethylformamide (0.1 M) was then added followed by N,N- diisopropylethylamine (10 equiv). This solution was added to the fritted syringe containing resin (1 equiv). The resin suspension was rotated end over end on a rotator for 2 h at room temperature. After 2 h, the resin was filtered and the capping procedure repeated. After the 66 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO second coupling, the resin was filtered and washed sequentially with N,N-dimethylformamide (5 × 10 mL/g resin) then dichloromethane (5 × 10 mL/g resin) and dried under a N2 stream for cleavage. [00289] Peptide cleavage was carried out as described above. [00290] The crude residue was then purified using prep HPLC (gradient, 0→70% MeCN/H₂O+0.1%TFA, 22 min) and concentrated under reduced pressure to give linear peptide 6b (2.5 mg, 32%) (SEQ ID NO: 10).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.09 (d, J = 6.1 Hz, 0.4H, NH), 4.77 (t, J = 6.3 Hz, 1H), 4.39–4.31 (m, 2H), 4.30–4.24 (m, 1H), 4.24–4.18 (m, 1H), 4.11–4.00 (m, 2H), 3.93 (d, J = 16.8 Hz, 1H), 3.77 (d, J = 16.7 Hz, 1H), 2.98–2.88 (m, 3H), 2.86 (dd, J = 16.9, 6.0 Hz, 1H), 2.36 (t, J = 7.1 Hz, 2H), 2.20–2.09 (m, 1H), 2.02–1.84 (m, 2H), 1.82–1.60 (m, 6H), 1.53–1.43 (m, 2H), 1.40 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 6.1 Hz, 3H), 0.93 (d, J = 6.1 Hz, 3H). ¹³C NMR (126 MHz, CD3OD, 25°C): δ 177.9, 177.6, 175.7, 175.5, 174.5, 174.4, 173.7, 173.6, 172.2, 62.7, 55.2, 54.5, 54.5, 50.9, 50.3, 44.1, 40.7, 40.6, 36.4, 32.3, 32.0, 28.0, 27.9, 25.9, 23.6, 23.3, 21.9, 18.1. FTIR (thin film) cm^-1: 3288 (br), 2930 (w), 1659 (s), 1536 (m), 1439 (w), 1200 (m), 1137 (m), 839 (w), 801 (w), 723 (w). HRMS (ESI) (m/z): calc’d for C₂₈H₅₀N₉O₁₁ [M+H]⁺: 688.3624, found: 688.3627. [00291] NMR comparison of control linear peptide 6b to SmI₂ linearized macrocycle 6 (FIG. 5B) [00292] ¹H NMR and ¹³C NMR comparison between linear peptides 6 and 6b generated directly by SPPS and by SmI₂ mediated linearization of macrocycle 5, respectively. Peaks present between 7 and 9 ppm correspond to amide NH protons that exchange with deuterated methanol over time. [00293] This data confirms that the structure of the linearized peptide is generated through the cleavage of the N–O bond. [00294] Example 17: Reactivity of various amino acids with N-terminal electrophiles [00295] Treatment of control peptides containing Lysine under cyclization conditions [00296] Synthesis of C-terminal amide peptides containing Lysine using SPPS 67 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00297] Rink amide resin (0.51 mmol/g) was swelled in

dimethylformamide for 1 h before use. SPPS was performed with amide coupling times of 1 h and at room temperature using conditions described above. N-terminal capping was carried out as described in the general procedure for peptide N-terminal capping with different electrophiles described above. Resin cleavage was carried out as described in the general procedure for peptide resin cleavage described above. The crude peptides were then purified by automated reverse phase chromatography (gradient 0→40% MeCN/H2O+0.1%TFA (10 CV)) and concentrated under vacuum. [00298] HRMS analysis of linear peptides: [00299] Iodoacetamide capped DQLGKA S5a (SEQ ID NO: 49): HRMS (ESI) (m/z): calc’d for C28H49IN9O10 [M+H]⁺: 798.2642, found: 798.2644. [00300] Bromoacetamide capped DQLGKA S5b (SEQ ID NO: 50): HRMS (ESI) (m/z): calc’d for C₂₈H₄₉BrN₉O₁₀ [M+H]⁺: 750.2780, found: 750.2785. [00301] Chloroacetamide capped DQLGKA S5c (SEQ ID NO: 51): HRMS (ESI) (m/z): calc’d for C28H49ClN9O10 [M+H]⁺: 706.3285, found: 706.3289. [00302] Treatment of control peptides containing Lysine under cyclization conditions [00303] Iodoacetamide capped DQLGKA S5a (SEQ ID NO: 49)

68 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00304] The reactivity of lysine toward the N-terminal iodoacetamide on peptide S5a (0.4 mg, 0.50 µmol) (SEQ ID NO: 49) was evaluated in 25 mM MOPS buffer (0.5 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method C: 0→60% MeCN. Approximately 87% peptide S5a (SEQ ID NO: 49) remained after 24 h. t = 24 h

[00305] Bromoacetamide capped DQLGKA S5b (SEQ ID NO: 50)

[00306] The reactivity of lysine toward the N-terminal bromoacetamide peptide S5b (0.9 mg, 1.20 µmol) (SEQ ID NO: 50) was evaluated in 25 mM MOPS buffer (1.2 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method C: 0→60% MeCN. Approximately 57% peptide S5b (SEQ ID NO: 50) remained after 24 h. t = 0 h – Pure S5b

t = 24 h

[00307] Chloroacetamide capped DQLGKA S5c (SEQ ID NO: 51) 69 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00308] The reactivity of lysine toward the N-terminal chloroacetamide peptide S5c (0.6 mg, 0.85 µmol) (SEQ ID NO: 51) was evaluated in 25 mM MOPS buffer (0.85 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. Approximately 97% S5c (SEQ ID NO: 51) remained after 24 h. t = 0 h – Pure S5c

t = 48 h

[00309] The reactivity of the N-terminal electrophile towards lysine residues is greatest for the bromoacetamide, with very little reactivity observed for the chloroacetamide after 48 h. The iodoacetamide was found to have some reactivity; however, for the C-terminal hydroxamic acid containing peptides developed in this work, the reactivity of the hydroxamic acid was found to be greater than that of the lysine amine under these conditions, hence lysine cyclized peptides are a minor side product in some cases. [00310] Treatment of control peptides containing Arginine under cyclization conditions [00311] Synthesis of C-terminal amide peptide containing Arginine using SPPS

[00312] Rink amide resin (0.51 mmol/g) was swelled in

dimethylformamide for 1 h before use. SPPS was performed with amide coupling times of 1 h and at room temperature using conditions described above. N-terminal capping was carried out as described in the 70 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO general procedure for peptide N-terminal capping with different electrophiles described above. Resin cleavage was carried out as described in the general procedure for peptide resin cleavage described above. The crude peptides were then purified by automated reverse phase chromatography (gradient 0→40% MeCN/H2O+0.1%TFA (10 CV)) and concentrated under vacuum. [00313] HRMS analysis of linear peptide: [00314] Iodoacetamide capped D^DF-NMe-V-R S6 (SEQ ID NO: 52): HRMS (ESI) (m/z): calc’d for C27H42IN8O7 [M+H]⁺: 717.2216, found: 717.2219. [00315] Treatment of control peptide containing Arginine under cyclization conditions S6 [00316] Iodoacetamide capped D^DF-NMe-V-R S6 (SEQ ID NO: 52)

[00317] The reactivity of arginine toward the N-terminal iodoacetamide on peptide S6 (0.9 mg, 1.25 µmol) (SEQ ID NO: 52) was evaluated in 25 mM MOPS buffer (1.25 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method C: 0→60% MeCN. No product with a mass corresponding to the cyclized product was observed (S12 (SEQ ID NO: 53)). Approximately 3% degradation of peptide S6 (SEQ ID NO: 52) was observed over 24 h and 6.5% over 48 h. t = 0 h – Pure S6

t = 24 h

t = 48 h

71 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00318] Treatment of control peptides containing Methionine under cyclization conditions S7a-c [00319] Synthesis of C-terminal amide peptides containing Methionine using SPPS

[00320] Rink amide resin (0.51 mmol/g) was swelled in

dimethylformamide for 1 h before use. SPPS was performed with amide coupling times of 1 h and at room temperature using conditions described above. N-terminal capping was carried out as described in the general procedure for peptide N-terminal capping with different electrophiles described above. Resin cleavage was carried out as described in the general procedure for peptide resin cleavage described above. The crude peptides were then purified by automated reverse phase chromatography (gradient 0→30% MeCN/H2O+0.1%TFA (10 CV)) and concentrated under vacuum. [00321] HRMS analysis of linear peptides: [00322] Iodoacetamide capped NMKAGF^D S7a (SEQ ID NO: 54): HRMS (ESI) (m/z): calc’d for C₃₁H₄₉IN₉O₈S [M+H]⁺: 834.2464, found: 834.2468. [00323] Bromoacetamide capped NMKAGF^D S7b (SEQ ID NO: 55): HRMS (ESI) (m/z): calc’d for C31H49BrN9O8S [M+H]⁺: 786.2603, found: 786.2606. [00324] Chloroacetamide capped NMKAGF^D S7c (SEQ ID NO: 56): HRMS (ESI) (m/z): calc’d for C₃₁H₄₉ClN₉O₈S [M+H]⁺: 742.3108, found: 742.3111. [00325] Treatment of control peptides containing Methionine under cyclization conditions [00326] Iodoacetamide capped NMKAGF^D S7a (SEQ ID NO: 54) 72 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00327] The reactivity of methionine toward the N-terminal iodoacetamide on peptide S7a (0.5 mg, 0.60 µmol) (SEQ ID NO: 54) was evaluated in 25 mM MOPS buffer (0.60 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. Approximately 49% starting material remained after 24 h at 38°C. Multiple cyclized product peaks were formed. t = 0 h – Pure S7a

t = 24 h

[00328] Bromoacetamide capped NMKAGF^D S7b

[00329] The reactivity of methionine toward the N-terminal iodoacetamide on peptide S7b (0.5 mg, 0.64 µmol) (SEQ ID NO: 55) was evaluated in 25 mM MOPS buffer (0.64 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. Significant levels of numerous peptidic products were formed after 24 h at 38°C. t = 0 h – Pure S7b

73 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

t = 24 h

[00330] Chloroacetamide capped NMKAGF^D S7c

[00331] The reactivity of methionine toward the N-terminal iodoacetamide on peptide S7c (0.5 mg, 0.67 µmol) (SEQ ID NO: 56) was evaluated in 25 mM MOPS buffer (0.67 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. Approximately 17% degradation of peptide S7c (SEQ ID NO: 56) was observed after 96 h at 38°C. t = 0 h – Pure S7c

t = 24 h

t = 96 h 74 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00332] Treatment of control peptides containing Histidine under cyclization conditions S8a- c [00333] Synthesis of C-terminal amide peptides containing Histidine using SPPS

[00334] Rink amide resin (0.51 mmol/g) was swelled in

dimethylformamide for 1 h before use. SPPS was performed with amide coupling times of 1 h and at room temperature using conditions described above. N-terminal capping was carried out as described in the general procedure for peptide N-terminal capping with different electrophiles described above. Resin cleavage was carried out as described in the general procedure for peptide resin cleavage described above. The crude peptides were then purified by automated reverse phase chromatography (gradient 0→40% MeCN/H2O+0.1%TFA (10 CV)) and concentrated under vacuum. [00335] HRMS analysis of linear peptides: [00336] Iodoacetamide capped LTGFHA S8a (SEQ ID NO: 57): HRMS (ESI) (m/z): calc’d for C₃₂H₄₇IN₉O₈ [M+H]⁺: 812.2587, found: 812.2591. [00337] Bromoacetamide capped LTGFHA S8b (SEQ ID NO: 58): HRMS (ESI) (m/z): calc’d for C32H46BrN9O8 [M+H]⁺: 764.2725, found: 764.2730. [00338] Chloroacetamide capped LTGFHA S8c (SEQ ID NO: 59): HRMS (ESI) (m/z): calc’d for C₃₂H₄₇ClN₉O₈ [M+H]⁺: 720.3231, found: 720.3234. [00339] Treatment of control peptides containing Histidine under cyclization conditions [00340] Iodoacetamide capped LTGFHA S8a (SEQ ID NO: 57) 75 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00341] The reactivity of histidine toward the N-terminal iodoacetamide on peptide S8a (0.7 mg, 0.86 µmol) (SEQ ID NO: 57) was evaluated in 25 mM MOPS buffer (0.86 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method C: 0→60% MeCN, 10 min. Approximately 85% cyclized product S12 (SEQ ID NO: 53) was formed after 24 h at 38°C. The product was validated using mass spectrometry. t = 24 h

[00342] Bromoacetamide capped LTGFHA S8b (SEQ ID NO: 57)

[00343] The reactivity of histidine toward the N-terminal bromoacetamide on peptide S8b (0.6 mg, 0.79 µmol) (SEQ ID NO: 58) was evaluated in 25 mM MOPS buffer (0.79 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method C: 0→60% MeCN. Approximately 100% cyclized product S12 (SEQ ID NO: 53) was formed after 24 h at 38°C. The product was validated using mass spectrometry. t = 0 h – Pure S8b

t = 24 h

76 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00344] Chloroacetamide capped LTGFHA S8c (SEQ ID NO: 59)

[00345] The reactivity of histidine toward the N-terminal chloroacetamide on peptide S8c (0.6 mg, 0.83 µmol) (SEQ ID NO: 59) was evaluated in 25 mM MOPS buffer (0.83 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. Approximately 40% cyclized product S12 (SEQ ID NO: 53) was formed after 48 h at 38°C. 0 h – Pure S8c

48 h

[00346] Example 18: Macrocyclizations in the presence of nucleophilic amino acids [00347] Histidine containing peptide cyclization optimization (Table 4) [00348] Purified peptide 20a (0.5–1 mg) (SEQ ID NO: 60) was dissolved in the specified buffer at the specified concentration with sonication. The reaction mixture was then heated and stirred for the specified amount of time. Samples were taken at t = 0 h and at the end of the reaction for analysis by HPLC using HPLC Analytical Method A: 0→60% MeCN. 77 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO Table 4. Optimization of the macrocyclization conditions for histidine-containing peptides

E_{ntry Solvent pH} ^Temperature Time 20b 21 Isolated yield

(%) 1 MOPS 8 38 96 56 34 - 2 MOPS 8 50 72 49 36 - 3 ^{100 mM Pi} b_{uffer 7.5 50 72 46 32 -} _{4 PBS 7 50 96 27 57} ^20b:32% 2_1:57% 5 PBS 9 38 72 14 73 - 6 PBS 10 38 48 83 9 - [00349] HPLC analysis t = 0 h – Pure 20a

78 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

t = 72 h, entry 2

t = 72 h, entry 3

t = 96 h, entry 4

t = 72 h, entry 5

t = 48 h, entry 6

[00350] NMR Characterization of Histidine containing peptides [00351] SPPS of chloroacetamide capped peptide 20a 79 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00352] SPPS was performed using general procedures described above on 50 μmol scale. Crude peptide was purified using prep HPLC (gradient 0→70% MeCN/H₂O+0.1% TFA, 22 min) to give chloroacetamide capped peptide 20a (16 mg, 43%) (SEQ ID NO: 60).¹H NMR (500 MHz, CD3OD, 25°C): δ 8.76 (d, J = 1.4 Hz, 1H), 8.22 (d, J = 6.2 Hz, 0.7H, NH), 8.08 (d, J = 7.8 Hz, 0.5 H, NH), 7.90 (d, J = 7.5 Hz, 0.4H, NH), 7.32 (s, 1H), 7.27–7.24 (m, 2H), 7.22–7.17 (m, 3H), 4.65–4.62 (m, 1H), 4.50 (ddd, J = 17.1, 8.6, 6.1 Hz, 2H), 4.32–4.30 (m, 1H), 4.25–4.20 (m, 2H), 4.14–4.06 (m, 2H), 3.88 (d, J = 16.8 Hz, 1H), 3.81 (d, J = 16.8 Hz, 1H), 3.28 (dd, J = 15.4, 5.5 Hz, 1H), 3.11 (td, J = 14.6, 13.8, 6.5 Hz, 2H), 2.95 (dd, J = 13.9, 8.8 Hz, 1H), 1.75–1.61 (m, 3H), 1.37 (d, J = 7.1 Hz, 3H), 1.20 (d, J = 6.4 Hz, 3H), 0.96 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H).¹³C NMR (126 MHz, CD₃OD, 25°C): δ: 174.8, 173.3, 173.1, 172.1, 171.7, 171.0, 169.9, 161.0 (d, J = 36 Hz, TFA), 138.2, 135.0, 130.2, 129.6, 129.5, 127.9, 119.1, 68.3, 60.3, 56.6, 54.0, 53.1, 49.8, 43.5, 43.0, 41.3, 38.1, 28.2, 25.9, 23.5, 21.8, 19.9, 18.2. FTIR (thin film) cm^-1: 3280 (br), 1629 (s), 1536 (m), 1454 (m), 1200 (m), 1137 (m), 1029 (w). HRMS (ESI) (m/z): calc’d for C32H47ClN9O9 [M+H]⁺: 736.3180, found: 736.3183. [00353] Macrocyclization of peptide 20a [00354] Macrocyclization of peptide 20a (8.3 mg, 11.3 µmol) (SEQ ID NO: 60) was carried out in PBS, pH 7 (11.3 mL). Following complete starting material conversion, products 20b (SEQ ID NO: 61) and 21 (SEQ ID NO: 62) were purified and isolated using automated flash column chromatography (Automated flash column chromatography: Procedure B desalt) and prep HPLC purification (gradient 0→85% MeCN/H₂O+0.1% TFA, 22 min) to yield macrocycle 20b (2.5 mg, 32%) (SEQ ID NO: 61) and histidine linked macrocycle 21 (4.5 mg, 57%) (SEQ ID NO: 62). [00355] Head-to-tail linked LTGFHA 20b (SEQ ID NO: 61) 80 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00356] ¹H NMR (500 MHz, CD3OD, 25°C): δ 8.81 (d, J = 1.4 Hz, 1H, 25), 8.35 (br s, 0.5H, NH), 8.15 (br s, 0.5H, NH), 7.73 (d, J = 6.6 Hz, 0.5H, NH), 7.30–7.17 (m, 6H, 18–20, 26), 4.58 (d, J = 16.5 Hz, 1H, 1), 4.54 (dd, J = 10.4, 4.2 Hz, 1H, 3), 4.49 (dd, J = 10.2, 4.8 Hz, 1H, 14), 4.38 (dd, J = 9.6, 5.1 Hz, 1H, 21), 4.33–4.26 (m, 2H, 1, 27), 4.16–4.14 (m, 2H, 10, 8), 3.93 (dd, J = 16.9, 5.8 Hz, 1H, 12), 3.65 (dd, J = 17.0, 4.2 Hz, 1H), 3.41 (dd, J = 15.6, 5.1 Hz, 1H, 23), 3.30–3.20 (m, 2H, 16, 23), 3.10 (dd, J = 14.2, 10.1 Hz, 1H, 16), 1.83–1.71 (m, 2H, 5, 6), 1.70– 1.65 (m, 1H, 5), 1.34 (d, J = 6.8 Hz, 3H, 29), 1.22 (d, J = 6.2 Hz, 3H, 11), 0.95 (d, J = 6.2 Hz, 3H, 7), 0.92 (d, J = 6.2 Hz, 3H, 7).¹³C NMR (126 MHz, CD3OD, 25°C): δ: 175.6 (4), 173.7 (overlapping 2C, 13, 15), 172.0 (overlapping 2C, 26, 9), 171.6 (2), 171.1 (22), 138.5 (17), 134.9 (25), 131.7 (24), 130.2 (18), 129.5 (19), 127.9 (20), 118.2 (26), 77.2 (1), 68.1 (10/8), 62.0 (10/8), 56.7 (14), 54.6 (21), 53.3 (3), 48.5 (overlapping CD3OD, 27), 43.9 (12), 41.9 (5), 38.0 (16), 26.7 (23), 26.0 (6), 23.5 (7), 21.7 (7), 20.3 (11), 17.7 (29). FTIR (thin film) cm^-1: 3276 (br), 2930 (w), 1670 (s), 1536 (w), 1454 (w), 1204 (m), 1141 (m). HRMS (ESI) (m/z): calc’d for C32H46N9O9 [M+H]⁺: 700.3413, found: 700.3416. 81 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00357] Histidine-linked LTGFHA 21 (SEQ ID NO: 62)

[00358] ¹H NMR (500 MHz, CD3OD, 25°C): δ 8.83 (d, J = 1.5 Hz, 1H, 26), 7.36 (s, 1H, 28), 7.28–7.25 (m, 2H, 11), 7.21–7.18 (m, 3H, 10, 12), 5.12 (d, J = 16.4 Hz, 1H, 25), 5.06 (d, J = 16.4 Hz, 1H, 25), 4.77 (dd, J = 7.5, 3.6 Hz, 1H, 5), 4.41 (dd, J = 9.5, 5.5 Hz, 1H, 7), 4.37–4.32 (m, 1H, 17), 4.31–4.19 (m, 3H, 2, 16, 20), 4.07 (d, J = 17.0 Hz, 1H, 14), 3.53 (d, J = 16.9 Hz, 1H, 14), 3.34–3.32 (m, 1H, 29), 3.17–3.11 (m, 2H, 8, 29), 2.98 (dd, J = 13.6, 9.6 Hz, 1H, 8), 1.85–1.76 (m, 1H, 22), 1.70 (t, J = 7.4 Hz, 2H, 21), 1.40 (d, J = 7.2 Hz, 3H, 3), 1.22 (d, J = 6.4 Hz, 3H, 18), 0.99 (d, J = 6.6 Hz, 3H, 23), 0.96 (d, J = 6.5 Hz, 3H, 23). [00359] ¹³C NMR (126 MHz, CD3OD, 25°C): δ: 175.1 (19), 173.5 (6), 172.8 (15), 172.3 (1), 171.3 (14), 171.1 (5), 168.6 (24), 138.6 (9), 137.5 (26), 130.5 (27), 130.3 (10), 129.5 (11), 127.8 (12), 122.7 (28), 67.9 (17), 60.7 (16), 57.0 (7), 55.4 (16), 52.2 (5), 51.8 (25), 48.5 (overlapping CD3OD, 2), 43.1 (14), 41.6 (21), 37.7 (8), 28.6 (29), 26.0 (22), 23.3 (23), 21.6 (23), 20.3 (18), 18.2 (3). FTIR (thin film) cm^-1: 3288 (br), 2926 (w), 1666 (s), 1539 (w), 1454 (w), 1204 (m), 1137 (m). HRMS (ESI) (m/z): calc’d for C₃₂H₄₆N₉O₉ [M+H]⁺: 700.3413, found: 700.3416. [00360] HMBC highlighting interactions between C25H and Histidine ring (FIG.7) [00361] N-terminal chloroacetamide macrocyclization and linearization scope (Table 4) 82 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00362] LTGFHA (20a–c): Macrocyclization

[00363] Macrocyclization of peptide 20a (0.5 mg, 0.68 µmol) (SEQ ID NO: 60) was carried out in PBS, pH 10 (0.68 mL) at 38°C for 48 h. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. [00364] LTGFHA (20a–c): Linearization

[00365] Samarium(II) iodide (0.1 M in tetrahydrofuran, 0.2 mL) was added dropwise to a solution of macrocycle 20b (3.5 mg, 5.00 µmol) (SEQ ID NO: 61) in a degassed mixture of tetrahydrofuran/methanol (3/1 v/v, 0.13 mL) until a deep blue solution was formed. The reaction mixture was then stirred under N₂ at room temperature for 1 h before analyzing with HPLC (HPLC Analytical Method B: 0→60% MeCN). The reaction was then quenched by stirring in air for 1 h and concentrated under a N2 stream. The crude residue was purified by prep HPLC (gradient 0→70% MeCN/H₂O+0.1% TFA) to give linear peptide 20c (2 mg, 57% yield) (SEQ ID NO: 63).¹H NMR (500 MHz, CD3OD, 25°C): δ 8.76 (d, J = 1.4 Hz, 1H, 25), 8.27 (t, J = 5.7 Hz, 0.4H, NH), 8.19–8.12 (m, 0.9H, NH), 8.06 (d, J = 7.9 Hz, 0.7H, NH), 7.91 (d, J = 7.4 Hz, 0.4H, NH), 7.35 (d, J = 1.4 Hz, 1H, 26), 7.32–7.08 (m, 5H, 18–20), 4.63 (dd, J = 6.5, 5.6 Hz, 1H, 3), 4.57–4.47 (m, 2H, 14, 21), 4.38–4.27 (m, 2H, 8, 27), 4.25–4.19 (m, 1H, 10), 4.04 (s, 2H, 1), 3.88 (d, J = 16.9 Hz, 1H, 12), 3.80 (d, J = 16.7 Hz, 1H, 12), 3.30–3.27 (m, 83 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 1H, 23), 3.17–3.07 (m, 2H, 16, 23), 2.95 (dd, J = 13.9, 8.9 Hz, 1H, 16), 1.71–1.65 (m, 3H, 5, 6), 1.40 (d, J = 7.2 Hz, 3H, 29), 1.20 (d, J = 6.4 Hz, 3H, 11), 0.96 (d, J = 6.0 Hz, 3H, 7), 0.93 (d, J = 6.0 Hz, 3H, 7). ¹³C NMR (126 MHz, CD₃OD, 25°C): δ 177.9 (28), 175.7 (2), 175.2 (22), 173.4 (15), 173.2 (9), 171.7 (13), 171.1 (4), 138.2 (17), 135.0 (25), 130.3 (24), 130.2 (18), 129.6 (19), 127.9 (20), 119.1 (26), 68.3 (10), 62.6 (1), 60.4 (8), 56.6 (14), 53.2 (3), 53.1 (21), 50.2 (27), 43.5 (12), 41.5 (5), 38.1 (16), 28.2 (23), 25.9 (6), 23.5 (7), 21.9 (7), 19.9 (11), 18.2 (29). FTIR (thin film) cm^-1: 3295 (br), 2960 (w), 2926 (w), 1662 (s), 1537 (w), 1454 (w), 1204 (m), 1141 (m), 839 (w), 801 (w), 723 (w). HRMS (ESI) (m/z): calc’d for C32H48N9O9 [M+H]⁺: 702.3570, found: 702.3572. HPLC analysis

[00366] NMKAG-^DF (22a-c): SPPS

[00367] SPPS performed using general procedures reported above on 28 μmol scale. Crude peptide was purified using automated flash column chromatography (gradient 0→45% MeCN/H₂O+0.1% TFA, 12 CV) to give chloroacetamide capped peptide 22a (10 mg, 48%) (SEQ ID NO: 64). ¹H NMR (500 MHz, CD₃OD, 25°C): δ: 8.51 (d, J = 6.5 Hz, 0.5H, NH), 8.33 (t, J = 6.0 Hz, 0.5H, NH), 8.24 (d, J = 7.3 Hz, 0.8H, NH), 8.09 (d, J = 4.9 Hz, 0.6H, NH), 7.89 (d, J = 8.3 Hz, 0.5H, NH), 7.31–7.25 (m, 4H), 7.21–7.18 (m, 1H), 4.70 (dd, J = 7.7, 5.4 Hz, 1H), 4.56–4.51 (m, 1H), 4.45–4.41 (m, 1H), 4.38–4.31 (m, 1H), 4.26–4.17 (m, 1H), 4.17– 4.07 (m, 2H), 3.82 (d, J = 17.1 Hz, 1H), 3.74 (d, J = 17.1 Hz, 1H), 3.20 (dd, J = 13.8, 5.7 Hz, 1H), 3.04 (dd, J = 13.8, 9.4 Hz, 1H), 2.99–2.94 (m, 2H), 2.85 (dd, J = 15.7, 7.6 Hz, 1H), 2.74 (dd, J = 15.6, 5.4 Hz, 1H), 2.65–2.56 (m, 1H), 2.54–2.49 (m, 1H), 2.17–2.09 (m, 1H), 2.04 (s, 3H), 2.00–1.92 (m, 2H), 1.87–1.79 (m, 1H), 1.74–1.68 (m, 2H), 1.54–1.47 (m, 2H), 1.40 (d, J 84 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO = 7.2 Hz, 3H). ¹³C NMR (126 MHz, CD3OD, 25°C): δ: 176.2, 174.7, 174.6, 174.6, 173.5, 171.4, 170.2, 169.2, 160.8 (q, J = 38.4 Hz, TFA), 138.5, 130.4, 129.5, 127.8, 54.9, 54.8, 54.4, 51.9, 51.7, 43.6, 43.1, 40.5, 38.6, 37.3, 31.8, 31.5, 31.1, 27.8, 23.5, 17.1, 15.2. FTIR (thin film) cm^-1: 3291 (br), 2926 (w), 2855 (w), 1666 (s), 1536 (m), 1439 (s), 1204 (m), 1137 (m), 839 (w), 801 (w), 723 (w). HRMS (ESI) (m/z): calc’d for C31H49ClN9O9S [M+H]⁺: 758.3057, found: 758.3061. [00368] NMKAG-^DF (22a-c): Macrocyclization

[00369] Macrocyclization of peptide 22a (8.7 mg, 11.5 µmol) (SEQ ID NO: 64) was carried out in degassed 25 mM MOPS buffer (11.5 mL) under an N2 atmosphere using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. After complete reaction, the product was purified using automated flash column chromatography (isocratic 100% H2O+0.1% TFA, 4 CV, gradient 0→35% MeCN/H2O+0.1% TFA, 10 CV) to give macrocycle 22b (7.8 mg, 94%) (SEQ ID NO: 65).¹H NMR (500 MHz, CD₃OD, 25°C): δ: 8.76 (br s, 0.2H, NH), 8.60 (br s, 0.3H, NH), 8.35–8.27 (m, 0.6H, NH), 8.12 (br s, 0.3H, NH), 8.06 (d, J = 8.4 Hz, 0.4H, NH), 7.32–7.25 (m, 4H, 27, 28), 7.23–7.20 (m, 1H, 29), 4.75 (t, J = 5.9 Hz, 1H, 3), 4.57–4.51 (m, 1H, 23), 4.50–4.40 (m, 2H, 12, 29), 4.37–4.27 (m, 2H, 7, 18), 4.11 (d, J = 16.3 Hz, 1H, 1), 4.02 (d, J = 16.9 Hz, 1H, 21), 3.59 (d, J = 16.8 Hz, 1H, 20), 3.17 (dd, J = 13.7, 6.3 Hz, 1H, 25), 3.12–3.03 (m, 1H, 25), 3.01–2.91 (m, 2H, 17), 2.92–2.81 (m, 2H, 5), 2.69–2.62 (m, 1H, 10), 2.58–2.52 (m, 1H, 10), 2.28–2.20 (m, 1H, 9), 2.19–2.12 (m, 1H, 9), 2.06 (s, 3H, 11), 1.97–1.83 (m, 2H, 14), 1.77–1.63 (m, 2H, 16), 1.52–1.46 (m, 2H, 15), 1.37 (d, J = 7.0 Hz, 3H, 20).¹³C NMR (126 MHz, CD₃OD, 25°C): δ: 175.6 (19), 175.0 (6/3), 174.4 (13), 173.9 (8), 173.2 (6/3), 173.0 (24), 171.6 (22), 170.9 (2), 160.7 (q, J = 38 Hz, TFA), 138.2 (26), 130.4 (27), 129.6 (28), 127.9 (29), 76.4 (1), 55.2 (7), 54.7 (12), 54.0 (23), 51.4 (3), 51.4 (18), 43.6 (21), 40.6 (17), 38.9 (25), 37.6 (5), 32.2 85 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO (15), 31.4 (10), 30.4 (9), 27.8 (16), 23.4 (15), 17.5 (20), 15.1 (11). FTIR (thin film) cm^-1: 3280 (br), 2930 (w), 1640 (s), 1528 (m), 1431 (m), 1338 (m), 1178 (s), 1133 (s), 1018 (m), 835 (m), 797 (m). HRMS (ESI) (m/z): calc’d for C₃₁H₄₈N₉O₉S [M+H]⁺: 722.3290, found: 722.3293. [00370] HPLC analysis t = 0 h – Pure 22a

Peak 1: 10% oxidized methionine Peak 2: linear peptide 22a+H mass of 758.3 t = 96 h

Peak 1: 10% oxidized methionine Peak 2: cyclized peptide 22b+H mass of 722.3 [00371] NMKAG-^DF (22a-c): Linearization

[00372] Linearization of macrocycle 22b (0.8 mg, 1.11 µmol) (SEQ ID NO: 65) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.15 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.20 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. Linear peptide 22c (SEQ ID NO: 66) HRMS (ESI) (m/z): calc’d for C31H50N9O9S [M+H]⁺: 724.3447, found: 724.3448. HPLC analysis

86 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00373] DQLGKA (4–6): Macrocyclization

[00374] Macrocyclization of peptide 4c (0.9 mg, 1.25 µmol) (SEQ ID NO: 8) was carried out in PBS, pH 9 (1.25 mL) at 50°C for 48 h. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN for t = 0 h and HPLC Analytical Method B: 0→40% MeCN for t = 48 h. t = 0 h – Pure 4c

t = 48 h

87 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00375] A-Ar-YHDQLGKA (23a–c): Macrocyclization

[00376] Macrocyclization of peptide 23a (15.0 mg, 12.2 µmol) (SEQ ID NO: 67) was carried out in PBS, pH 9 (12.2 mL) at 50°C for 72 h. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN. After complete reaction, the product was purified first using automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 4 CV, gradient 0→40% MeCN/H2O+0.1% TFA, 8 CV) then further purified by prep HPLC (gradient 0→65% MeCN/H₂O+0.1% TFA) to give macrocycle 23b (6.0 mg, 41%) (SEQ ID NO: 68).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.69 (d, J = 1.4 Hz, 1H), 7.68 (t, J = 2.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.29 – 7.21 (m, 2H), 7.05–6.99 (m, 2H), 6.97 (d, J = 7.6 Hz, 1H), 6.68 (d, J = 6.7 Hz, 2H), 4.58–4.53 (m, 3H), 4.47 (d, J = 15.6 Hz, 1H), 4.41 (dd, J = 9.1, 6.2 Hz, 1H), 4.38– 4.22 (m, 5H), 3.96 (d, J = 16.7 Hz, 1H), 3.74 (d, J = 16.7 Hz, 1H), 3.56 (d, J = 14.5 Hz, 1H), 3.49 (d, J = 14.4 Hz, 1H), 3.26 (d, J = 15.5, 5.6 Hz, 1H), 3.09 (dd, J = 15.5, 7.6 Hz, 1H), 3.02 (dd, J = 14.1, 6.2 Hz, 1H), 2.97–2.80 (m, 5H), 2.39–2.32 (m, 2H), 2.15–1.99 (m, 2H), 1.83– 1.75 (m, 1H), 1.72–1.62 (m, 4H), 1.60–1.52 (m, 2H), 1.48 (d, J = 7.2 Hz, 3H), 1.39 (d, J = 7.1 Hz, 3H), 1.35–1.28 (m, 2H), 0.95 (d, J = 5.8 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H).¹³C NMR (126 88 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO MHz, CD3OD, 25°C): δ 178.0, 174.5, 174.4, 174.4, 174.3, 174.1, 174.0, 173.8, 173.3, 173.2, 172.0, 171.9, 170.7, 161.4 (q, J = 37 Hz, TFA), 157.4, 139.6, 137.5, 135.0, 131.2, 130.7, 130.1, 128.7, 126.7, 122.3, 120.1, 118.6, 116.4, 76.2, 57.4, 55.3, 54.4, 54.1, 54.0, 52.0, 51.0, 48.5 (overlapping CD3OD), 43.8, 43.2, 40.9, 40.6, 37.2, 35.9, 32.5, 32.1, 28.2, 28.0, 27.3, 25.9, 23.4, 23.4, 21.9, 18.3, 17.4. FTIR (thin film) cm^-1: 3299 (br), 2930 (w), 1662 (s), 1539 (m), 1446 (m), 1200 (m), 1141 (m), 1059 (w), 835 (w), 801 (w), 723 (w). Macrocycle 23b (SEQ ID NO: 68) HRMS (ESI) (m/z): calc’d for C₅₄H₇₆N₁₅O₁₆ [M+H]⁺: 1190.5589, found: 1190.5593. Chloroacetamide capped peptide 23a (SEQ ID NO: 67): HRMS (ESI) (m/z): calc’d for C₅₄H₇₇ClN₁₅O₁₆ [M+H]⁺: 1226.5356, found: 1226.5359. [00377] HPLC analysis t = 0 h – Pure 23a; HPLC Analytical Method A: 0→60% MeCN

[00378] A-Ar-YHDQLGKA (23a-c): Linearization

[00379] Samarium(II) iodide (0.1 M in tetrahydrofuran, 0.7 mL) was added dropwise to a solution of macrocycle 23b (4.0 mg, 3.4 µmol) (SEQ ID NO: 68) in a degassed mixture of tetrahydrofuran/methanol (3/1 v/v, 0.3 mL) until a deep blue solution was formed. The reaction mixture was then stirred under N2 at room temperature for 1 h before analyzing with HPLC 89 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO (HPLC Analytical Method B: 0→60% MeCN). The reaction was then quenched by stirring in air for 1 h and concentrated under a N2 stream. The crude product was purified first using automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 4 CV, gradient 0→35% MeCN/H2O+0.1% TFA, 8 CV) then further purified by prep HPLC (gradient 0→50% MeCN/H2O+0.1% TFA) to give linear peptide 23c (2.6 mg, 65% yield) (SEQ ID NO: 69).¹H NMR (500 MHz, CD₃OD, 25°C): δ 8.64 (s, 1H), 8.53 (d, J = 6.4 Hz, 0.3H, NH), 8.28 (t, J = 5.0 Hz, 0.3H, NH), 8.15 (d, J = 6.4 Hz, 0.3H, NH), 8.10–8.03 (m, 1H, NH), 7.96 (d, J = 7.2 Hz, 0.3H, NH), 7.56 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.27–7.21 (m, 2H), 6.99 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 7.6 Hz, 1H), 6.67 (d, J = 8.2 Hz, 2H), 4.64–4.53 (m, 3H), 4.44–4.30 (m, 5H), 4.05 (s, 2H), 3.94 (d, J = 16.7 Hz, 1H), 3.77 (d, J = 16.6 Hz, 1H), 3.51 (s, 2H), 3.23 (dd, J = 15.5, 5.4 Hz, 1H), 3.07 (dd, J = 15.4, 7.6 Hz, 1H), 2.99 (dd, J = 14.0, 6.4 Hz, 1H), 2.94–2.79 (m, 5H), 2.36 (t, J = 7.1 Hz, 2H), 2.16–2.09 (m, 1H), 2.04–1.96 (m, 1H), 1.91–1.84 (m, 1H), 1.80–1.74 (m, 1H), 1.71–1.63 (m, 5H), 1.49–1.42 (m, 5H), 1.39 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 5.7 Hz, 3H), 0.91 (d, J = 5.8 Hz, 3H).¹³C NMR (126 MHz, CD3OD, 25°C): δ 178.0, 177.6, 175.6, 174.9, 174.4, 174.3, 174.2, 174.1, 173.7, 173.5, 173.2, 172.1, 171.9, 157.4, 139.6, 137.4, 134.9, 131.2, 130.6, 130.1, 128.6, 126.4, 122.4, 120.1, 118.7, 116.4, 62.6, 57.2, 55.3, 54.5, 54.1, 53.7, 51.8, 50.6, 50.3, 44.0, 43.3, 40.9, 40.6, 37.4, 36.1, 32.4, 32.1, 28.2, 27.9, 27.6, 25.9, 23.6, 23.4, 21.9, 18.6, 18.1. FTIR (thin film) cm^-1: 3288 (br), 2933 (w), 1655 (s), 1536 (m), 1439 (m), 1200 (s), 1141 (s), 839 (w), 801 (w), 723 (w). HRMS (ESI) (m/z): calc’d for C₅₄H₇₉N₁₅O₁₆ [M+2H]²⁺: 596.7909, found: 596.7911. [00380] HPLC analysis

[00381] Protecting group strategy for accessing cysteine containing macrocycles [00382] Synthesis of protected hydroxamic acid functionalized peptides

90 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00383] Resin functionalization, SPPS, and N-terminal capping were carried out using general procedures as described above. [00384] Resin cleavage conditions leaving protecting groups intact: Cleavage solution (1% TFA in dichloromethane, 10 mL/g resin) was added to the fritted syringe containing resin. The resin suspension was rotated end over end on a rotator for 1 h at room temperature. After 1 h, the filtrate was collected, and the resin was washed with dichloromethane (2 × 20 mL/g resin) then N,N-dimethylformamide (3 × 20 mL/g resin). The combined filtrates were concentrated under vacuum and used directly in the following steps with no further purification to give iodoacetamide capped peptide 24 (32.3 mg, 54%) (SEQ ID NO: 70). Iodoacetamide capped peptide 24 (SEQ ID NO: 70) HRMS (ESI) (m/z): calc’d for C₄₄H₅₂IN₆O₇S [M+H]⁺: 935.2657, found: 935.2661. HPLC analysis of crude residue: HPLC Analytical Method D 0→95% MeCN

[00386] A round-bottom flask was sequentially charged with iodoacetamide capped peptide 24 (19.0 mg, 20.3 µmol, 1.0 equiv) (SEQ ID NO: 70) and N,N-dimethylformamide (20 mL) at 91 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO room temperature. Cesium carbonate (66.3 mg, 0.203 mmol, 10 equiv) was then added to the solution and the suspension was sonicated for 10 min. The reaction mixture was then stirred at room temperature for 2 h before being filtered through celite and concentrated under reduced pressure. The crude residue was then treated with cleavage solution (1.5 mL, 92.5/2.5/2.5/2.5/2.5 v/v/v/v, TFA/TIPS/H2O/dichloromethane) for 1 h at room temperature before being partially concentrated under a N₂ stream (to ~0.15 mL). The peptide was precipitated by addition of ice-cold diethyl ether (1.5 mL). The peptide was filtered, dried under a N2 stream, and purified by prep HPLC (gradient, 0→90% MeCN/H2O+0.1% TFA) to give macrocycle 25 (5.0 mg, 49% yield) (SEQ ID NO: 72).¹H NMR (500 MHz, CD₃OD, 25°C): δ 7.30–7.25 (m, 4H, 12, 13), 7.23–7.18 (m, 1H, 14), 4.70 (dd, J = 8.9, 5.5 Hz, 1H, 8), 4.58 (d, J = 16.8 Hz, 1H, 1), 4.45 (q, J = 6.7 Hz, 1H, 20), 4.20 (dd, J = 10, 5.4 Hz, 1H, 3), 4.14 (d, J = 14.8 Hz, 1H, 1), 4.11 (d, J = 15.3 Hz, 1H, 18), 3.96 (t, J = 7.2 Hz, 1H, 15), 3.63 (d, J = 17.2 Hz, 1H, 18), 3.23 (dd, J = 13.7, 5.6 Hz, 1H, 10), 2.95–2.78 (m, 3H, 10, 17), 1.59–1.47 (m, 2H, 5), 1.42–1.36 (m, 1H, 6), 1.22 (d, J = 6.7 Hz, 3H, 22), 0.93 (d, J = 6.5 Hz, 3H, 7), 0.86 (d, J = 6.5 Hz, 3H, 7); (minor oxidized peptide present – overlap in Leu CH3 region).¹³C NMR (126 MHz, CD₃OD, 25°C): δ: 174.1 (4), 173.8 (9), 173.3 (16), 172.1 (21), 171.7 (2), 171.4 (19), 137.7 (11), 130.9 (12), 129.5 (13), 127.9 (14), 77.3 (1), 60.2 (15), 54.5 (3), 54.1 (8), 48.1 (20), 44.0 (18), 42.1 (5), 40.0 (10), 26.0 (6), 24.8 (17), 23.1 (7), 21.9 (7), 17.0 (22). FTIR (thin film) cm^-1: 3299 (br), 2950 (w), 2930 (w), 1662 (s), 1536 (w), 1454 (w), 1204 (w), 1141 (w), 835 (w), 801 (w). HRMS (ESI) (m/z): calc’d for C₂₅H₃₇N₆O₇S [M+H]⁺: 565.2439, found: 565.2444. HPLC analysis of crude global deprotection: HPLC Analytical Method A 0→60% MeCN

[00387] LFCGA: Linearization

AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00388] Linearization of macrocycle 25 (3.5 mg, 6.20 µmol) (SEQ ID NO: 72) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.30 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.30 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→60% MeCN. Linear peptide 26 (SEQ ID NO: 73) HRMS (ESI) (m/z): calc’d for C25H39N6O7S [M+H]⁺: 567.2595, found: 567.2597. HPLC Analysis

[00389] Example 19: Applications of the macrocyclization and linearization technology [00390] In situ post-cyclization functionalization of macrocycles

[00391] Macrocyclization of peptide 4a (4.0 mg, 4.9 µmol, 1 equiv) (SEQ ID NO: 6) was carried out in 25 mM MOPS buffer (4.9 mL) using General Macrocyclization Procedure B. After 24 h at 38°C, a solution of (+)-biotin N-hydroxysuccinimide ester (13.4 mg, 39.2 µmol, 8.0 equiv) in methanol (2 mL) was added to the reaction mixture at room temperature. The reaction mixture was stirred for 24 h, and upon complete reaction determined by HPLC, was purified first by automated flash column chromatography (isocratic 100% H2O+0.1% TFA, 5 CV, then gradient 0→30% MeCN/H2O+0.1% TFA, 10 CV) and further purified on the prep HPLC (gradient, 0→75% MeCN/H₂O+0.1% TFA) to give functionalized macrocycle 27 (4.3 mg, 96%) (SEQ ID NO: 74). HRMS (ESI) (m/z): calc’d for C₃₈H₆₂N₁₁O₁₃S [M+H]⁺: 912.4244, found: 912.4249. HPLC Analysis: Crude reaction mixture – HPLC Analytical Method A 0→60% MeCN

HPLC Analysis: Purified peptide – HPLC Analytical Method A 0→60% MeCN 93 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00392] pH stability of DQLGKA macrocycle 5 (result summarized at the end of Example 5) [00393] PBS pH 2.5 stability [00394] Cyclized peptide 5 (0.25 mg, 0.36 µL) (SEQ ID NO: 5) was dissolved in PBS pH 2.5 (0.36 mL) with sonication and heated to 37°C in an incubator for 28 days. At the specified timepoints, 20 µL of reaction mixture was diluted with 8 µL of caffeine internal standard (0.1 mg/mL in H₂O) and 25 µL of sample was injected into the HPLC and analyzed using HPLC Analytical Method B: 0→40% MeCN. The samples were run in duplicate, and the average area-under-curve (AUC) values were compared with the t = 0 h timepoint to determine percent peptide remaining, caffeine standard runs at t_R = 9.55 min and peptide starting material runs at tR = 10.21 min. Additional peak formed at 28 days at tR = 11.3 min corresponds to a mass of 5-H2O, indicating possible amide bond formation between aspartic acid and lysine residues (FIG.11). [00395] PBS pH 10 stability [00396] Cyclized peptide 5 (0.25 mg, 0.36 µL) (SEQ ID NO: 5) was dissolved in PBS pH 10 (0.36 mL) with sonication and heated to 37°C in an incubator for 28 days. At the specified timepoints, 20 µL of reaction mixture was diluted with 8 µL of caffeine internal standard (0.1 mg/mL in H2O) and 25 µL of sample was injected into the HPLC and analyzed using HPLC Analytical Method B: 0→40% MeCN. The samples were run in duplicate, and the average area-under-curve (AUC) values were compared with the t = 0 h timepoint to determine % peptide remaining, caffeine standard runs at tR = 9.55 min and peptide starting material runs at tR = 10.21 min (FIG.12). [00397] PBS pH 7.4 stability [00398] Cyclized peptide 5 (0.25 mg, 0.36 µL) (SEQ ID NO: 5) was dissolved in PBS pH 7.4 (0.36 mL) with sonication and heated to 37°C in an incubator for 14 days. At the specified timepoints, 20 µL of reaction mixture was diluted with 8 µL of caffeine internal standard (0.1 mg/mL in H₂O) and 25 µL of sample was injected into the HPLC and analyzed using HPLC Analytical Method B: 0→40% MeCN. The samples were run in duplicate, and the average area-under-curve (AUC) values were compared with the t = 0 h timepoint to determine % peptide remaining, caffeine standard runs at t_R = 9.55 min and peptide starting material runs at tR = 10.21 min (FIG.13). 94 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00399] Beyond 14d significant levels of both Asn hydrolysis and epimerization prevented further analysis. These impurities were not related to the hydroxamate linkage stability. [00400] Proteolytic and oxidative stability of DQLGKA macrocycle 5 (results summarized at the end of Example 5) [00401] H2O2 stability [00402] A vial was sequentially charged with cyclized peptide 5 (SEQ ID NO: 5) (4.5 µL, 40 mM dimethylsulfoxide (DMSO) stock), Tris pH 8 (15 µL, 1 M), H₂O (10.5 µL) and H₂O₂ (120 µL, 100 mM in H2O) and heated to 37°C in an incubator for 48 h. At the specified timepoints, 20 µL of reaction mixture was diluted with 40 µL of acetonitrile containing caffeine internal standard (0.03 mg/mL) and 50 µL of sample was injected into the HPLC and analyzed using HPLC Analytical Method B: 0→40% MeCN. The samples were run in duplicate, and the average area-under-curve (AUC) values were compared with the t = 0 h timepoint to determine %peptide remaining, caffeine standard runs at t_R = 9.55 min and peptide starting material runs at tR = 10.21 min (FIG.14). [00403] Protease stability: Proteinase K [00404] A vial was sequentially charged with cyclized peptide 5 (SEQ ID NO: 5) (3.75 µL, 40 mM DMSO stock), PBS pH 7.4 (194.3 µL), and Proteinase K (2 µL, 0.05 mg/mL in PBS) and heated to 37°C in an incubator for 48 h. At the specified timepoints, 35 µL of reaction mixture was diluted with 35 µL of acetonitrile containing caffeine internal standard (0.05 mg/mL) and cooled at –20°C for 10 min then centrifuged (20,000×g, 0°C) for 15 min. The supernatant was transferred to a clean HPLC vial for analysis (60 µL injection) using HPLC Analytical Method B: 0→40% MeCN. Caffeine standard runs at t_R = 9.58 min and peptide starting material runs at t_R = 10.24 min. [00405] HPLC Analysis t = 0 h

t = 48 h

[00406] Protease stability: Pepsin 95 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No. 046094-781001WO [00407] A vial was sequentially charged with cyclized peptide 5 (SEQ ID NO: 5) (3.75 µL, 40 mM DMSO stock), PBS pH 2 (194.3 µL), and Proteinase K (2 µL, 1.0 mg/mL in PBS pH 2) and heated to 37°C in an incubator for 48 h. At the specified timepoints, 35 µL of reaction mixture was diluted with 35 µL of acetonitrile containing caffeine internal standard (0.05 mg/mL) and cooled at –20°C for 10 min then centrifuged (20,000×g, 0°C) for 15 min. The supernatant was transferred to a clean HPLC vial for analysis (60 µL injection) using HPLC Analytical Method B: 0→40% MeCN. Caffeine standard runs at t_R = 9.55 min and peptide starting material runs at tR = 10.21 min. [00408] HPLC Analysis t = 0 h

t = 48 h

[00409] Example 20: Split-and-pool synthesis of 8-member peptide library (FIG. 2)

[00410] Resin functionalization, SPPS, N-terminal capping, and cleavage were all carried out using general procedures described above using 24 h amino acid coupling times. Split-and- pool synthesis protocol: Fmoc-deprotected resin was suspended in N,N-dimethylformamide (30 mL/g resin) and divided equally into two separate fritted syringes. Each syringe was drained and then treated with the respective activated amino acid solution (prepared as described above) and rotated for 24 h at room temperature. The resins were then drained and washed with N,N-dimethylformamide (5 × 20 mL/g resin) and dichloromethane (5 × 20 mL/g 96 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO resin) before the resins were combined into a single fritted syringe for the subsequent deprotection procedure. [00411] Following peptide precipitation as described in the General procedure for peptide resin cleavage described above, the crude peptide was used directly in the macrocyclization procedure without further purification. HPLC-MS analysis: HPLC Analytical Method A 0→60% MeCN

[00412] Macrocyclization of 8-member peptide library (FIG.2) 97 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00413] Macrocyclization of the pooled peptide library (14.7 mg, 18.3 µmol) was carried out in 25 mM MOPS buffer (18 mL) using General Macrocyclization Procedure B. After 48 h at 38°C, the reaction mixture was analyzed using HPLC Analytical Method B 0→40% MeCN. Upon complete reaction, the mixture was purified by automated flash column chromatography (isocratic 100% H2O+0.1% TFA, 4 CV, then 70% MeCN/H2O+0.1% TFA, 5 CV). The organic fractions were combined and concentrated under vacuum to give macrocycle library (8.5 mg, 69%). HPLC-MS analysis: HPLC Analytical Method B 0→40% MeCN

98 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00415] Linearization of the pooled peptide library (1.5 mg, 2.22 µmol) was carried out in tetrahydrofuran/methanol (3/1 v/v, 0.20 mL) with a solution of samarium(II) iodide (0.1 M in tetrahydrofuran, 0.30 mL) using the General Linearization Procedure. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. HPLC-MS analysis: HPLC Analytical Method B 0→40% MeCN

99 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00416] Example 21: RGD containing macrocycles targeting integrin family proteins (FIG. 3 and Table 5) [00417] Macrocyclizations of RGD containing peptides (FIG.3 and Table 5) [00418] cyclo-RGD (29a): SPPS

[00419] Resin functionalization, SPPS, and N-terminal capping were carried out using general procedures on a 43 µmol scale as described above. [00420] Resin cleavage leaving protecting groups intact: Cleavage solution (1% TFA in dichloromethane, 10 mL/g resin) was added to the fritted syringe containing resin. The resin suspension was rotated end over end on a rotator for 1 h at room temperature. After 1 h, the filtrate was collected, and the resin washed with dichloromethane (2 × 20 mL/g resin). The combined filtrates were concentrated under vacuum and iodoacetamide capped peptide S14 (34 mg, 94%) was used directly in the following steps with no further purification. Iodoacetamide capped peptide S14 HRMS (ESI) (m/z): calc’d for C31H50IN7O10S [M+H]⁺: 838.2301; found: 838.2305. HPLC analysis: HPLC Analytical Method D 0→95% MeCN

100 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00421] cyclo-RGD (29a): macrocyclization

[00422] A round-bottom flask was sequentially charged with iodoacetamide capped peptide S14 (11.0 mg, 13.1 µmol, 1.0 equiv) and N,N-dimethylformamide (13 mL) at room temperature. Cesium carbonate (43.0 mg, 0.131 mmol, 10 equiv) was then added to the solution and the suspension was sonicated for 10 min. The reaction mixture was then stirred at room temperature for 2 h before being filtered through celite and concentrated under reduced pressure. Reaction analysis was carried out using HPLC Analytical Method D: 0→95% MeCN. The two peaks formed were attributed to monomer and dimer formation. The residue was purified by automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 2 CV, then gradient 0→80% MeCN/H2O+0.1% TFA, 8 CV) to isolate the monomer S15 (5.0 mg, 54%). [00423] The crude residue was then treated with cleavage solution (1.5 mL, 92.5/2.5/2.5/2.5/2.5 v/v/v/v, TFA/TIPS/H₂O/dichloromethane) for 1 h at room temperature before being concentrated under a N2 stream. The peptide was filtered, dried under a N2 stream, and purified by prep HPLC (gradient, 0→15% MeCN/H₂O+0.1% TFA) to give 29a (1.5 mg, 54% yield). Macrocycle 29a HRMS (ESI) (m/z): calc’d for C₁₄H₂₄N₇O₇ [M+H]⁺: 402.1732, found: 402.1731. [00424] HPLC Analysis Post-macrocyclization: HPLC Analytical Method D 0→95% MeCN

101 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00425] cyclo-RGDA (29b) (SEQ ID NO: 84): macrocyclization

[00426] Macrocyclization of peptide 28b (5.0 mg, 8.3 µmol) (SEQ ID NO: 83) was carried out in 25 mM MOPS buffer (8.3 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. After complete conversion, the reaction mixture was desalted using automated flash column chromatography (isocratic 100% H2O+0.1% TFA, 4 CV, then gradient 0→20% MeCN/H2O+0.1% TFA, 8 CV) and further purified on the prep HPLC (gradient, 0→15% MeCN/H₂O+0.1% TFA) to give macrocycle 29b (3.6 mg, MOPS salt, 63%) (SEQ ID NO: 84). Iodoacetamide capped peptide 28b (SEQ ID NO: 83) HRMS (ESI) (m/z): calc’d for C17H30IN8O8 [M+H]⁺: 601.1226, found: 601.1228. Macrocycle 29b (SEQ ID NO: 84) HRMS (ESI) (m/z): calc’d for C₁₇H₂₉N₈O₈ [M+H]⁺: 473.2103, found: 473.2104. [00427] HPLC Analysis t = 0 h – Pure 28b

t = 48 h

Following HPLC purification: HPLC Analytical Method B: 0→40% MeCN

102 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00428] cyclo-RGDAA (29c) (SEQ ID NO: 1): macrocyclization

[00429] Macrocyclization of peptide 28c (4.0 mg, 5.96 µmol) (SEQ ID NO: 85) was carried out in 25 mM MOPS buffer (6.0 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. After complete conversion, the reaction mixture was desalted using automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 4 CV, then gradient 0→30% MeCN/H₂O+0.1% TFA, 8 CV) to give macrocycle 29c (4.1 mg, MOPS salt, 91%) (SEQ ID NO: 1). [00430] Iodoacetamide capped peptide 28c (SEQ ID NO: 85) HRMS (ESI) (m/z): calc’d for C₂₀H₃₅IN₉O₉ [M+H]⁺: 672.1597, found: 672.1600. Macrocycle 29c (SEQ ID NO: 1) HRMS (ESI) (m/z): calc’d for C₂₀H₃₄N₉O₉ [M+H]⁺: 544.2474, found: 544.2475. [00431] HPLC Analysis t = 0 h – Pure 28c

t = 48 h

Following automated flash column chromatography purification: HPLC Analytical Method B: 0→40% MeCN

103 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00432] cyclo-ARGDAA (29d) (SEQ ID NO: 2): macrocyclization

[00433] Macrocyclization of peptide 28d (6.5 mg, 8.76 µmol) (SEQ ID NO: 86) was carried out in 25 mM MOPS buffer (8.8 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. After complete conversion, the reaction mixture was desalted using automated flash column chromatography (isocratic 100% H2O+0.1% TFA, 4 CV, then gradient 0→30% MeCN/H2O+0.1% TFA, 8 CV) and further purified on the prep HPLC (gradient, 0→40% MeCN/H2O+0.1% TFA) to give macrocycle 29d (2.0 mg, MOPS salt, 28%) (SEQ ID NO: 2). [00434] Iodoacetamide capped peptide 28d (SEQ ID NO: 86) HRMS (ESI) (m/z): calc’d for C23H40IN10O10 [M+H]⁺: 743.1968, found: 743.1973. Macrocycle 29d (SEQ ID NO: 2) HRMS (ESI) (m/z): calc’d for C₂₃H₃₉N₁₀O₁₀ [M+H]⁺: 615.2845, found: 615.2847. [00435] HPLC Analysis t = 0 h – Pure 28d

t = 70 h

104 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00436] cyclo-AARGDAA (29e) (SEQ ID NO: 88): macrocyclization

[00437] Macrocyclization of peptide 28e (4.0 mg, 4.92 µmol) (SEQ ID NO: 87) was carried out in 25 mM MOPS buffer (4.9 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method A: 0→60% MeCN for t = 0 h, and HPLC Analytical Method B: 0→40% MeCN for t = 48 h. After complete conversion, the reaction mixture was desalted and purified using automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 4 CV, then gradient 0→30% MeCN/H₂O+0.1% TFA, 10 CV) to give macrocycle 29e (3.9 mg, MOPS salt, 89%) (SEQ ID NO: 88). [00438] Iodoacetamide capped peptide 28e (SEQ ID NO: 87) HRMS (ESI) (m/z): calc’d for C₂₆H₄₅IN₁₁O₁₁ [M+H]⁺: 814.2339, found: 814.2344. Macrocycle 29e (SEQ ID NO: 88) HRMS (ESI) (m/z): calc’d for C26H44N11O11 [M+H]⁺: 686.3216, found: 686.3220. [00439] HPLC Analysis t = 0 h – Pure 28e

105 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

[00440] cyclo-AAARGDAA (29f) (SEQ ID NO: 90): macrocyclization

[00441] Macrocyclization of peptide 28f (2.0 mg, 2.26 µmol) (SEQ ID NO: 89) was carried out in 25 mM MOPS buffer (2.3 mL) using General Macrocyclization Procedure B. Reaction analysis was carried out using HPLC Analytical Method B: 0→40% MeCN. After complete conversion, the reaction mixture was desalted and purified using automated flash column chromatography (isocratic 100% H₂O+0.1% TFA, 4 CV, then gradient 0→30% MeCN/H2O+0.1% TFA, 10 CV) to give macrocycle 29f (1.7 mg, MOPS salt, 78%) (SEQ ID NO: 90). [00442] Iodoacetamide capped peptide 28f (SEQ ID NO: 89) HRMS (ESI) (m/z): calc’d for C29H50IN12O12 [M+H]⁺: 885.2710, found: 885.2716. Macrocycle 29f (SEQ ID NO: 90) HRMS (ESI) (m/z): calc’d for C29H49N12O12 [M+H]⁺: 757.3587, found: 757.3590. [00443] HPLC Analysis t = 0 h – Pure 28f 106 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

t = 72 h

[00444] Example 22: Biological Methodology [00445] ELISA protocol Sources of proteins: Integrin α_Vβ₃ ELISA materials Extracellular matrix (ECM) protein: 1.0 μg/mL, Vitronectin Protein, Human, Recombinant (His Tag); SinoBiological Human Integrin protein: 1.0 µg/mL, Integrin alpha V beta 3 Protein, Human, Recombinant (His Tag), SinoBiological Primary antibody: 2.0 µg/mL, Integrin αV/β3/CD51/CD61 Antibody (23C6): sc-7312, scbt Secondary antibody: 1.0 µg/mL, goat anti-Mouse IgG (H+L) Secondary Antibody, HRP ThermoFisher Scientific Positive control peptide: Cilengitide (purchased from Cayman Chemicals) Integrin α_Vβ₆ ELISA materials ECM protein: 0.4 μg/mL, TGF beta 1 Protein, Human, Recombinant (His Tag); SinoBiological Human Integrin protein: 0.5 μg/mL, Integrin alpha V beta 6 Protein, Human, Recombinant (His Tag), SinoBiological Primary antibody: 1:750 dilution, Anti-Integrin alpha V beta 6 Antibody, Mouse Monoclonal, SinoBiological Secondary antibody: 2.0 μg/mL, goat anti-Mouse IgG (H+L) Secondary Antibody, HRP ThermoFisher Scientific Positive control peptide: RTDLDSLRT (SEQ ID NO: 91) (S16 synthesized by SPPS; HRMS (ESI) (m/z): calc’d for C₄₃H₇₈N₁₅O₁₇ [M+H]⁺: 1076.5695, found: 1076.5698.) HPLC analysis: HPLC Analytical Method A 0→60% MeCN 107 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

Integrin α_IIbβ₃ ELISA materials ECM protein: 10.0 μg/mL; human fibrinogen, ThermoFisher Human Integrin protein: 5.0 μg/mL, native human platelet integrin α_IIbβ₃ (Activation site) (ab95130), Abcam Primary antibody: 2.0 μg/mL, Integrin αIIb/β3/CD41/CD61 Antibody (A 2A9/6): sc-21783, SCBT Secondary antibody: 1.0 μg/mL, goat anti-Mouse IgG (H+L) Secondary Antibody, HRP ThermoFisher Scientific Positive control compound: Tirofiban (purchased from MedChemExpress) [00446] General ELISA procedure (following Kapp et al. protocols (Kapp et al., Sci. Rep. 2017, 7(1):39805)): [00447] 96-well clear, flat-bottom assay plates were coated overnight at 4°C with ECM protein (100 µL per well) in carbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6). Each well was then drained and washed three times with PBS-T-buffer (phosphate-buffered saline/Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween20, pH 7.4; 200 µL). The plate was then blocked for 1 h at room temperature with TS- B-buffer (Tris saline/BSA buffer; 150 µL/well; 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA). A dilution series for each cyclic peptide was then prepared in TS-B-Buffer, alongside a positive control compound. [00448] The plate was then drained, washed thrice with PBS-T buffer (200 µL), then inhibitors were transferred to each well (50 µL), followed by recombinant Human Integrin protein (50 µL, PBS-T buffer). The plate was then incubated for 1 h at room temperature, before draining and washing thrice with PBS-T buffer (200 µL) and treating with primary antibody (100 µL per well) for 1 h at room temperature. The plate was then drained, washed thrice with PBS-T buffer (200 µL), then treated with secondary antibody (100 µL per well) for 1 h at room temperature. The plate was then drained, washed thrice with PBS-T buffer (200 µL), then developed by quick addition of 1-Step Ultra TMB-ELISA Substrate Solution (100 µL per well) and incubated for 30 min at room temperature in the dark. The reaction was stopped with 2 M H₂SO₄ (100 µL/well), and the absorbance was measured at 450 nm with a Clariostar plate reader. 108 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO [00449] The inhibitory activity of each compound was tested in duplicate, and the resulting inhibition curves were analyzed using GraphPad software. The inflection point describes the IC₅₀ value. All determined IC₅₀ values were referenced to the activity of the internal standard. Table 5. Sequences, cyclization yields, and binding affinities of RGD containing hydroxamate linked macrocycles against integrin αVβ3, αVβ6 and αIIbβ3.

^aMacrocyclization carried out using protecting group strategy with arginine and aspartic acid protected linear peptide. [00450] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [00451] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims. 109 AFDOCS:200413442.1

Claims

Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO What is claimed is: 1. A compound having a structure represented by formula I:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: X is an electron withdrawing group; each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. 2. A compound (macrocyclic peptide) having a structure represented by formula II:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. 3. A compound having a structure represented by formula III:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. 110 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO 4. The compound of anyone of claim 1-3, wherein n is an integer from 13-46. 5. The compound of anyone of claim 1-4, wherein X is halo. 6. The compound of claim 5, wherein X is iodo or chloro. 7. The compound of any one of claims 1-6, wherein the non-naturally occurring amino acid contains N-methylation, an aromatic backbone, or a tag. 8. The compound of claim 7, wherein the tag is biotin or a derivative thereof. 9. The compound of any one of claims 1-6, wherein the non-naturally occurring amino acid is a D-amino acid or β-amino acids. 10. A process of preparing a compound of formula I:

stereoisomer thereof, comprising: (i) functionalizing a resin with an O-alkylhydroxylamine group; (ii) coupling the functionalized resin with an optionally substituted naturally or non- naturally occurring amino acid having a terminal amine group; (iii) deprotecting the amine group of the optionally substituted naturally or non-naturally occurring amino acid from step (ii); (iv) repeating steps (ii) and (iii) a number of times until the desired length of n is reached; (v) capping the resin; and (vi) cleaving the compound of formula I off the resin, wherein: X is an electron withdrawing group; each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. 11. A process of preparing a compound of formula II: 111 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

wherein: X is an electron withdrawing group; each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. 12. The process of claim 11, wherein the solvent is a buffered aqueous solvent. 13. The process of claim 12, wherein the buffered aqueous solvent is phosphate-buffered saline. 14. The process of any one of claims 11-13, wherein the mixing is carried out at a temperature of about 0-100°C. 15. The process of claim 14, wherein the mixing is carried out at a temperature of about 30- 60°C. 16. The process of any one of claims 11-15, wherein the pH is about 7.5 to about 10. 17. The process of any one of claims 11-16, wherein the mixing is carried out for about 0.25 to about 240 hours. 18. A process of preparing a compound of formula III:

stereoisomer thereof, comprising reacting a compound of formula II: 112 AFDOCS:200413442.1 Date of Deposit: June 21, 2024 Attorney Docket No.046094-781001WO

SmI₂ in the presence of a solvent, wherein: each A is independently an optionally substituted naturally or non-naturally occurring amino acid; and n is an integer from 3-50. 19. The process of claim 18, wherein the solvent is a solvent mixture. 20. The process of claim 19, wherein the solvent mixture is THF/MeOH. 21. The process of any one of claims 18-20, wherein SmI₂ is in excess. 22. The process of any one of claims 18-21, wherein the reacting is carried out at a temperature of about 0-100°C. 23. The process of claim 22, wherein the reacting is carried out at about room temperature (20-25°C. 24. The process of any one of claims 18-23, wherein the reacting is conducted for about 1 hour. 113 AFDOCS:200413442.1