Radical sam enzyme catalysis for polycyclic peptide library generation

Abstract

The present disclosure provides a method for biocatalytic production of a polycyclic peptide comprising providing a peptide sequence containing a recognition motif for a radical S-adenosylmethionine metallocnzyme (SAM enzyme), exposing the peptide sequence to an enzymatic reaction including catalysis under standard enzymatic reaction conditions with the radical SAM enzyme, and generating a further cyclized peptide by modifying a product of the enzymatic reaction chemically and / or via enzymes. The radical SAM enzyme catalysis generates macrocyclic modifications using unactivated carbon centers. The product of the enzymatic reaction may be modified using haloalkane- or haloacetyl-containing compounds, macrocyclases, or additional radical SAM enzymes. The peptide sequence may be altered at permissive sites to generate libraries of polycyclic peptide structures. The enzymatic reaction may occur at ambient temperature and pH under buffered aqueous conditions.

Description

[0001] Princeton - 107076

[0002] RADICAL SAM ENZYME CATALYSIS FOR POLYCYCLIC PEPTIDE LIBRARY GENERATION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Application No. 63 / 761,520, titled "Radical SAM Enzyme Catalysis for Creation of Polycy clic Peptide and Peptoid Libraries", filed February 21, 2025, which is hereby incorporated by reference in its entirety.

[0005] TECHNICAL FIELD

[0006] The present disclosure relates to biocatalytic methods for peptide modification, and more particularly to the use of radical S-adenosylmethionine metalloenzymes in combination with downstream chemical and enzymatic modifications to generate polycyclic peptide and peptoid libraries.

[0007] BACKGROUND

[0008] Peptides and peptide-based therapeutics represent a growing area of pharmaceutical development due to their ability to interact with biological targets in ways that small molecules cannot. Macrocyclic and polycyclic peptides are of particular interest because their constrained structures can provide enhanced binding specificity, improved metabolic stability, and better cell permeability compared to their linear counterparts.

[0009] The creation of cyclic peptide structures typically involves forming covalent bonds between amino acid residues within the peptide chain. Conventional approaches to peptide cyclization often rely on reactions between activated functional groups, such as the formation of disulfide bonds between cysteine residues, lactam bridges between lysine and glutamic acid side chains, or head-to-tail cyclization through the peptide termini. While these methods have proven useful, they are generally limited to forming bonds at positions bearing reactive functional groups.

[0010] Polycyclic peptides, which contain multiple ring systems within a single molecule, present additional synthetic challenges. The construction of such architectures often requires multiple sequential cyclization steps, each of which may impose constraints on the types of amino acids and positions that can participate in ring formation. Furthermore, the formation of carbon-carbon bonds at unactivated carbon centers within peptide side chains remains difficult to achieve using conventional synthetic chemistry.Princeton - 107076

[0011] Radical S-adenosylmethionine (SAM) metalloenzymes represent a diverse superfamily of enzymes that catalyze a wide range of chemical transformations. These enzymes utilize a [4Fe-4S] cluster and S-adenosylmethionine as a cofactor to generate highly reactive radical intermediates capable of performing challenging chemical reactions under mild conditions. Some members of this enzyme family have been identified as participating in the biosynthesis of Ribosomally synthesized and Post-translationally modified Peptides (RiPPs), where they install unusual structural modifications including crosslinks between amino acid side chains.

[0012] The development of peptide libraries for screening against biological targets has become an established approach in drug discovery'. Such libraries allow researchers to identify peptide sequences with desired binding properties or biological activities. However, the structural diversity accessible within peptide libraries is often constrained by the available methods for introducing modifications and cyclizations.

[0013] There remains interest in developing methodologies that can expand the chemical space accessible to peptide libraries, particularly approaches that can introduce structural features not readily achievable through conventional synthetic methods.

[0014] SUMMARY

[0015] This summary' is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0016] According to an aspect of the present disclosure, a method for biocatalytic production of a polycyclic peptide is provided. The method comprises providing a peptide sequence containing a recognition motif for a radical S-adenosylmethionine metalloenzyme (SAM enzyme). The method further comprises exposing the peptide sequence to an enzymatic reaction, the enzymatic reaction including catalysis under standard enzy matic reaction conditions with the radical SAM enzyme. The method further comprises generating a further cyclized peptide by modifying a product of the enzymatic reaction chemically and / or via enzymes. This approach enables the creation of new-to-nature polycyclic peptides that are not accessible with existing technologies, and the reaction occurs at ambient temperature and pH under buffered aqueous conditions, making it easily scalable for preparation of larger quantities of a desired peptide.

[0017] According to other aspects of the present disclosure, the enzymatic reaction may form at least one carbon-carbon, carbon-sulfur, carbon-nitrogen and / or carbon-oxygen bond at onePrinceton - 107076

[0018] or more unactivated carbon centers of the peptide sequence, regardless of the nature of the sidechain. The ability to form bonds at unactivated carbon centers expands the chemical space accessible to peptide libraries beyond what is achievable through conventional synthetic methods that rely on activated functional groups.

[0019] According to other aspects of the present disclosure, non-canonical amino acids may¬ be incorporated into the peptide. The incorporation of non-canonical amino acids allows for the introduction of structural diversity and functional groups not present in natural amino acids, thereby expanding the range of polycyclic peptide structures that can be generated.

[0020] According to other aspects of the present disclosure, non-canonical amino acid sidechain^) may be crosslinked using a radical SAM enzyme. This capability- enables the formation of crosslinks involving non-natural side chains, further expanding the structural diversity of the resulting polycyclic peptide libraries.

[0021] According to other aspects of the present disclosure, the enzy matic reaction may form at least one ly sine-try ptophan crosslink at an unactivated carbon center of the peptide sequence. The lysine-tryptophan crosslink represents a specific type of carbon-carbon bond formation at unactivated positions that creates constrained macrocyclic structures with defined conformational properties.

[0022] According to other aspects of the present disclosure, the radical SAM enzyme may be but is not limited to SuiB, WgkB, GggB, TqqB, ItfD, HghC, HghB, RrrB, NxxcB. KgrB, FwwB. WprB. OscB, XncB, RscB. and HtkB or orthologs thereof. These enzymes install a variety of carbon-carbon, carbon-sulfur (e.g. thioethers), carbon-nitrogen and carbon-oxygen (e.g. aliphatic ethers) bonds at unactivated carbon centers, providing multiple options for introducing different ty pes of crosslinks into peptide substrates.

[0023] According to other aspects of the present disclosure, the product of the enzymatic reaction may be modified chemically using a haloalkane- or haloacetyl-containing compound. Chemical modification of the enzymatic product enables the introduction of additional macrocyclic constraints, creating polycyclic architectures with increased structural complexity-.

[0024] According to other aspects of the present disclosure, the haloalkane- or haloacetyl-containing compound may include 1,3-dibromoacetone and / or 4,4'-Bis(bromomethyl)-2-2'-bipyridine. These specific reagents react with cysteine-containing peptides to form additional macrocycles, with the bipyridine compound providing a rigid aromatic linker and the dibromoacetone providing a ketone-containing bridge.

[0025] According to other aspects of the present disclosure, the product of the enzymatic reaction may be modified chemically using a radical SAM enzyme or a macrocyclase. The usePrinceton - 107076

[0026] of additional enzymatic modification steps allows for the sequential installation of multiple crosslinks, yielding polycyclic peptides with increased complexity.

[0027] According to other aspects of the present disclosure, the macrocyclase (SurE) may be a member of the thioesterase family or a homolog thereof. The use of SurE or related macrocyclases provides a biocatalytic route to additional macrocyclization that operates under mild aqueous conditions compatible with the radical SAM enzyme products.

[0028] According to other aspects of the present disclosure, modifying a product of the enzymatic reaction may include generating a proteolyzed product by proteolyzing the product and then chemically or enzy matically modify ing the proteolyzed product. Proteolysis allows for the removal of portions of the peptide sequence, such as recognition motifs, to yield truncated polycyclic peptides with reduced molecular weight while retaining the crosslinked core structure.

[0029] According to other aspects of the present disclosure, the proteolyzed product may be modified chemically using a haloalkane- or haloacetyl-containing compound. This combination of proteolysis followed by chemical cyclization enables the generation of compact polycyclic peptide structures with multiple ring systems.

[0030] According to other aspects of the present disclosure, the proteolyzed product of the enzymatic reaction may be modified chemically using a radical SAM enzyme or a macrocyclase. This approach provides flexibility in the order and type of modifications applied to generate diverse polycyclic peptide architectures.

[0031] According to other aspects of the present disclosure, the method may further comprise generating at least one additional peptide sequence by altering the peptide sequence at permissive sites. The method may further comprise exposing the at least one additional peptide to an enzymatic reaction, the enzymatic reaction including catalysis under standard enzymatic reaction conditions with the radical SAM enzyme. The method may further comprise generating a further cyclized peptide by modifying a product of the enzymatic reaction chemically and / or via enzymes. This approach enables the systematic generation of libraries of polycyclic peptides with varied sequences while maintaining the crosslinked architecture.

[0032] According to other aspects of the present disclosure, the method may further comprise generating a library of crosslinked peptides by exposing a library of peptides, consisting as a mixture, to radical SAM enzy me catalysis. Processing peptide libraries as mixtures provides an efficient route to generating diverse collections of crosslinked peptides for subsequent screening applications.Princeton - 107076

[0033] According to other aspects of the present disclosure, the crosslinked library may be further modified chemically or enzymatically as described above. The ability to apply downstream modifications to entire libraries enables the generation of polycyclic peptide collections with enhanced structural diversity for screening against desired phenotypes.

[0034] According to another aspect of the present disclosure, a kit is provided. The kit contains a plurality of further cyclized peptides generated using the method described above. Such a kit provides ready access to collections of polycyclic peptides for screening in any desired assay without requiring the end user to perform the biocatalytic synthesis.

[0035] According to another aspect of the present disclosure, a composition of matter is provided. The composition of matter has a structure according to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6. These compounds represent specific polycyclic peptide structures generated through the disclosed methodology, each featuring distinct combinations of enzymatic and chemical crosslinks that create constrained bicyclic architectures suitable for applications as inhibitors of protein-protein interactions and as specific modulators of biological phenomena.

[0036] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.

[0037] BRIEF DESCRIPTION OF FIGURES

[0038] Non-limiting and non-exhaustive examples are described with reference to the following figures.

[0039] Figure 1 is a chemical structure of Compound 1, a polycyclic peptide.

[0040] Figure 2 is a chemical structure of Compound 2, a polycyclic peptide.

[0041] Figure 3 is a chemical structure of Compound 3, a polycyclic peptide.

[0042] Figure 4 is a chemical structure of Compound 4, a polycyclic peptide.

[0043] Figure 5 is a chemical structure of Compound 5, a polycyclic peptide.

[0044] Figure 6 is a chemical structure of Compound 6, a polycyclic peptide.

[0045] Figure 7 is an image of a computational model of a polycyclic peptide cyclized by SuiB, proteolyzed by trypsin, and then cyclized by 1,3 dibromoacetone. The peptide bears the characteristic C-C bond between the lysine beta carbon to the 7 position of the indole of tryptophan 4 residues downstream, as well as the acetone moiety cyclizing two cysteine residues.Princeton - 107076

[0046] Figure 8 is an image of a computational model of the peptide in Figure 7 bound to Cathepsin B.

[0047] Figure 9 is an image of a computation model of the peptide in Figure 7 bound to Cathepsin B, but with Cathepsin B shown as a space filling model.

[0048] Figure 10 is a graph showing inhibition of Cathepsin B using the peptide of Figure 7, where the peptide displays an ICso of 2.33uM against Cathepsin B.

[0049] Figure 11 is an image of a computational model of polycyclic peptide cyclized by WgkB, proteolyzed by trypsin, and then cyclized by 1,3 dibromoacetone. Peptide bears the characteristic C-C bonds between the tryptophan 5 and 6 positions and the lysine alpha and delta positions respectively two residues downstream, as well as the acetone moiety cyclizing two cysteine residues.

[0050] Figure 12 is an image of a computational model of the peptide in Figure 11 bound to FKBP12.

[0051] Figure 13 is an image of a computational model of the peptide in Figure 11 bound to FKBP12. with FKBP12 shown as a space filling model.

[0052] DETAILED DESCRIPTION

[0053] The following description sets forth exemplary7aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.

[0054] In one general embodiment, a method for biocatalytic production of a polycyclic peptide comprises providing a peptide sequence containing a recognition motif for a radical S-adenosylmethionine metalloenzyme (SAM enzyme), exposing the peptide sequence to an enzymatic reaction, the enzymatic reaction including catalysis under standard enzymatic reaction conditions with the radical SAM enzy me, and generating a further cyclized peptide by modifying a product of the enzymatic reaction chemically and / or via enzymes. The biocatalytic reaction may occur at ambient temperature and pH in aqueous solvent and may be devoid of toxic or dangerous components.

[0055] In some cases, the radical SAM metalloenzymes generate macrocyclic modifications at unactivated carbon centers of the peptide sequence. The enzymatic reaction may introduce crosslinks between amino acid side chains that are not accessible through conventional chemical synthesis methods. The product of the enzymatic reaction may then be subjected toPrinceton - 107076

[0056] downstream chemical modification and / or enzymatic modification to create polycyclic peptides having multiple ring systems.

[0057] A plurality of radical SAM enzymes may catalyze reactions to introduce heterocyclic and macrocyclic modifications into peptide substrates. Over a dozen radical SAM enzymes have been identified that catalyze such reactions. The radical SAM enzymes may install a variety of bond types at unactivated carbon centers, including carbon-carbon bonds, carbonsulfur bonds, carbon-nitrogen and carbon-oxy gen bonds.

[0058] The methodology may be used to generate mono-, bi-, and polycyclic peptides. By combining radical SAM enzyme catalysis with downstream chemical and / or enzymatic modification, new-to-nature polycyclic and macrocyclic peptides may be generated. Libraries of macro- and polycyclic peptide structures may be generated by modifying the peptide sequence at permissive sites, as described above. The sequence of the peptide may be modified via peptide synthesis, allowing generation of a library' of polycyclic peptide molecules from a common enzymatic transformation. Such polycyclic peptides may serve as inhibitors of protein-protein interactions or as modulators of biological phenomena.

[0059] As used herein, the term "radical S-adenosylmethiomne metalloenzyme" or "radical SAM enzyme" refers to any enzyme belonging to the radical SAM superfamily that utilizes a [4Fe-4S] cluster and S-adenosylmethionine as a cofactor to generate radical intermediates capable of catalyzing chemical transformations. This term encompasses naturally occurring enzymes, engineered variants, mutants, homologs, and functional equivalents thereof that retain the ability7to catalyze bond formation at unactivated carbon centers within peptide substrates. Examples include, but are not limited to, SuiB, WgkB, GggB, TqqB, and enzymes sharing sequence homology or functional similarity thereto (see above).

[0060] As used herein, the term "unactivated carbon center" refers to a carbon atom within an amino acid side chain or peptide backbone that does not bear a functional group conventionally reactive under standard synthetic conditions, such as a leaving group, electrophilic carbonyl, or nucleophilic heteroatom. Unactivated carbon centers include, but are not limited to, aliphatic carbons in lysine, leucine, isoleucine, and valine side chains, as well as aromatic carbons in tryptophan, phenylalanine, and tyrosine residues that are not inherently activated toward nucleophilic or electrophilic attack.

[0061] As used herein, the term "permissive sites" refers to positions within a peptide sequence where amino acid substitutions, insertions, or deletions may be made without abolishing the ability of a radical SAM enzyme to recognize and modify the peptide substrate. PermissivePrinceton - 107076

[0062] sites may be identified through sequence analysis, structural modeling, or empirical testing, and may vary’ depending on the specific radical SAM enzyme employed.

[0063] As used herein, the term "recognition motif' refers to a sequence of amino acids within a peptide substrate that is recognized by a radical SAM enzy me and is necessary for enzymatic activity. The recognition motif may include a leader peptide, a core peptide region, or specific amino acid residues that interact with the enzyme active site or auxiliary domains. Recognition motifs may be native sequences or engineered variants that retain the ability to direct enzymatic modification.

[0064] As used herein, the term "macrocyclase" refers to any enzyme capable of catalyzing the formation of a macrocyclic ring within a peptide substrate. Macrocyclases include, but are not limited to, members of the thioesterase family such as SurE. as well as homologs, variants, and functional equivalents thereof that catalyze head-to-tail cyclization, side chain-to-terminus cyclization, or other macrocyclization reactions under aqueous conditions.

[0065] As used herein, the term "polycyclic peptide" refers to a peptide molecule containing two or more ring systems formed through covalent bonds between amino acid residues. The ring systems may be formed through enzymatic crosslinks, chemical crosslinks, or combinations thereof, and may include macrocyclic rings, fused rings, bridged rings, or spiro ring systems. Polycyclic peptides encompass bicyclic peptides as well as peptides containing three or more ring systems.

[0066] As used herein, the term "non-canonical amino acids" refers to amino acids other than the twenty standard proteinogenic amino acids encoded by the genetic code. Non-canonical amino acids include, but are not limited to, amino acids bearing modified side chains, unnatural stereochemistry’, backbone modifications, isotopic labels, reactive handles, or functional groups not present in standard amino acids. Non-canonical amino acids may be incorporated into peptides through chemical synthesis, genetic code expansion, or post-translational modification.

[0067] As used herein, the term "homolog" refers to a polypeptide that shares a common ancestral sequence with a reference polypeptide, encompassing both orthologs (arising via speciation) and paralogs (arising via gene duplication).

[0068] As used herein, the term "ortholog" refers to a polypeptide in a first species that is the functional equivalent of a corresponding polypeptide in a second species, having evolved from a common ancestral gene via speciation. In the specific context of Radical S-adenosylmethionine (SAM) enzymes of the SPASM / Twitch subfamily (e.g., SuiB), an ortholog is defined as a polypeptide that: (i) comprises an amino acid sequence having at leastPrinceton - 107076

[0069] 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity to a reference sequence; (ii) maintains the canonical radical SAM CX3CX2C motif and a C-terminal SPASM / Twitch domain characterized by a seven-cysteine motif (CX9 15GX4C-CX2CX5CX3C-C) capable of binding auxiliary [4Fe-4S] clusters; (iii) catalyzes the formation of an intramolecular carbon-carbon or carbon-sulfur bond (e.g., aLys-Trp cross-link) on a ribosomally synthesized precursor peptide; and / or (iv) is identified as a Reciprocal Best Hit (RBH) using a sequence alignment algorithm such as BLASTP.

[0070] As used herein, the term "paralog" refers to a gene or polypeptide within the same genome as a reference sequence that has evolved from a common ancestral gene via a duplication event. For the purposes of this disclosure, a paralog of a reference enzyme is defined as a polypeptide that: (i) shares at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the reference enzyme, particularly within conserved catalytic domains; (ii) is encoded by a distinct genetic locus within the same species or strain as the reference enzyme; (iii) catalyzes a reaction that is chemically related to the reference enzyme but may differ in substrate preference, optimal pH, temperature stability , or kinetic parameters (Km or Vmax); and (iv) retains the fundamental protein architecture of the reference enzyme family (e g., the radical SAM core) while potentially possessing variations in auxiliary domains (e.g., modified SPASM / Twitch domains) that confer distinct substrate binding affinities.

[0071] The definitions provided herein are intended to encompass functional equivalents and variants within the scope of the disclosure, including molecules and enzymes that perform substantially the same function in substantially the same way to achieve substantially the same result as those explicitly described.

[0072] The radical SAM enzymes suitable for the disclosed method include but are not limited to SuiB, WgkB, GggB, ItfD. HghC, HghB, RrrB, NxxcB KgrB and TqqB. In some cases, the radical SAM enzyme is SuiB, WgkB, GggB, ItfD, HghC, HghB, RrrB, NxxcB KgrB or TqqB. Each of these enzymes belongs to the radical SAM superfamily and utilizes a [4Fe-4S] cluster in conjunction with S-adenosylmethionine to generate radical intermediates that catalyze sidechain crosslinking reactions within peptide substrates.

[0073] SuiB is a radical SAM enzyme that catalyzes the formation of a lysine-tryptophan crosslink at unactivated carbon centers of a peptide sequence. The enzymatic reaction catalyzed by SuiB forms at least one lysine-tryptophan crosslink at an unactivated carbon center of the peptide sequence. The lysine-tr ptophan crosslink involves bond formation between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions.Princeton - 107076

[0074] WgkB is a radical SAM enzyme that catalyzes crosslinking reactions between amino acid side chains at unactivated carbon centers. WgkB installs two carbon-carbon bonds between the alpha and delta carbons of a lysine residue to the unactivated C5 and C6-position on the indole sidechain of tryptophan. Homologs of WgkB sharing sequence identity or functional similarity may also be employed in the disclosed method.

[0075] GggB is a radical SAM enzyme that catalyzes the formation of crosslinks at unactivated carbon centers within peptide substrates. GggB catalyzes thioether linkages between cysteine sidechains and unactivated carbon centers Functional equivalents of GggB that retain the ability to catalyze sidechain crosslinking reactions at unactivated carbon centers may be substituted in the disclosed method.

[0076] TqqB is a radical SAM enzyme that installs crosslinks between amino acid residues at unactivated carbon centers. TqqB catalyzes the formation of aliphatic ether bonds at unactivated carbon centers

[0077] The enzymatic reaction may form at least one carbon-carbon, carbon-sulfur, carbonnitrogen and / or carbon-oxygen bond at one or more unactivated carbon centers of the peptide sequence, regardless of the nature of the side-chain. Carbon-sulfur bonds formed by radical SAM enzymes include thioether linkages, wherein a sulfur atom of a cysteine or methionine residue is covalently bonded to a carbon atom of another amino acid side chain. Carbon-oxy gen bonds formed by radical SAM enzymes include aliphatic ether linkages, wherein an oxygen atom of a serine, threonine, or tyrosine residue is covalently bonded to a carbon atom of another amino acid side chain.

[0078] Alternative radical SAM enzymes and functional equivalents that catalyze sidechain crosslinking reactions may be employed in the disclosed method. Such alternative enzymes include homologs of SuiB, WgkB, GggB, TqqB ItfD, HghC, HghB. RrrB, NxxcB. KgrB, FwwB, WprB, OscB, XncB, RscB, and HtkB identified through sequence homology searches, structural analysis, or functional screening. Enzymes sharing at least about 30% to about 90% sequence identity with SuiB, WgkB, GggB, or TqqB, and more typically from about 40% to about 80% sequence identity, may retain the ability to catalyze crosslinking reactions at unactivated carbon centers. Engineered variants of radical SAM enzymes bearing mutations that alter substrate specificity, catalytic efficiency, or stability may also be employed. Chimeric enzymes comprising domains from multiple radical SAM enzymes may be constructed to achieve desired crosslinking specificities.

[0079] It is expected that the ability of these rSAM enzymes, including SuiB. WgkB. GggB, TqqB ItfD, HghC, HghB, RrrB, NxxcB and KgrB, and their homologs, to catalyze thePrinceton - 107076

[0080] formation of carbon-carbon (C-C) or carbon-sulfur (C-S) bonds at unactivated carbon centers is rooted in their conserved Radical SAM core and specialized SPASM / T witch domains. Each of these enzymes contains a signature CX3CX2C motif that coordinates a [4Fe-4S] cluster. This cluster facilitates the reductive cleavage of S-adenosylmethionine (SAM) to generate a highly reactive 5'-deoxyadenosyl radical (5'-dA-). This radical is chemically potent enough to abstract a hydrogen atom from chemically inert sp3-hybridized carbons — such as those on the side chains of lysine, tryptophan, or valine — thereby "activating" the substrate for subsequent cross-linking.

[0081] What distinguishes this specific group of enzymes (e.g., SuiB, WgkB, GggB, TqqB, etc.) is their classification within the SPASM / T witch subfamily, characterized by an extended C -terminal sequence. While SuiB and WgkB belong to the SPASM class (containing two auxiliary [4Fe~4S] clusters, Aux I and Aux II), GggB and TqqB typically fall into the Twitch class (containing only one auxiliary cluster). These auxiliary clusters may be coordinated by a series of precisely spaced cysteine residues — such as the CX9-15GX4C and CX2CX5C motifs — and act as essential redox conduits. Alternative coordinations, e.g. by Glu or Asp, are possible as well. They may facilitate the abstraction of a second electron from the substrate radical intermediate, a critical step that may prevent the reaction from stalling and ensures the formation of a stable macrocyclic cross-link.

[0082] The architectural precision of these reactions may be further governed by the RiPP Recognition Element (RRE) at the N-terminus of their sequences. The RRE may provide the binding energy necessary to recruit the precursor peptide substrate (like SuiA or WgkA) and orient it within the catalytic pocket. By anchoring the substrate's leader peptide, the RRE ensures that the target unactivated carbon centers are held in the exact proximity and orientation required to be struck by the 5'-dA- radical. This sequence-dependent positioning is why these enzymes can achieve near-perfect regioselectivity across different RiPP (Ribosomally synthesized and Post-translationally modified Peptide) families, despite the extreme reactivity7of the radical intermediates involved.

[0083] The radical SAM enzymes may be obtained from natural sources, produced recombinantly in heterologous expression systems, or synthesized through cell-free protein synthesis methods. Expression systems suitable for production of radical SAM enzymes include bacterial hosts such as Escherichia coli, yeast hosts such as Saccharomyces cerevisiae or Pichia pastoris, insect cell systems, and mammalian cell systems. The enzymes may bePrinceton - 107076

[0084] purified using affinity chromatography, ion exchange chromatography, size exclusion chromatography, or combinations thereof.

[0085] The peptide substrate containing the recognition motif for a radical SAM enzyme may be prepared using standard solid-phase peptide synthesis or solution-phase peptide synthesis methods. In some cases, the peptide substrate is a SuiA peptide or a variant thereof containing the recognition motif for SuiB. The peptide substrate may be synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, Boc (tert-butyloxycarbonyl) chemistry, or other protecting group strategies suitable for peptide assembly.

[0086] The C-terminus of the peptide substrate may be activated prior to radical SAM enzyme catalysis. In one embodiment, the SuiA peptide is suitably prepared with an activated C-terminus prior to SuiB catalysis. Activation of the C-terminus may be achieved through formation of a thioester, an ester, an amide with a leaving group, or other activated carboxyl derivatives. The activated C-terminus may facilitate downstream enzymatic reactions, such as macrocyclization by a macrocyclase enzyme, or may serve as a handle for further chemical modification. Methods for C-terminal activation include, but are not limited to, coupling with thiol-containing reagents to form thioesters, activation with carbodiimide reagents, or incorporation of C-terminal amino acids bearing activated side chains during peptide synthesis.

[0087] In some cases, the peptide substrate may be modified to contain two cysteine residues for subsequent chemical cyclization with haloalkane-containing compounds or haloacetyl-containing compounds. The SuiA peptide or other peptides bearing permissive residues for radical SAM catalysis may be modified to incorporate two cysteine residues at positions that allow formation of a macrocyclic ring upon reaction with a bifunctional electrophile. The cysteine residues may be positioned at permissive sites within the peptide sequence such that incorporation of the cysteine residues does not abolish recognition by the radical SAM enzyme. The thiol groups of the two cysteine residues may serve as nucleophiles for reaction with haloalkane-containing compounds, including 4,4'-Bis(bromomethyl)-2,2'-bipyridine, or haloacetyl-containing compounds, including 1,3-dibromoacetone, to form thioether linkages that create an additional macrocyclic ring within the polycyclic peptide product.

[0088] The peptide substrate may be purified following synthesis using reverse-phase high-performance liquid chromatography (RP-HPLC), preparative HPLC, or other chromatographic methods. The identity and purity' of the peptide substrate may be confirmed using mass spectrometry, amino acid analysis, or other analytical techniques. The peptide substrate may be lyophilized and stored under conditions that preserve the integrity of the peptide, such asPrinceton - 107076

[0089] storage at temperatures from about -80°C to about -20°C under inert atmosphere or in the presence of desiccants.

[0090] The enzymatic reaction process for radical SAM enzyme catalysis may be performed according to a reproducible protocol under defined reaction conditions. The enzymatic reaction may be conducted in an aqueous buffer system at ambient temperature and physiological pH, providing conditions compatible with enz me activity while avoiding harsh chemical reagents or elevated temperatures.

[0091] The buffer system for the enzymatic reaction may comprise HEPES (4-(2-hydroxyethyl)-! -piperazineethanesulfonic acid) buffer at a concentration from about 10 mM to about 100 mM, and more ty pically from about 25 mM to about 75 mM, and in one embodiment at about 50 mM. The pH of the buffer may be maintained from about 7.0 to about 8.0, and more preferably from about 7.4 to about 7.8, and in one embodiment at about pH 7.6. Alternative buffer systems that maintain the pH within the specified range and are compatible yvith radical SAM enzyme activity may be substituted, including Tris-HCl buffer, phosphate buffer, or MOPS buffer at comparable concentrations and pH values.

[0092] The enzymatic reaction is performed in the presence of cofactor S-adenosylmethionine (SAM), which serves as the source of the 5'-deoxyadenosyl radical generated by the [4Fe-4S] cluster of the radical SAM enzyme. The concentration of S-adenosylmethionine in the reaction mixture may range from about 0.1 mM to about 10 mM, and more typically from about 0.5 mM to about 5 mM, and in one embodiment from about 1 mM to about 2 mM. S-adenosylmethionine may be provided as a chloride salt, sulfate salt, or other pharmaceutically acceptable salt form. The cofactor may be added to the reaction mixture immediately prior to initiation of the enzymatic reaction to minimize degradation.

[0093] A reductant is included in the reaction mixture to maintain the [4Fe-4S] cluster of the radical SAM enzyme in the reduced state required for catalytic activity. The reductant may be sodium dithionite or titanium(III) citrate. Sodium dithionite may be employed at a concentration from about 1 mM to about 20 mM, and more ty pically from about 2 mM to about 10 mM, and in one embodiment at about 5 mM. Titanium(III) citrate may be employed at a concentration from about 0.5 mM to about 10 mM, and more typically from about 0.5 mM to about 2.5 mM. Other reducing agents capable of maintaining the [4Fe-4S] cluster in the reduced state may be substituted, including dithiothreitol, flavodoxin / flavodoxin reductase systems, or ferredoxin / ferredoxin reductase systems.

[0094] The enzymatic reaction occurs at ambient temperature, typically from about 20°C to about 30°C, and more preferably from about 22°C to about 28°C, and in one embodiment atPrinceton - 107076

[0095] about 25°C. The reaction may be performed under anaerobic conditions to prevent oxidation of the [4Fe-4S] cluster and the reductant. Anaerobic conditions may be achieved by conducting the reaction in an anaerobic chamber, by purging the reaction vessel with an inert gas such as nitrogen or argon, or by using degassed buffers and reagents.

[0096] The peptide substrate containing the recognition motif for the radical SAM enzyme is combined with the enzyme in the buffered reaction mixture containing the cofactor and reductant. The concentration of the peptide substrate may range from about 10 pM to about 10 mM, and more typically from about 50 pM to about 2 mM, and in one embodiment from about 100 pM to about 500 pM. The concentration of the radical SAM enzyme may range from about 0.1 pM to about 100 pM, and more typically from about 1 pM to about 50 pM, depending on the desired reaction rate and substrate conversion.

[0097] The reaction mixture may be incubated for a period from about 30 minutes to about 24 hours, and more typically from about 1 hour to about 12 hours, and in one embodiment from about 2 hours to about 6 hours, to allow the enz matic reaction to proceed to completion or to a desired level of conversion. The progress of the reaction may be monitored by removing aliquots at defined time intervals and analyzing the aliquots using liquid chromatography-mass spectrometry (LC-MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), or other analytical techniques capable of distinguishing the substrate peptide from the crosslinked product.

[0098] Upon completion of the enzymatic reaction, the product containing the crosslink installed by the radical SAM enzyme may be isolated from the reaction mixture. Isolation methods may include quenching the reaction by heat, addition of acid or organic solvent, followed by centrifugation to remove precipitated protein, and purification of the crosslinked peptide product using reverse-phase high-performance liquid chromatography and / or solidphase extraction. The isolated product may be characterized by mass spectrometry to confirm the presence of the crosslink and by tandem mass spectrometry or nuclear magnetic resonance spectroscopy to determine the site of crosslink formation.

[0099] The enzymatic reaction conditions described herein may be adapted for use with radical SAM enzymes other than SuiB, including WgkB, GggB, ItfD, HghC, HghB, RrrB, NxxcB, KgrB, TqqB, FwwB, WprB, OscB, XncB, RscB, and HtkB. Each radical SAM enzyme may have optimal reaction conditions that differ from those described for SuiB, and the buffer concentration, pH, cofactor concentration, reductant type and concentration, temperature, and reaction time may be adjusted accordingly. Optimization of reaction conditions for a particularPrinceton - 107076

[0100] radical SAM enzyme may be performed by systematic variation of individual parameters while monitoring product formation.

[0101] The product of the enzymatic reaction catalyzed by a radical SAM enzyme may be modified using a macrocyclase to generate a further cyclized peptide. In some cases, the product of the enzy matic reaction is modified chemically using a radical SAM enzyme or a macrocyclase. The macrocyclase catalyzes the formation of an additional macrocyclic ring within the peptide substrate, thereby converting a monocyclic peptide product of radical SAM enzyme catalysis into a bicyclic or polycyclic peptide.

[0102] SurE is a macrocyclase that may be employed for enzymatic modification of radical SAM enzyme products. The macrocyclase SurE is a member of the thioesterase family or a homolog thereof. SurE catalyzes head-to-tail macrocyclization or side chain-to-terminus cyclization reactions, depending on the peptide substrate and the positioning of reactive functional groups within the peptide sequence. The bonds installed by SurE create an additional macrocycle through enzymatic catalysis, yielding polycyclic peptide products having increased structural complexity’ relative to the monocyclic products of radical SAM enzyme catalysis alone.

[0103] Homologs of SurE belonging to the thioesterase family may be substituted for SurE in the disclosed method. Such homologs include enzymes sharing sequence identity’ or functional similarity with SurE that retain the ability to catalyze macrocyclization reactions under aqueous conditions. Enzymes sharing from about 25% to about 95% sequence identity with SurE, and more typically from about 35% to about 85% sequence identity, may retain macrocyclase activity’ suitable for the disclosed method. Functional equivalents of SurE that catalyze comparable macrocyclization reactions through alternative catalytic mechanisms may also be employed.

[0104] The ability of SurE or its homologs to perform macrocyclization in aqueous conditions — rather than simple proteolysis (hydrolysis) — may7be primarily driven by a specific structural feature known as the "Hydrophobic Lid" or the Acyl-Binding Pocket (ABP). In some instances, this ability7derives from its C-terminal a / p-hydrolase domain, which contains the catalytic triad (Ser419, Asp528, His554). However, it is expected that two specific elements are responsible for the cyclization chemistry7:

[0105] 1. The Hydrophobic Capping Loop (The "Lid"). In standard proteases, the active site is open to water, allowing H2O to attack the acyl-enzyme intermediate and release a linear peptide (hydrolysis). In SurE, a specific loop sequence creates a hydrophobic enclosure around the substrate. This loop is part of the fl- pro pel I er domain that sits over the catalytic site. ItPrinceton - 107076

[0106] physically excludes bulk water from the active site once the surugamide precursor is bound. By "drying out" the active site, it prevents hydrolysis and forces the N-terminus of the peptide to act as the nucleophile instead of water.

[0107] 2. The Specificity Pocket (Acyl-Binding) / . The sequence surrounding the catalytic Ser419 is tuned to accommodate the specific hydrophobic side chains of the surugamide peptide (which is rich in Isoleucine and Leucine). The "G-X-S-X-G" motif common to serine proteases is present, but in SurE, the flanking residues create a narrow, greasy channel. Because SurE is a prolyl oligopeptidase, it specifically recognizes the Proline residue at the C-terminus of the core peptide (e.g., a "Proline trap"). This positioning is what precisely orients the peptide chain so that the N-terminus is perfectly aligned to attack the ester bond, forming the macrocycle.

[0108] The enzymatic modification using a macrocyclase may be performed sequentially following radical SAM enzyme catalysis. In one embodiment, the peptide substrate is first exposed to radical SAM enzyme catalysis to install a crosslink at unactivated carbon centers, and the crosslinked product is subsequently mixed with SurE or another macrocyclase to establish an additional macrocyclic ring. The sequential combination of radical SAM enzyme catalysis followed by macrocyclase catalysis yields bicyclic peptide products containing two distinct ring systems formed through enzy matic transformations.

[0109] Products of radical SAM enzymes may be combined alternatively or in conjunction with other radical SAM enzymes and / or with macrocyclases such as SurE to yield additional complexity and polycyclic peptides. In some cases, the product of a first radical SAM enzyme reaction may be subjected to catalysis by a second radical SAM enzyme to install an additional crosslink, followed by macrocyclase catalysis to form a further macrocyclic ring. Such sequential or combinatorial enzyme treatments may generate polycyclic peptides containing three or more ring systems. The order of enzymatic modifications may be varied to access different polycyclic architectures from a common peptide substrate.

[0110] The reaction conditions for macrocyclase catalysis may be compatible with those employed for radical SAM enzyme catalysis, allowing the enzymatic modifications to be performed in a single reaction vessel or in sequential reactions without extensive purification between steps. The macrocyclase reaction may be conducted in an aqueous buffer system at ambient temperature and physiological pH. The concentration of the macrocyclase in the reaction mixture may range from about 0.1 pM to about 100 pM. and more typically from about 1 pM to about 50 pM. The reaction may be incubated for a period from about 30 minutes toPrinceton - 107076

[0111] about 24 hours to allow macrocyclization to proceed to completion or to a desired level of conversion.

[0112] In some cases, modifying a product of the enzymatic reaction includes generating a proteolyzed product by proteolyzing the product and then chemically or enzymatically modifying the proteolyzed product. Proteolysis may be employed to remove leader peptide sequences, recognition motifs, or other portions of the peptide substrate that are not desired in the final polycyclic peptide product. The proteolyzed product retains the crosslink installed by the radical SAM enzyme while having a reduced molecular weight and altered peptide sequence relative to the full-length crosslinked product.

[0113] Proteolysis may be performed using endoproteases, exoproteases, or combinations thereof. Suitable proteases include trypsin, chymotrypsin, pepsin, thermolysin, proteinase K, and other proteases having defined cleavage specificities. The protease and cleavage conditions may be selected based on the amino acid sequence of the crosslinked product to achieve cleavage at desired positions while preserving the crosslinked core structure. Proteolysis may¬ be performed prior to macrocyclase treatment, after macrocyclase treatment, or in place of macrocyclase treatment depending on the desired polycyclic peptide product.

[0114] The proteolyzed product of the enzy matic reaction may7be modified chemically using a radical SAM enzyme or a macrocyclase. In one embodiment, the crosslinked product of radical SAM enzyme catalysis is proteolyzed to generate a truncated crosslinked peptide, and the proteolyzed product is subsequently reacted with SurE or another macrocyclase to form an additional macrocyclic ring. The combination of proteolysis and macrocyclase catalysis may yield polycyclic peptides having different ring sizes, peptide sequences, or structural features relative to polycyclic peptides generated without proteolysis.

[0115] Alternatively, the proteolyzed product may be subjected to catalysis by a second radical SAM enzyme to install an additional crosslink. The proteolyzed product may retain sufficient recognition motif elements to serve as a substrate for a radical SAM enzyme, or the proteolyzed product may be ligated to a new recognition motif sequence prior to the second enzymatic reaction. Sequential radical SAM enzyme reactions on proteolyzed intermediates may generate polycyclic peptides containing multiple crosslinks installed at different positions within the peptide sequence.

[0116] The product of the enzy matic reaction catalyzed by a radical SAM enzy me may be modified chemically using a haloalkane- or haloacetyl-containing compound. Chemical modification of radical SAM enzyme products provides an alternative or complementary approach to enzymatic macrocyclization for generating polycyclic peptides. The chemicalPrinceton - 107076

[0117] cyclization reaction may be performed on peptide substrates that have been modified to contain two or more cysteine residues, wherein the thiol groups of the cysteine residues serve as nucleophiles for reaction with bifunctional electrophilic reagents.

[0118] The haloalkane- or haloacetyl-containing compound includes 1,3 -dibromoacetone and / or 4,4'-Bis(bromomethyl)-2,2'-bipyridine. These bifunctional reagents contain two electrophilic centers capable of reacting with the thiol groups of two cysteine residues within a peptide substrate to form thioether linkages that create an additional macrocyclic ring. The selection of the haloalkane- or haloacetyl-containing compound may be based on the desired ring size, the chemical properties of the resulting macrocycle, and the intended application of the polycyclic peptide product.

[0119] 4,4'-Bis(bromomethyl)-2,2'-bipyridine is a bifunctional haloalkane-containing compound bearing two bromomethyl groups attached to a bipyridine scaffold. Reaction of 4,4'-Bis(bromomethyl)-2,2'-bipyridine with a cysteine-containing peptide product of radical SAM enzyme catalysis creates an additional macrocycle containing a bipyridine moiety. The bipyridine moiety may confer additional properties to the polycyclic peptide product, including the ability to coordinate metal ions, altered conformational rigidity, altered cell permeability or modified physicochemical characteristics. The thioether linkages formed between the cysteine thiol groups and the bromomethyl carbons of 4,4'-Bis(bromomethyl)-2,2'-bipyridine are stable under physiological conditions and resistant to hydrolysis.

[0120] 1,3 -Dibromoacetone is a bifunctional haloacetyl-containing compound bearing two bromomethyl groups flanking a central ketone functional group. Reaction of 1,3-dibromoacetone with a cysteine-containing peptide product of radical SAM enzyme catalysis creates an additional macrocycle containing a ketone moiety. The ketone moiety within the macrocyclic ring may serve as a site for further chemical modification, including reductive amination, oxime formation, or hydrazone formation. The thioether linkages formed between the cysteine thiol groups and the bromomethyl carbons of 1,3-dibromoacetone provide stable covalent connections that define the macrocyclic ring structure.

[0121] The chemical cyclization reaction may be performed under aqueous or mixed aqueous-organic solvent conditions. The reaction may be conducted in a buffer system at a pH from about 6.5 to about 9.0, and more typically from about 7.0 to about 8.5, to maintain the cysteine thiol groups in a nucleophilic state while minimizing side reactions. Suitable buffer systems include phosphate buffer, HEPES buffer, Tris-HCl buffer, or bicarbonate buffer at concentrations from about 10 mM to about 200 mM, and more typically from about 25 mM to about 100 mM.Princeton - 107076

[0122] The concentration of the haloalkane- or haloacetyl-containing compound in the reaction mixture may range from about 0.5 equivalents to about 10 equivalents relative to the cysteine-containing peptide substrate, and more typically from about 1 equivalent to about 5 equivalents, and in one embodiment from about 5 equivalents to about 8 equivalents. The concentration of the peptide substrate may range from about 10 pM to about 10 mM, and more typically from about 50 LIM to about 2 rnM. The reaction may be performed at a temperature from about 4°C to about 40°C, and more typically from about 20°C to about 30°C, and in one embodiment at about 25°C.

[0123] The reaction time for chemical cyclization may range from about 30 minutes to about 24 hours, and more ty pically from about 1 hour to about 12 hours, depending on the reactivity of the haloalkane- or haloacetyl-containing compound and the accessibility of the cysteine thiol groups within the peptide substrate. The progress of the reaction may be monitored by liquid chromatography-mass spectrometry or other analytical techniques to determine the extent of cyclization and to identify the optimal reaction time for a particular substrate and reagent combination.

[0124] A reducing agent may be included in the reaction mixture to maintain the cysteine thiol groups in the reduced state and to prevent disulfide bond formation between cysteine residues. Suitable reducing agents include tris(2-carboxyethyl)phosphine (TCEP) at a concentration from about 0.5 mM to about 10 mM, dithiothreitol (DTT) at a concentration from about 1 mM to about 20 mM. or P-mercaptoethanol at a concentration from about 1 mM to about 50 mM. The reducing agent may be added to the reaction mixture prior to addition of the haloalkane-or haloacetyl-containing compound to ensure that the cysteine thiol groups are available for reaction.

[0125] The proteolyzed product may be modified chemically using a haloalkane- or haloacetyl-containing compound. As described above, proteolysis may be employed to remove leader peptide sequences or other portions of the peptide substrate following radical SAM enzy me catalysis. The proteolyzed product retaining the crosslink installed by the radical SAM enzyme and containing two cysteine residues may be subjected to chemical cyclization with 4, 4'-Bis(bromomethyl)-2,2'-bi pyridine or 1,3-dibromoacetone to generate a polycyclic peptide product. The reaction conditions for chemical cyclization of the proteolyzed product may be comparable to those described above for chemical cyclization of the full-length crosslinked product.

[0126] Alternative haloalkane-containing compounds and haloacetyl-containing compounds may be employed for chemical cyclization of cysteine-containing peptide products of radicalPrinceton - 107076

[0127] SAM enzyme catalysis. Such alternative reagents include a,a'-dibromo-m-xylene, a,a'-dibromo-p-xylene, 1.2-bis(bromomethyl)benzene. bis(2-bromoethyl) ether, 1,4-dibromobutane, 1,5 -dibromopentane, 1,6-dibromohexane, bromoacetyl bromide derivatives, and other bifunctional electrophiles bearing two halogen-containing reactive groups. The selection of the bifunctional reagent may be based on the desired ring size, conformational properties, and functional group content of the resulting macrocyclic ring.

[0128] Referring to FIG. 1, Compound 1 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB and SurE. Compound 1 comprises a bicyclic architecture formed through the combination of radical SAM enzyme catalysis followed by macrocyclase catalysis. The structure of Compound 1 displays bonds installed by SuiB 102, which represent the lysinetryptophan crosslink formed at unactivated carbon centers through radical SAM enzyme catalysis. The structure of Compound 1 also displays bonds installed by SurE 104, which represent the additional macrocyclic linkage introduced by the macrocyclase enzyme.

[0129] The bonds installed by SuiB 102 in Compound 1 result from the enzy matic reaction of the SuiA peptide substrate with SuiB in the presence of cofactor S-adenosylmethionine and a reductant such as sodium dithionite or titanium(III) citrate in HEPES buffer at pH 7.6. The bonds installed by SuiB 102 comprise a covalent linkage between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions. The lysine-try ptophan crosslink represented by the bonds installed by SuiB 102 creates a first macrocyclic ring within the peptide structure.

[0130] With continued reference to FIG. 1, the bonds installed by SurE 104 in Compound 1 result from the subsequent reaction of the SuiB product with the macrocyclase SurE. The bonds installed by SurE 104 create a second macrocyclic ring through head-to-tail cyclization or side chain-to-terminus cyclization catalyzed by the thioesterase activity of SurE. The combination of the bonds installed by SuiB 102 and the bonds installed by SurE 104 yields the bicyclic architecture of Compound 1, wherein two distinct ring systems are interconnected through the peptide backbone and crosslinked side chains.

[0131] The peptide backbone of Compound 1 includes multiple amino acid residues connected through amide linkages forming the macrocyclic framework. As further shown in FIG. 1, the structure of Compound 1 contains various functional groups including amino groups (NH2), hydroxyl groups (OH), and carboxylic acid groups. The structure also includes a sulfur-containing moiety, which may be present as part of a cysteine or methionine residue within thePrinceton - 107076

[0132] peptide sequence. A lysine residue with a characteristic side chain containing a terminal amino group is present within the macrocyclic framework defined by the bonds installed by SuiB 102.

[0133] The polycyclic architecture of Compound 1 results from the sequential enzymatic modifications, wherein the SuiB enzyme first installs the crosslink indicated by the bonds installed by SuiB 102, and the SurE enzyme subsequently installs the additional macrocyclic linkage indicated by the bonds installed by SurE 104. The bicyclic peptide structure of Compound 1 may exhibit conformational rigidity and defined three-dimensional geometry arising from the constraints imposed by the two ring systems. The structural features of Compound 1 may confer properties suitable for inhibition of protein-protein interactions or modulation of biological phenomena.

[0134] Referring to FIG. 2, Compound 2 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB, followed by proteolysis and subsequent modification with SurE. Compound 2 comprises a bicyclic architecture formed through the combination of radical SAM enzyme catalysis, proteolytic cleavage, and macrocyclase catalysis. The structure of Compound 2 displays bonds installed by SuiB 102. which represent the lysine-tryptophan crosslink formed at unactivated carbon centers through radical SAM enzyme catalysis. The structure of Compound 2 also displays bonds installed by SurE 104, which represent the additional macrocyclic linkage introduced by the macrocyclase enzyme following proteolysis.

[0135] The bonds installed by SuiB 102 in Compound 2 result from the enzymatic reaction of the SuiA peptide substrate with SuiB in the presence of cofactor S-adenosylmethionine and a reductant such as sodium dithionite or titanium(III) citrate in HEPES buffer at pH 7.6. The bonds installed by SuiB 102 comprise a covalent linkage between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions. The lysine-tryptophan crosslink represented by the bonds installed by SuiB 102 creates a first macrocyclic ring within the peptide structure that is retained following proteolytic cleavage.

[0136] With continued reference to FIG. 2, the generation of Compound 2 involves proteolyzing the SuiB product prior to reaction with SurE. Proteolysis removes portions of the peptide substrate, such as leader peptide sequences or recognition motif elements, that are not desired in the final polycyclic peptide product. The proteolyzed product retains the crosslink installed by the radical SAM enzy me, as indicated by the bonds installed by SuiB 102, while having a reduced molecular weight and altered peptide sequence relative to the full-length crosslinked product. The proteolytic cleavage may be performed using endoproteases, exoproteases, or combinations thereof, with the protease and cleavage conditions selectedPrinceton - 107076

[0137] based on the amino acid sequence of the crosslinked product to achieve cleavage at desired positions while preserving the crosslinked core structure.

[0138] The bonds installed by SurE 104 in Compound 2 result from the reaction of the proteolyzed SuiB product with the macrocyclase SurE. The bonds installed by SurE 104 create a second macrocyclic ring through head-to-tail cyclization or side chain-to-terminus cyclization catalyzed by the thioesterase activity of SurE. The combination of the bonds installed by SuiB 102 and the bonds installed by SurE 104 yields the bicyclic architecture of Compound 2, wherein two distinct ring systems are interconnected through the peptide backbone and crosslinked side chains.

[0139] As further shown in FIG. 2, the peptide backbone of Compound 2 includes multiple amino acid residues arranged in a cyclic configuration connected through amide linkages. The structure of Compound 2 contains various functional groups including hydroxyl groups, amino groups, and amide linkages. The structure also includes a thioether moiety containing a sulfur atom, which may be present as part of a cysteine or methionine residue within the peptide sequence. An imidazole ring system characteristic of a histidine residue is present within the macrocyclic framework. A terminal carboxylic acid group is visible at one end of the structure, while various side chains extend from the peptide backbone throughout the macrocyclic framework.

[0140] The polycyclic architecture of Compound 2 demonstrates the combination of enzymatic modifications with an intervening proteolysis step. The SuiB enzyme first installs the crosslink indicated by the bonds installed by SuiB 102 on the full-length peptide substrate. Proteolysis then generates a truncated crosslinked peptide having a reduced size relative to the full-length crosslinked product. The SurE enzyme subsequently installs the additional macrocyclic linkage indicated by the bonds installed by SurE 104 on the proteolyzed intermediate. The resulting bicyclic peptide structure of Compound 2 may exhibit different conformational properties, ring sizes, or structural features relative to Compound 1, which is generated without the intervening proteolysis step.

[0141] The truncated polycyclic peptide structure of Compound 2 may confer properties distinct from those of the full-length bicyclic peptide Compound 1. The reduced molecular weight of Compound 2 may provide advantages in terms of cell permeability, metabolic stability', or target binding characteristics. The altered peptide sequence resulting from proteolytic cleavage may expose different functional groups or modify the surface properties of the polycyclic peptide. The structural features of Compound 2 may be suitable for inhibition of protein-protein interactions or modulation of biological phenomena, and the proteolysis-Princeton - 107076

[0142] based approach provides access to polycyclic peptide structures that are not accessible through direct enzymatic modification of the full-length peptide substrate.

[0143] Referring to FIG. 3, Compound 3 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB followed by chemical cyclization with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. Compound 3 comprises a bicyclic architecture formed through the combination of radical SAM enzyme catalysis and chemical modification using a haloalkane-containing compound. The structure of Compound 3 displays the bonds installed by SuiB 102. which represent the lysine-tryptophan crosslink formed at unactivated carbon centers through radical SAM enzyme catalysis. The structure of Compound 3 also displays bonds installed by chemical cyclization 106, which represent the additional macrocyclic linkage introduced through reaction with the bifunctional haloalkane-containing compound.

[0144] The bonds installed by SuiB 102 in Compound 3 result from the enzymatic reaction of a modified SuiA peptide substrate with SuiB in the presence of cofactor S-adenosylmethionine and a reductant such as sodium dithionite or titanium(III) citrate in HEPES buffer at pH 7.6. The modified SuiA peptide substrate contains two cysteine residues incorporated at positions that allow formation of a macrocyclic ring upon reaction with a bifunctional electrophile. The bonds installed by SuiB 102 comprise a covalent linkage between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions. The lysine-tryptophan crosslink represented by the bonds installed by SuiB 102 creates a first macrocyclic ring within the peptide structure.

[0145] With continued reference to FIG. 3, the bonds installed by chemical cyclization 106 in Compound 3 result from the reaction of the cysteine-containing SuiB product with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. The bonds installed by chemical cyclization 106 comprise thioether linkages formed between the thiol groups of the two cysteine residues and the bromomethyl carbons of the bipyridine reagent. The bipyridine moiety serves as a rigid aromatic linker that bridges the two cysteine residues, creating a second macrocyclic ring within the polycyclic peptide structure. The thioether linkages represented by the bonds installed by chemical cyclization 106 are stable under physiological conditions and resistant to hydrolysis.

[0146] The bipyridine scaffold of 4,4'-Bis(bromomethyl)-2,2'-bipyridine provides a conformationally constrained linker element within the macrocyclic ring formed by the bonds installed by chemical cyclization 106. The bipyridine ring system contains two nitrogen atoms within aromatic rings that may confer additional properties to the polycyclic peptide product.Princeton - 107076

[0147] The bipyridine moiety may coordinate metal ions, which may be useful for applications involving metal-mediated binding interactions or for incorporation of imaging or therapeutic metal complexes. The bipyridine moiety may change the cell-permeability profile of the polycyclic peptide. The rigid aromatic character of the bipyridine linker may impose conformational constraints on the macrocyclic ring, potentially enhancing the structural definition and binding selectivity of the polycyclic peptide.

[0148] As further shown in FIG. 3, the peptide backbone of Compound 3 includes multiple amino acid residues connected through amide linkages forming the macrocyclic framework. The structure of Compound 3 contains various functional groups including hydroxyl groups, amino groups, and amide linkages. The structure includes sulfur atoms that form part of the thioether moieties connecting the cysteine residues to the bipyridine linker, as indicated by the bonds installed by chemical cyclization 106. Terminal amino groups and carboxylic acid groups are present at various positions throughout the macrocyclic framework, along with multiple side chains extending from the peptide backbone.

[0149] The polycyclic architecture of Compound 3 results from the combination of enzymatic and chemical modifications. The SuiB enzyme first installs the crosslink indicated by the bonds installed by SuiB 102 on the modified SuiA peptide containing two cysteine residues. The crosslinked product is subsequently reacted with 4,4'-Bis(bromomethyl)-2,2'-bipyridine to form the additional cyclization indicated by the bonds installed by chemical cyclization 106. The combination of the bonds installed by SuiB 102 and the bonds installed by chemical cyclization 106 yields the bicyclic architecture of Compound 3, wherein two distinct ring systems are interconnected through the peptide backbone, the lysine-tryptophan crosslink, and the bipyridine-bridged thioether linkages.

[0150] The structural features of Compound 3 may confer properties suitable for various applications. The bipyridine moiety introduced through the bonds installed by chemical cyclization 106 may provide a site for metal coordination, enabling the generation of metallopeptide complexes with defined geometry'. The conformational rigidity imparted by both the lysine-tryptophan crosslink and the bipyridine-bridged macrocycle may enhance the binding affinity and selectivity- of Compound 3 for target proteins or other biomolecules. The polycyclic peptide structure of Compound 3 may exhibit improved metabolic stability relative to linear or monocyclic peptide analogs due to the constrained architecture that may resist proteolytic degradation.

[0151] Referring to FIG. 4, Compound 4 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB followed by chemical cyclization with 1,3 -dibromoacetone.Princeton - 107076

[0152] Compound 4 comprises a bicyclic architecture formed through the combination of radical SAM enzyme catalysis and chemical modification using a haloacetyl-containing compound. The structure of Compound 4 displays bonds installed by SuiB 102, which represent the lysinetryptophan crosslink formed at unactivated carbon centers through radical SAM enzyme catalysis. The structure of Compound 4 also displays bonds installed by chemical cyclization 106, which represent the additional macrocyclic linkage introduced through reaction with the bifunctional haloacetyl-containing compound.

[0153] The bonds installed by SuiB 102 in Compound 4 result from the enzymatic reaction of a modified SuiA peptide substrate with SuiB in the presence of cofactor S-adenosylmethionine and a reductant such as sodium dithionite or titanium(III) citrate in HEPES buffer at pH 7.6. The modified SuiA peptide substrate contains two cysteine residues incorporated at positions that allow formation of a macrocyclic ring upon reaction with a bifunctional electrophile. The bonds installed by SuiB 102 comprise a covalent linkage between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions. The lysine-try ptophan crosslink represented by the bonds installed by SuiB 102 creates a first macrocyclic ring within the peptide structure.

[0154] With continued reference to FIG. 4, the bonds installed by chemical cyclization 106 in Compound 4 result from the reaction of the cysteine-containing SuiB product with 1,3-di bromoacetone. The bonds installed by chemical cyclization 106 comprise thioether linkages formed between the thiol groups of the two cysteine residues and the bromomethyl carbons of the dibromoacetone reagent. The 1,3-dibromoacetone reagent serves as a bifunctional linker that bridges the two cysteine residues through a ketone-containing bridge, creating a second macrocyclic ring within the polycyclic peptide structure. The thioether linkages represented by the bonds installed by chemical cyclization 106 are stable under physiological conditions and resistant to hydrolysis.

[0155] The ketone-containing bridge connecting the sulfur atoms of the cysteine residues in Compound 4 comprises a three-carbon unit with a central carbonyl functional group flanked by two methylene groups. Each methylene group is covalently bonded to a sulfur atom of a cysteine residue through a thioether linkage. The ketone moiety is positioned between the two sulfur atoms, providing a defined spacing and geometry within the macrocyclic ring formed by the bonds installed by chemical cyclization 106. The carbonyl group of the ketone-containing bridge may serve as a site for further chemical modification, including reductive amination to introduce amine functionality, oxime formation through reaction with hydroxylamine orPrinceton - 107076

[0156] hydroxylamine derivatives, or hydrazone formation through reaction with hydrazine or hydrazide reagents.

[0157] As further shown in FIG. 4, the peptide backbone of Compound 4 includes multiple amino acid residues connected through amide linkages forming the macrocyclic framework. The structure of Compound 4 contains various functional groups including hy droxy l groups, amino groups, and amide linkages. The structure features a tryptophan residue with a characteristic indole ring system that participates in the lysine-tryptophan crosslink indicated by the bonds installed by SuiB 102. The structure includes sulfur atoms that form part of the thioether moieties connecting the cysteine residues to the ketone-containing bridge, as indicated by the bonds installed by chemical cyclization 106. Terminal carboxylic acid groups are present at the ends of the structure, and multiple side chains extend from the peptide backbone throughout the macrocyclic framework.

[0158] The polycyclic architecture of Compound 4 results from the combination of enzymatic and chemical modifications. The SuiB enzyme first installs the crosslink indicated by the bonds installed by SuiB 102 on the modified SuiA peptide containing two cysteine residues. The crosslinked product is subsequently reacted with 1,3-dibromoacetone to form the additional cyclization indicated by the bonds installed by chemical cyclization 106. The combination of the bonds installed by SuiB 102 and the bonds installed by chemical cyclization 106 yields the bicyclic architecture of Compound 4, wherein two distinct ring systems are interconnected through the peptide backbone, the lysine-tryptophan crosslink, and the ketone-bridged thioether linkages.

[0159] The structural features of Compound 4 may confer properties distinct from those of Compound 3. The ketone-containing bridge introduced through the bonds installed by chemical cyclization 106 provides a shorter and more flexible linker element relative to the bipyridine scaffold of Compound 3. The ketone functional group within the macrocyclic ring may provide a reactive handle for conjugation to other molecules, including fluorescent labels, affinity tags, or therapeutic payloads. The conformational properties of the macrocyclic ring formed by the ketone-containing bridge may differ from those of the bipyridine-bridged macrocycle, potentially providing access to different binding conformations or target interactions. The polycyclic peptide structure of Compound 4 may exhibit metabolic stability arising from the constrained architecture that may resist proteolytic degradation.

[0160] Referring to FIG. 5, Compound 5 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB. followed by proteolysis and subsequent chemical cyclization with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. Compound 5 comprises a polycyclic architecturePrinceton - 107076

[0161] formed through the combination of radical SAM enzy me catalysis, proteolytic cleavage, and chemical modification using a haloalkane-containing compound. The structure of Compound 5 displays bonds installed by SuiB 102, which represent the lysine-tryptophan crosslink formed at unactivated carbon centers through radical SAM enzyme catalysis. The structure of Compound 5 also displays bonds installed by chemical cyclization 106 at multiple locations within the structure, which represent the additional macrocyclic linkages introduced through reaction with the bifunctional haloalkane-containing compound.

[0162] The bonds installed by SuiB 102 in Compound 5 result from the enzymatic reaction of a modified SuiA peptide substrate with SuiB in the presence of cofactor S-adenosylmethionine and a reductant such as sodium dithionite or titanium(III) citrate in HEPES buffer at pH 7.6. The modified SuiA peptide substrate contains two cysteine residues incorporated at positions that allow formation of a macrocyclic ring upon reaction with a bifunctional electrophile. The bonds installed by SuiB 102 comprise a covalent linkage between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions. The lysine-tryptophan crosslink represented by the bonds installed by SuiB 102 creates a first macrocyclic ring within the peptide structure that is retained following proteolytic cleavage.

[0163] With continued reference to FIG. 5, the generation of Compound 5 involves proteolyzing the SuiB product prior to chemical cyclization with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. Proteolysis removes portions of the peptide substrate, such as leader peptide sequences or recognition motif elements, that are not desired in the final polycyclic peptide product. The proteolyzed product retains the crosslink installed by the radical SAM enzyme, as indicated by the bonds installed by SuiB 102, while having a reduced molecular weight and altered peptide sequence relative to the full-length crosslinked product. The proteolytic cleavage may be performed using endoproteases, exoproteases, or combinations thereof, with the protease and cleavage conditions selected based on the amino acid sequence of the crosslinked product to achieve cleavage at desired positions while preserving the crosslinked core structure containing the two cysteine residues.

[0164] The bonds installed by chemical cyclization 106 in Compound 5 result from the reaction of the proteolyzed cysteine-containing SuiB product with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. The bonds installed by chemical cyclization 106 comprise thioether linkages formed between the thiol groups of the two cysteine residues and the bromomethyl carbons of the bipyridine reagent. The bipyridine moiety serves as a rigid aromatic linker that bridges the two cysteine residues within the truncated peptide framework, creating an additionalPrinceton - 107076

[0165] macrocyclic ring within the polycyclic peptide structure. The thioether linkages represented by the bonds installed by chemical cyclization 106 are stable under physiological conditions and resistant to hydrolysis.

[0166] As further shown in FIG. 5, the peptide backbone of Compound 5 includes multiple amino acid residues connected through amide linkages forming the macrocyclic framework. The structure of Compound 5 contains various functional groups including hydroxyl groups, amino groups, and amide linkages. The structure features a bipyridine moiety containing two nitrogen atoms within aromatic rings, which is characteristic of cyclization using 4,4'-Bis(bromomethyl)-2,2'-bipyridine. The structure also contains an imidazole ring system and sulfur-containing thioether moieties that connect the cysteine residues to the bipyridine linker, as indicated by the bonds installed by chemical cyclization 106. A terminal carboxylic acid group is present at one end of the structure, and a terminal amino group is present at another position within the macrocyclic framework.

[0167] The polycyclic architecture of Compound 5 results from the combination of enzymatic modification by SuiB, an intervening proteolysis step, and subsequent chemical cyclization. The SuiB enzyme first installs the crosslink indicated by the bonds installed by SuiB 102 on the modified SuiA peptide containing two cysteine residues. Proteolysis then generates a truncated crosslinked peptide having a reduced size relative to the full-length crosslinked product while retaining the lysine-tryptophan crosslink and the two cysteine residues. The proteolyzed product is subsequently reacted with 4,4'-Bis(bromomethyl)-2,2'-bipyridine to form the additional cyclization indicated by the bonds installed by chemical cyclization 106. The combination of the bonds installed by SuiB 102 and the bonds installed by chemical cyclization 106 yields the compact polycyclic architecture of Compound 5.

[0168] The compact polycyclic architecture of Compound 5 may confer properties distinct from those of Compound 3, which is generated without the intervening proteolysis step. The reduced molecular weight of Compound 5 resulting from proteolytic cleavage may provide advantages in terms of cell permeability, metabolic stability, or target binding characteristics. The truncated peptide framework may expose different functional groups or modify the surface properties of the polycyclic peptide relative to the full-length analog. The bipyridine moiety introduced through the bonds installed by chemical cyclization 106 may provide a site for metal coordination within the compact polycyclic structure, enabling the generation of metallopeptide complexes with defined geometry. The conformational rigidity imparted by both the lysine-tryptophan crosslink and the bipyridine-bridged macrocycle within the truncated peptide framework may enhance the binding affinity and selectivity of Compound 5Princeton - 107076

[0169] for target proteins or other biomolecules. The polycyclic peptide structure of Compound 5 may exhibit improved metabolic stability relative to linear or monocyclic peptide analogs due to the constrained architecture that may resist proteolytic degradation.

[0170] Referring to FIG. 6, Compound 6 is a polycyclic peptide generated through sequential enzy matic catalysis by SuiB, followed by proteolysis and subsequent chemical cyclization with 1,3 -dibromoacetone. Compound 6 comprises a polycyclic architecture formed through the combination of radical SAM enzyme catalysis, proteolytic cleavage, and chemical modification using a haloacetyl-conlaining compound. The structure of Compound 6 displays bonds installed by SuiB 102, which represent the ly sine-try ptophan crosslink formed at unactivated carbon centers through radical SAM enzyme catalysis. The structure of Compound 6 also displays bonds installed by chemical cyclization 106, which represent the additional macrocyclic linkage introduced through reaction with the bifunctional haloacetyl-containing compound.

[0171] The bonds installed by SuiB 102 in Compound 6 result from the enzy matic reaction of a modified SuiA peptide substrate with SuiB in the presence of cofactor S-adenosylmethionine and a reductant such as sodium dithionite or titanium(III) citrate in HEPES buffer at pH 7.6. The modified SuiA peptide substrate contains two cysteine residues incorporated at positions that allow formation of a macrocyclic ring upon reaction with a bifunctional electrophile. The bonds installed by SuiB 102 comprise a covalent linkage between the beta carbon of a lysine residue and a carbon atom of the indole ring of a tryptophan residue, both of which are unactivated carbon centers under conventional synthetic conditions. The lysine-tryptophan crosslink represented by the bonds installed by SuiB 102 creates a first macrocyclic ring within the peptide structure that is retained following proteolytic cleavage.

[0172] With continued reference to FIG. 6, the generation of Compound 6 involves proteolyzing the SuiB product prior to chemical cyclization with 1,3-dibromoacetone. Proteolysis removes portions of the peptide substrate, such as leader peptide sequences or recognition motif elements, that are not desired in the final polycyclic peptide product. The proteolyzed product retains the crosslink installed by the radical SAM enzyme, as indicated by the bonds installed by SuiB 102, while having a reduced molecular weight and altered peptide sequence relative to the full-length crosslinked product. The proteolytic cleavage may be performed using endoproteases, exoproteases, or combinations thereof, with the protease and cleavage conditions selected based on the amino acid sequence of the crosslinked product to achieve cleavage at desired positions while preserving the crosslinked core structure containing the two cysteine residues.Princeton - 107076

[0173] The bonds installed by chemical cyclization 106 in Compound 6 result from the reaction of the proteolyzed cysteine-containing SuiB product with 1,3 -dibromoacetone. The bonds installed by chemical cyclization 106 comprise thioether linkages formed between the thiol groups of the two cysteine residues and the bromomethyl carbons of the dibromoacetone reagent. The 1,3-dibromoacetone reagent serves as a bifunctional linker that bridges the two cysteine residues within the truncated peptide framework through a ketone-containing bridge, creating an additional macrocyclic ring within the polycyclic peptide structure. The thioether linkages represented by the bonds installed by chemical cyclization 106 are stable under physiological conditions and resistant to hydrolysis.

[0174] As further shown in FIG. 6, the ketone functional group is positioned between the two sulfur atoms that connect to the cysteine residues within the macrocyclic ring formed by the bonds installed by chemical cyclization 106. The ketone-containing bridge comprises a three-carbon unit with a central carbonyl functional group flanked by two methylene groups. Each methylene group is covalently bonded to a sulfur atom of a cysteine residue through a thioether linkage, providing a defined spacing and geometry within the macrocyclic ring. The carbonyl group of the ketone-containing bridge may serve as a site for further chemical modification, including reductive amination to introduce amine functionality, oxime formation through reaction with hydroxylamine or hydroxylamine derivatives, or hydrazone formation through reaction with hydrazine or hy drazide reagents.

[0175] The peptide backbone of Compound 6 includes multiple amino acid residues arranged in a cyclic configuration connected through amide linkages. The structure of Compound 6 contains various functional groups including hydroxyl groups, amino groups, and amide linkages. The structure features an imidazole ring system characteristic of a histidine residue, as well as an aromatic ring system. The structure includes thioether moieties containing sulfur atoms that connect the cysteine residues to the ketone-containing bridge, as indicated by the bonds installed by chemical cyclization 106. Terminal carboxylic acid groups are present at the periphery of the structure, while various side chains extend from the peptide backbone throughout the macrocyclic framework.

[0176] The polycyclic architecture of Compound 6 results from the combination of enzymatic modification by SuiB, an intervening proteolysis step, and subsequent chemical cyclization with 1,3-dibromoacetone. The SuiB enzyme first installs the crosslink indicated by the bonds installed by SuiB 102 on the modified SuiA peptide containing two cysteine residues. Proteolysis then generates a truncated crosslinked peptide having a reduced size relative to the full-length crosslinked product while retaining the lysine-tryptophan crosslink and the twoPrinceton - 107076

[0177] cysteine residues. The proteolyzed product is subsequently reacted with 1,3-dibromoacetone to form the additional cyclization indicated by the bonds installed by chemical cyclization 106. The combination of the bonds installed by SuiB 102 and the bonds installed by chemical cyclization 106 yields the compact polycyclic architecture of Compound 6.

[0178] The compact polycyclic architecture of Compound 6 may confer properties distinct from those of Compound 4, which is generated without the intervening proteolysis step. The reduced molecular weight of Compound 6 resulting from proteolytic cleavage may provide advantages in terms of cell permeability, metabolic stability, or target binding characteristics. The truncated peptide framework may expose different functional groups or modify the surface properties of the polycyclic peptide relative to the full-length analog. The ketone-containing bridge introduced through the bonds installed by chemical cyclization 106 provides a shorter and more flexible linker element relative to the bipyridine scaffold of Compound 5, and the ketone functional group within the macrocyclic ring may provide a reactive handle for conjugation to other molecules, including fluorescent labels, affinity tags, or therapeutic payloads. The conformational properties of the macrocyclic ring formed by the ketone-containing budge within the truncated peptide framework may differ from those of the bipyridine-bridged macrocycle, potentially providing access to different binding conformations or target interactions. The polycyclic peptide structure of Compound 6 may exhibit metabolic stability arising from the constrained architecture that may resist proteolytic degradation.

[0179] The disclosed methodology enables generation of polycyclic peptide libraries by altering peptide sequences at permissive sites. As described above, permissive sites refer to positions within a peptide sequence where amino acid substitutions, insertions, or deletions may be made without abolishing the ability of a radical SAM enzyme to recognize and modify the peptide substrate. By generating at least one additional peptide sequence by altering the peptide sequence at permissive sites, a collection of peptide substrates having varied amino acid compositions may be prepared while retaining the recognition motif elements required for radical SAM enzyme catalysis.

[0180] The generation of at least one additional peptide sequence by altering the peptide sequence at permissive sites may be accomplished through standard peptide synthesis methods. Solid-phase peptide synthesis or solution-phase peptide synthesis may be employed to prepare peptide variants having amino acid substitutions at one or more permissive sites. The amino acid substitutions may include replacement of one amino acid with another amino acid having different physicochemical properties, such as substitution of a hydrophobic residue with aPrinceton - 107076

[0181] hydrophilic residue, substitution of a charged residue with an uncharged residue, or substitution of a small residue with a bulky residue. The amino acid substitutions may also include incorporation of non-canonical amino acids at permissive sites, as described above, to introduce functional groups or structural features not present in standard proteinogenic amino acids.

[0182] The at least one additional peptide sequence may be exposed to an enzymatic reaction, the enzymatic reaction including catalysis under standard enzymatic reaction conditions with the radical SAM enzyme. The enzy matic reaction conditions for processing the at least one additional peptide may be comparable to those described above for processing the parent peptide sequence, including the use of HEPES buffer at pH 7.6, cofactor S-adenosylmethionine. and a reductant such as sodium dithionite or titanium(III) citrate. The radical SAM enzyme may install crosslinks at unactivated carbon centers within the at least one additional peptide, generating crosslinked products having the same type of crosslink as the parent peptide but with varied amino acid compositions at the permissive sites.

[0183] A further cyclized peptide may be generated by modifying a product of the enzymatic reaction chemically and / or via enzymes. The crosslinked products derived from the at least one additional peptide sequence may be subjected to downstream chemical modification using haloalkane- or haloacetyl-containing compounds, as described above, or to enzymatic modification using macrocyclases such as SurE. The combination of radical SAM enzyme catalysis followed by downstream chemical or enzymatic modification yields polycyclic peptide products having varied amino acid compositions at the permissive sites while sharing a common polycyclic architecture defined by the crosslinks installed through enzymatic and chemical transformations.

[0184] The disclosed methodology further comprises generating a library of crosslinked peptides by exposing a library' of peptides, consisting as a mixture, to radical SAM enzyme catalysis. A library of peptides may be prepared by synthesizing multiple peptide variants having different amino acid sequences at permissive sites and combining the peptide variants into a single mixture. The library’ of peptides may contain any number of enzymes. For example, the library of peptides may include from, e.g., about 2 to about 1018distinct peptide sequences. In some implementations, the library of peptides may include from about 2 to about 10,000 distinct peptide sequences and more ty pically from about 10 to about 1,000 distinct peptide sequences, and in some cases from about 50 to about 500 distinct peptide sequences. In some implementations, the library of peptides may include from about 108to about 1016peptide sequences, such as from about 108to about 1014peptide sequences. These may also bePrinceton - 107076

[0185] simulated in silico prior to synthesis, which can significantly enhance the number of peptides that can be probed. Each peptide within the library retains the recognition motif required for radical SAM enzyme catalysis while having a varied amino acid composition at one or more permissive sites.

[0186] The library' of peptides, consisting as a mixture, may be exposed to radical SAM enzyme catalysis in a single reaction vessel. The radical SAM enzyme may process multiple peptide substrates within the mixture, installing crosslinks at unactivated carbon centers within each peptide that contains the appropriate recognition motif. The enzymatic reaction conditions for processing the library' of peptides may be comparable to those described above for processing individual peptide substrates, with the total peptide concentration in the reaction mixture adjusted to account for the presence of multiple peptide species. The radical SAM enzyme may exhibit comparable catalytic efficiency across the library of peptide substrates, provided that the amino acid variations at permissive sites do not interfere with enzyme recognition or catalysis.

[0187] The library of crosslinked peptides generated through radical SAM enzyme catalysis may be further modified chemically or enzymatically. The crosslinked library may be further modified chemically or enzymatically using the methods described above for individual crosslinked peptide products. Chemical modification of the crosslinked library' may include reaction with haloalkane- or haloacetyl-containing compounds, such as 4,4'-Bis(bromomethyl)-2,2'-bipyridine or 1.3 -di bromoacetone, to install additional macrocyclic rings within the crosslinked peptides. Enzymatic modification of the crosslinked library7may include treatment with macrocyclases such as SurE to catalyze head-to-tail cyclization or side chain-to-terminus cyclization reactions. The crosslinked library7may also be subjected to proteolysis prior to or following chemical or enzymatic modification, as described above, to generate truncated polycyclic peptide products.

[0188] The processing of peptide libraries as mixtures through radical SAM enzyme catalysis followed by downstream modification provides an efficient approach for generating diverse collections of polycyclic peptides. The library-based approach may reduce the number of individual enzymatic reactions required to generate a collection of polycyclic peptide variants, as multiple peptide substrates may be processed in a single reaction vessel. The crosslinked library may be subjected to downstream chemical or enzymatic modification as a mixture, further reducing the number of individual reactions required to generate the final polycyclic peptide library. Individual polycyclic peptides, or the resulting library of polycyclic peptidesPrinceton - 107076

[0189] may be screened against a desired phenotype using in vitro or in vivo assays to identify polycyclic peptides having desired biological activities.

[0190] The methodology is scalable and may be used to generate large quantities of a desired analog. The enzymatic reaction catalyzed by radical SAM enzymes occurs at ambient temperature and pH under buffered aqueous conditions, which facilitates scale-up from analytical-scale reactions to preparative-scale reactions. The reaction conditions are devoid of toxic or dangerous components, which simplifies handling and disposal considerations at larger scales. The scalability of the methodology enables preparation of milligram to gram quantities of a desired polycyclic peptide analog for downstream applications, including biological screening, structural characterization, and preclinical evaluation.

[0191] Scale-up of the enzymatic reaction may be achieved by proportionally increasing the volumes of the reaction components while maintaining the concentrations of the peptide substrate, radical SAM enzyme, cofactor, and reductant within the ranges described above. The reaction may be performed in larger reaction vessels, including round-bottom flasks, bioreactors, or fermentation vessels, depending on the desired scale. Mixing may be provided by magnetic stirring, mechanical stirring, or orbital shaking to ensure homogeneous distribution of the reaction components. The anaerobic conditions required for radical SAM enz me activity may be maintained at larger scales through the use of inert gas sparging, sealed reaction vessels, or anaerobic chambers designed for preparative-scale reactions.

[0192] The downstream chemical modification steps may also be scaled to generate large quantities of polycyclic peptide products. The chemical cyclization reactions using haloalkane-or haloacetyl-containing compounds may be performed at preparative scale by proportionally increasing the amounts of the crosslinked peptide substrate and the bifunctional reagent. The enzymatic modification steps using macrocyclases may be scaled by increasing the amounts of the crosslinked peptide substrate and the macrocyclase enzyme. Purification of the polycyclic peptide products at preparative scale may be achieved using preparative high-performance liquid chromatography , flash chromatography , or other chromatographic methods suitable for processing larger quantities of material.

[0193] A kit containing a plurality of further cyclized peptides generated using the disclosed method may be provided for various applications. The kit may contain a collection of polycyclic peptides generated through radical SAM enzyme catalysis followed by dow nstream chemical and / or enzymatic modification, as described above. The plurality of further cyclized peptides within the kit may include polycyclic peptides having varied amino acid compositions at permissive sites, polycyclic peptides generated using different radical SAM enzymes,Princeton - 107076

[0194] polycyclic peptides modified using different downstream chemical reagents or macrocyclases, or combinations thereof.

[0195] The kit may contain from about 2 to about 1018distinct polycyclic peptide species. The library of peptides may contain any number of enzy mes. For example, the library of peptides may include from, e.g., about 2 to about 1018distinct peptide sequences. In some implementations, the library of peptides may include from about 2 to about 10,000 distinct peptide sequences and more typically from about 10 to about 1,000 distinct peptide sequences, and in some cases from about 50 to about 500 distinct peptide sequences. In some implementations, the library of peptides may include from about 108to about 1016peptide sequences, such as from about 108to about 1014peptide sequences. Each polycyclic peptide within the kit may be provided in a defined quantity, such as from about 0.1 mg to about 100 g, about 0.1 mg to about 10 g, about 0.1 mg to about 1 g, about 0.1 mg to about 100 mg, or from about 0.5 mg to about 10 mg, depending on the intended application and the scale of screening to be performed. The polycyclic peptides within the kit may be provided in individual containers, such as vials, tubes, or wells of a multi-well plate, or may be provided as a mixture in a single container.

[0196] The kit may include polycyclic peptides having structures generated by a radical SAM. The polycyclic peptides may have structures according to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or combinations thereof. The kit may also include polycyclic peptide variants derived from Compounds 1-6 through amino acid substitutions at permissive sites, as described above. The polycyclic peptides within the kit may be provided in lyophilized form, in solution, or in other formulations suitable for storage and subsequent use in screening assays.

[0197] The kit may further include reagents, buffers, or instructions for use in screening the plurality of further cyclized peptides against a desired phenotype. The kit may include assay buffers, detection reagents, positive control compounds, negative control compounds, or other components useful for performing in vitro or in vivo screening assays. The kit may include instructions describing protocols for screening the polycyclic peptides against target proteins, cells, tissues, or organisms.

[0198] A composition of matter having a structure according to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6 may be provided. As described above, Compound 1 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB and SurE. Compound 2 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB, followed by proteolysis and subsequent modification with SurE. CompoundPrinceton - 107076

[0199] 3 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB followed by chemical cyclization with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. Compound 4 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB followed by chemical cyclization with 1,3 -dibromoacetone. Compound 5 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB, followed by proteolysis and subsequent chemical cyclization with 4,4'-Bis(bromomethyl)-2.2'-bipyridine. Compound 6 is a polycyclic peptide generated through sequential enzymatic catalysis by SuiB, followed by proteolysis and subsequent chemical cyclization with 1,3-dibromoacetone.

[0200] The composition of matter having a structure according to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6 may be provided in isolated form, in purified form, or as a component of a mixture. The composition of matter may be provided in a pharmaceutically acceptable salt form, as a solvate, as a hydrate, or in other solid-state forms. The composition of matter may be formulated with pharmaceutically acceptable excipients, carriers, or diluents for administration to a subject or for use in biological assays.

[0201] The kits and compositions of matter described herein may be used for screening against a desired phenotype in vitro or in vivo. The polycyclic peptides may be screened against target proteins to identify inhibitors of protein-protein interactions. The polycyclic peptides may be screened against enzymes to identify inhibitors or modulators of enzymatic activity7. The polycyclic peptides may be screened against receptors to identify agonists, antagonists, or allosteric modulators. The polycyclic peptides may be screened against cells to identify compounds that modulate cell proliferation, cell death, cell differentiation, cell migration, or other cellular phenotypes. The polycyclic peptides may be screened against tissues or organisms to identify compounds that modulate physiological processes or disease states.

[0202] In vitro screening assays may include biochemical assays, such as binding assays, enzyme activity assays, or competition assays, that measure the interaction of polycyclic peptides with purified target proteins or other biomolecules. In vitro screening assays may also include cell-based assays, such as reporter gene assays, proliferation assays, cytotoxicity assays, or phenotypic assays, that measure the effects of polycyclic peptides on cellular functions. The polycyclic peptides may be tested at concentrations from about 1 nM to about 100 pM, and more typically from about 10 nM to about 10 pM, to determine dose-response relationships and to identify polycyclic peptides having desired potency.

[0203] In vivo screening assays may include administration of polycyclic peptides to model organisms, such as mice, rats, zebrafish. Caenorhabditis elegans, or Drosophila melanogaster, to evaluate effects on disease models, physiological processes, or developmental phenotypes.Princeton - 107076

[0204] The polycyclic peptides may be administered by various routes, including intravenous, intraperitoneal, subcutaneous, oral, or topical administration, depending on the model organism and the phenotype being evaluated. In vivo screening may be used to identify polycyclic peptides having desired pharmacokinetic properties, biodistribution profiles, or therapeutic effects.

[0205] Pharmaceutical or biotech firms may acquire polycyclic peptide libraries generated using the disclosed methodology to screen in desired assays. Alternatively, such firms may generate polycyclic peptide libraries using the disclosed process for internal screening programs. Companies engaged in the production and properties of peptide libraries, including those containing strained macro- and polycyclic structures, may utilize the disclosed methodology to access polycyclic peptide chemical space that is not accessible through other methodologies.

[0206] The following example demonstrates the synthesis of a bicyclic peptide through sequential enzymatic catalysis using SuiB and SurE.

[0207] Example 1 - Synthesis of Bicyclic Peptide via SuiB and SurE

[0208] A peptide substrate corresponding to the SuiA precursor peptide was synthesized using standard Fmoc solid-phase peptide synthesis on 2-chlorotrityl resin. The peptide sequence contained the recognition motif for SuiB, including the leader peptide region and the core peptide region containing lysine and tryptophan residues at positions suitable for crosslink formation. The C-terminus of the peptide was prepared with an activated thioester functionality to enable subsequent macrocyclization by SurE. Following synthesis, the peptide was cleaved from the resin using a cleavage cocktail comprising trifluoroacetic acid, triisopropylsilane, and water in a ratio of 95:2.5:2.5 (v / v / v) for a period of about 2 hours at ambient temperature. The crude peptide was precipitated by addition of cold diethyl ether, collected by centrifugation, and purified by reverse-phase high-performance liquid chromatography using a Cl 8 column with a gradient of acetonitrile in water containing 0.1 % trifluoroacetic acid. The identity of the purified peptide substrate was confirmed by electrospray ionization mass spectrometry, and the purity was determined to be greater than about 95% by analytical HPLC.

[0209] The SuiB enzyme was expressed recombinantly in Escherichia coli BL21(DE3) cells transformed with a plasmid encoding the SuiB gene with an N-terminal hexahistidine tag. Cells were grown in M9 medium supplemented with 2 mM MgSO.bO.l mM CaCL, 20 m glucose, 25 pM (NH4)2Fe(SO4), 50 pg / mL Kan, and 50 pg / mL Amp at 37°C until the optical density at 600 nm reached about 0.3. Cultures were then supplemented with 2 m L-Cys, 25 pM (NH4)2Fe(S04), and 0.2% (w / v) i-(+)-arabinose and grown until opticalPrinceton - 107076

[0210] density at 600 nm reached about 0.6. Expression was induced by addition of isopropyl P-D-l-thiogalactopyranoside at a final concentration of about 0.5 mM. and the culture was incubated at 18°C for about 16 hours. Cells were harvested by centrifugation, resuspended in lysis buffer comprising 50 mM Tris-HCl at pH 8.0, 300 mM sodium chloride, 10 mM imidazole, and 5 mM dithiothreitol, and lysed by sonication under anaerobic conditions in an anaerobic chamber maintained under a nitrogen atmosphere. The lysate was clarified by centrifugation at about 20,000 x g for about 30 minutes at 4°C, and the supernatant was applied to a nickelnitrilotriacetic acid affinity column equilibrated with lysis buffer. The column was washed with lysis buffer containing 20 mM imidazole, and the enzyme was eluted with lysis buffer containing 250 mM imidazole. The eluted enzyme was buffer-exchanged into storage buffer comprising 50 mM HEPES at pH 7.6. 300 mM potassium chloride, 10% glycerol, and 5 mM dithiothreitol using a desalting column. The enzyme concentration was determined by Bradford assay, and the enzyme was stored at -80°C until use.

[0211] The SurE macrocyclase was expressed and purified using a comparable protocol. The SurE gene with an N-terminal hexahistidine tag was expressed in Escherichia coli BL21(DE3) cells, and the enzyme was purified by nickel affinity chromatography followed by size exclusion chromatography using a Superdex 200 column equilibrated with 50 mM HEPES at pH 7.6, 150 mM sodium chloride, and 5 mM dithiothreitol. The purified SurE enzy me was concentrated to about 10 mg / mL and stored at -80°C until use.

[0212] The enzymatic reaction with SuiB was performed under anaerobic conditions in an anaerobic chamber. The reaction mixture was prepared in a total volume of about 1 mL and contained the following components: 50 mM HEPES buffer at pH 7.6, 300 mM potassium chloride, 2 mM S-adenosylmethionine chloride salt, 5 mM sodium dithionite, 500 pM SuiA peptide substrate, and 20 pM SuiB enzyme. The reaction mixture was incubated at about 25°C for about 4 hours with gentle agitation. The progress of the reaction was monitored by removing 10 pL aliquots at 1-hour intervals and analyzing the aliquots by liquid chromatography -mass spectrometry using a C 18 analytical column with a gradient of acetonitrile in water containing 0.1% formic acid. The formation of the crosslinked product was confirmed by the appearance of a new peak with a mass corresponding to the loss of 2 Da relative to the starting peptide substrate, consistent with the formation of a carbon-carbon bond between the lysine and try ptophan residues.

[0213] Upon completion of the SuiB reaction, the crosslinked product was subjected to macrocyclization by SurE. The SurE enzyme was added directly to the SuiB reaction mixture at a final concentration of about 10 pM, and the reaction was incubated at about 25°C for anPrinceton - 107076

[0214] additional period of about 6 hours. The progress of the macrocyclization reaction was monitored by liquid chromatography -mass spectrometry, and the formation of the bicyclic product was confirmed by the appearance of anew peak with a mass corresponding to the loss of water (18 Da) relative to the monocyclic crosslinked intermediate, consistent with the formation of a macrolactone or macrolactam linkage catalyzed by SurE.

[0215] The molecular weight of the purified bicyclic peptide was confirmed by high-resolution electrospray ionization mass spectrometry. The observed mass was within about 5 ppm of the calculated mass for the bicyclic peptide structure, confirming the identity of the product. Tandem mass spectrometry (MS / MS) analysis was performed to confirm the sites of crosslink formation. Fragmentation of the bicyclic peptide produced a characteristic fragmentation pattern consistent with the presence of a lysine-tryptophan crosslink and a macrocyclic linkage formed by SurE. The fragmentation pattern showed the absence of fragment ions that would be expected from cleavage of the crosslinked region, confirming the covalent nature of the crosslinks.

[0216] The bicyclic peptide exhibited a single major peak in the chromatogram, with minor peaks accounting for less than about 5% of the total peak area. The retention time of the bicyclic peptide was distinct from that of the starting peptide substrate and the monocyclic crosslinked intermediate, confirming the formation of a new chemical entity.

[0217] The following example demonstrates the synthesis of a bicyclic peptide through enzymatic catalysis using SuiB followed by chemical cyclization with a haloalkane-containing compound.

[0218] Example 2 - Synthesis of Bicyclic Peptide via SuiB and Chemical Cyclization A modified SuiA peptide substrate was synthesized using standard Fmoc solid-phase peptide synthesis on Wang-cysteine resin. The peptide sequence contained the recognition motif for SuiB, including the leader peptide region and the core peptide region containing lysine and tryptophan residues at positions suitable for crosslink formation. The modified SuiA peptide was designed to incorporate two cysteine residues at permissive sites within the peptide sequence to enable subsequent chemical cyclization with bifunctional electrophilic reagents. The cysteine residues were positioned such that incorporation did not abolish recognition by the SuiB enzyme. Following synthesis, the peptide was cleaved from the resin using a cleavage cocktail comprising trifluoroacetic acid, triisopropylsilane, ethanedithiol, and water in a ratio of 92.5:2.5:2.5:2.5 (v / v / v / v) for a period of about 2.5 hours at ambient temperature. The inclusion of ethanedithiol in the cleavage cocktail served as a scavenger to prevent oxidation of the cysteine thiol groups during cleavage. The crude peptide was precipitated by addition ofPrinceton - 107076

[0219] cold diethyl ether, collected by centrifugation, and purified by reverse-phase high-performance liquid chromatography using a C18 column with a gradient of acetonitrile in water containing 0.1% trifluoroacetic acid.

[0220] The SuiB enzyme was expressed and purified as described in Example 1. The enzy matic reaction with SuiB was performed under anaerobic conditions in an anaerobic chamber. The reaction mixture was prepared in a total volume of about 2 mL and contained the following components: 50 mM HEPES buffer at pH 7.6, 300 mM potassium chloride, 2 mM S-adenosylmethionine chloride salt, 5 mM Tris (2-carboxyethyl) phosphine (TCEP), 500 pM modified SuiA peptide substrate containing two cysteine residues, and 25 pM SuiB enzy me. The reaction mixture was incubated at about 25°C for about 5 hours with gentle agitation. The progress of the reaction was monitored by removing 10 pL aliquots at 1-hour intervals and analyzing the aliquots by liquid chromatography -mass spectrometry using a C18 analytical column with a gradient of acetonitrile in water containing 0.1% formic acid. The formation of the crosslinked product was confirmed by the appearance of a new peak with a mass corresponding to the loss of 2 Da relative to the starting peptide substrate, consistent with the formation of a carbon-carbon bond between the lysine and tryptophan residues.

[0221] Upon completion of the SuiB reaction, the crosslinked product containing two cysteine residues was subjected to chemical cyclization with 4,4'-Bis(bromomethyl)-2,2'-bipyridine. The reaction mixture from the SuiB reaction was removed from the anaerobic chamber and diluted with an equal volume of 100 mM ammonium bicarbonate buffer at pH 8.0. TCEP was added at a final concentration of about 2 mM to ensure that the cysteine thiol groups were maintained in the reduced state and to prevent disulfide bond formation between the cysteine residues. The reaction mixture was incubated at ambient temperature for about 15 minutes to allow complete reduction of any disulfide bonds that may have formed during handling.

[0222] A stock solution of 4,4'-Bis(bromomethyl)-2,2'-bipyridine was prepared at a concentration of about 50 mM in 50 / 50 H2O-Acetonitrile. The 4,4'-Bis(bromomethyl)-2,2'-bipyridine stock solution was added to the reduced crosslinked peptide mixture at a final concentration of about 1.2 mM, corresponding to approximately 3 equivalents relative to the crosslinked peptide substrate. The reaction mixture was incubated at about 25°C for about 4 hours with gentle agitation. The progress of the chemical cyclization reaction was monitored by liquid chromatography-mass spectrometry7, and the formation of the bicyclic product was confirmed by the appearance of a new peak with a mass corresponding to the addition of the bipyridine linker (molecular weight increase of about 260 Da) minus two equivalents of hydrogen bromide (loss of about 162 Da), resulting in a net mass increase of about 98 DaPrinceton - 107076

[0223] relative to the monocyclic crosslinked intermediate. The mass increase was consistent with the formation of two thioether linkages between the cysteine thiol groups and the bromomethyl carbons of the bipyridine reagent.

[0224] The bicyclic peptide product was purified from the reaction mixture by reverse-phase high-performance liquid chromatography using a semi-preparative Cl 8 column with a gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. Fractions containing the bicyclic peptide were identified by mass spectrometry, pooled, and lyophilized to yield the purified bicyclic peptide as an off-white powder. The yield of the purified bicyclic peptide was about 35% based on the starting modified SuiA peptide substrate.

[0225] The molecular weight of the purified bicyclic peptide was confirmed by high-resolution electrospray ionization mass spectrometry using a quadrupole time-of-flight mass spectrometer. The observed mass was within about 3 ppm of the calculated mass for the bicyclic peptide structure containing the lysine-tryptophan crosslink and the bipyridine-bridged thioether macrocycle, confirming the identity of the product. Tandem mass spectrometry (MS / MS) analysis was performed to confirm the sites of crosslink formation and chemical cyclization. Fragmentation of the bicyclic peptide produced a characteristic fragmentation pattern consistent with the presence of a lysine-tryptophan crosslink installed by SuiB and thioether linkages connecting the cysteine residues to the bipyridine scaffold. The fragmentation pattern showed diagnostic ions corresponding to loss of the bipyridine moiety and cleavage of the thioether bonds, confirming the structure of the chemically installed macrocycle.

[0226] The purity of the bicyclic peptide was determined to be greater than about 92% by analytical HPLC using a Cl 8 column with UV detection at 220 nm and 280 nm. The bicyclic peptide exhibited a single major peak in the chromatogram, with minor peaks accounting for less than about 8% of the total peak area. The retention time of the bicyclic peptide was distinct from that of the starting modified SuiA peptide substrate, the monocyclic crosslinked intermediate, and unreacted 4,4'-Bis(bromomethyl)-2,2'-bipyridine, confirming the formation of a new chemical entity.

[0227] A parallel synthesis was performed using 1,3 -dibromoacetone as the chemical cyclization reagent in place of 4,4'-Bis(bromomethyl)-2,2'-bipyridine. The SuiB reaction was performed as described above using the modified SuiA peptide substrate containing two cysteine residues. Upon completion of the SuiB reaction, the crosslinked product was diluted with 100 mM ammonium bicarbonate buffer at pH 8.0, and TCEP was added at a final concentration of about 2 mM to maintain the cysteine thiol groups in the reduced state.Princeton - 107076

[0228] A stock solution of 1,3 -dibromoacetone was prepared at a concentration of about 100 mM in dimethyl sulfoxide. The 1.3 -dibromoacetone stock solution was added to the reduced crosslinked peptide mixture at a final concentration of about 2 mM, corresponding to approximately 5 equivalents relative to the crosslinked peptide substrate. The reaction mixture was incubated at about 25°C for about 3 hours with gentle agitation. The progress of the chemical cyclization reaction was monitored by liquid chromatography-mass spectrometry, and the formation of the bicyclic product was confirmed by the appearance of a new peak with a mass corresponding to the addition of the acetone linker (molecular weight increase of about 56 Da) minus two equivalents of hydrogen bromide (loss of about 162 Da), resulting in a net mass decrease of about 106 Da relative to the monocyclic crosslinked intermediate when accounting for the loss of two hydrogen atoms from the cysteine thiol groups. The observed mass change was consistent with the formation of two thioether linkages between the cysteine thiol groups and the bromomethyl carbons of the dibromoacetone reagent, with retention of the central ketone functional group.

[0229] The bicyclic peptide product containing the ketone-bridged macrocycle was purified from the reaction mixture by reverse-phase high-performance liquid chromatography using a semi-preparative Cl 8 column. Fractions containing the bicyclic peptide were identified by mass spectrometry', pooled, and lyophilized to yield the purified bicyclic peptide as a white powder. The yield of the purified bicyclic peptide was about 35% based on the starting modified SuiA peptide substrate.

[0230] The molecular weight of the purified bicyclic peptide containing the ketone-bridged macrocycle was confirmed by high-resolution electrospray ionization mass spectrometry'. The observed mass was within about 4 ppm of the calculated mass for the bicyclic peptide structure containing the lysine-tryptophan crosslink and the ketone-bridged thioether macrocycle, confirming the identity of the product. Tandem mass spectrometry analysis confirmed the presence of the lysine-tryptophan crosslink and the ketone-containing linker connecting the two cysteine residues through thioether bonds.

[0231] The physical properties of the purified bicyclic peptides were characterized. The bicyclic peptide containing the bipyridine-bridged macrocycle was soluble in aqueous buffers at concentrations up to about 5 mM and in dimethyl sulfoxide at concentrations up to about 40 mM. The bicyclic peptide containing the ketone-bridged macrocycle was soluble in aqueous buffers at concentrations up to about 8 mM and in dimethyl sulfoxide at concentrations up to about 50 mM. Both bicyclic peptides exhibited stability when stored as lyophilized powders at -20°C for periods of at least about 6 months, as determined by HPLC analysis showing noPrinceton - 107076

[0232] detectable degradation. The bicyclic peptides also exhibited stability in aqueous buffer at pH 7.4 and 37°C for periods of at least about 24 hours, as determined by HPLC analysis showing greater than about 88% of the starting material remaining after incubation.

[0233] The bicyclic peptide containing the bipyridine-bridged macrocycle exhibited UV absorbance maxima at about 220 nm, about 280 nm, and about 305 nm, with the absorbance at about 305 nm being characteristic of the bipyridine chromophore. The molar extinction coefficient at about 305 nm was determined to be approximately 12,000 M^cm'1. The bicyclic peptide containing the ketone-bridged macrocycle exhibited UV absorbance maxima at about 220 nm and about 280 nm, with no additional absorbance features attributable to the ketone-containing linker.

[0234] Referring to FIGS. 7-10, Cathepsin B inhibition was studied. FIG. 7 shows a model of a binder peptide, without a target shown. FIGS. 8 and 9 show a model of that binder peptide bound to Cathepsin B. FIG. 10 shows how concentration of the binder peptide affects Cathepsin B activity, with an ICso of 2.33 pM against Cathepsin B.

[0235] Referring to FIGS. 11-13, FKBP12 inhibition was studied. FIG. 11 shows a model of a binder peptide, without a target shown. The binder peptide was a WGK bicycle FKBP12 hit (S at lysine alpha, R at lysine delta). FIGS. 12 and 13 show a model of that binder peptide bound to FKBP12.

[0236] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 wt.%" is intended to mean "about 40 wt.%".

[0237] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

Claims

Princeton - 107076CLAIMS1. A method for biocatalytic production of a polycyclic peptide, comprising:providing a peptide sequence containing a recognition motif for a radical S-adenosylmethionine mctalloenzyme (radical SAM enzyme);exposing a peptide sequence to an enzymatic reaction, the enzymatic reaction including catalysis under standard enzymatic reaction conditions with the radical SAM enzyme; and generating a further cyclized peptide by modifying a product of the enzymatic reaction chemically and / or via enzymes.

2. The method of claim 1, wherein the enzymatic reaction forms at least one carbon-carbon, carbon-sulfur, carbon-nitrogen and / or carbon-oxy gen bond at one or more unactivated carbon centers of the peptide sequence, regardless of the nature of a side-chain.

3. The method of claim 1, wherein non-canonical amino acids are incorporated into the peptide sequence.

4. The method of claim 1, wherein non-canonical amino acid side-chain(s) are crosslinked using a radical SAM enzyme.

5. The method of claim 1, wherein the enzymatic reaction forms at least one lysinetryptophan crosslink at an unactivated carbon center of the peptide sequence.

6. The method of claim 1, wherein the radical SAM enzyme is SuiB, WgkB, GggB, or TqqB ItfD, HghC, HghB, RrrB, NxxcB, KgrB, FwwB, WprB, OscB, XncB, RscB, and HtkB.

7. The method of claim 1, wherein the product of the enzymatic reaction is modified chemically using a haloalkane- or haloacetyl-containing compound.

8. The method of claim 7, wherein the haloalkane- or haloacetyl-containing compound includes 1,3-di bromoacetone and / or 4,4'-Bis(bromomethyl)-2-2'-bipyridine.

9. The method of claim 1, wherein the product of the enzymatic reaction is modified chemically using a radical SAM enzyme or a macrocyclase.Princeton - 10707610. The method of claim 9, wherein the macrocyclase is a thioesterase or a homolog thereof.

11. The method of claim 1 , wherein modifying a product of the enzymatic reaction includes generating a proteolyzed product by proteolyzing the product and then chemically or enzymatically modifying the proteolyzed product.

12. The method of claim 11, wherein the proteolyzed product is modified chemically using a haloalkane- or haloacefyl-containing compound.

13. The method of claim 11. wherein the proteolyzed product of the enzymatic reaction is modified chemically using a radical SAM enzyme or a macrocyclase.

14. The method of claim 1, further comprising:generating at least one additional peptide sequence by altering the peptide sequence at permissive sites;exposing at least one additional peptide encoded by the at least one additional peptide sequence to an enz matic reaction, the enzy matic reaction including catalysis under standard enzymatic reaction conditions with the radical SAM enzyme; andgenerating a further cyclized peptide by modifying a product of the enzymatic reaction chemically and / or via enzymes.

15. The method of claim 1, further comprising generating a library of crosslinked peptides by exposing a library of peptides, consisting as a mixture, to radical SAM enzyme catalysis.

16. The method of claim 15, wherein the library7of crosslinked peptides is further modified chemical or enzymatically as described in claims 7-13.

17. A kit containing a plurality of further cyclized peptides generated using a method of claim 1.

18. A composition of matter having a structure according to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5. or Compound 6.

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