PapB as a two-moiety-dependent thioether introduction tool

Abstract

The present disclosure relates to a method for chemically modifying a peptide sequence to introduce a thioether bond, the method comprising reacting the peptide sequence with PapB. Also disclosed are compounds produced by such methods that may be useful, for example, in peptide therapeutic applications. This summary is intended as a scanning tool for searching in a particular technical field and is not intended to limit the invention.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 446,589, filed February 17, 2023, U.S. Provisional Application No. 63 / 393,174, filed July 28, 2022, U.S. Provisional Application No. 63 / 337,029, filed April 29, 2022, and U.S. Provisional Application No. 63 / 331,393, filed April 15, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] Statement regarding federally funded research This invention was made with Government support under Grant No. GM126956 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing Reference The sequence listing submitted as an xml file named "21101.0436P1.xml", created on April 14, 2023 and having a size of 16,384 bytes, is incorporated herein by reference in accordance with 37 C.FR § 1.52(e)(5). [Background technology]

[0004] Peptide-based therapeutics are growing due to their unique structure and ability to be produced by solid-phase peptide synthesis (SPPS) or recombinant DNA. Many peptide therapeutics contain disulfide bonds in their active form. Disulfide bonds are susceptible to breakage via biological reducing agents such as glutathione. In addition, many peptide therapeutics contain bulky or basic amino acid side chains that make them vulnerable to degradation by proteases. These factors contribute to their short serum half-life. Strategies such as amino acid exchange between L-D, derivatization of the N- and C-termini, terminal cyclization between N-C, introduction of non-proteinogenic amino acids, and metal chelation have both increased peptide half-life and diverse therapeutic targets. The extent of these modifications is limited to the chemical space afforded by organic synthesis and SPPS.

[0005] Nature has access to a vast chemical space through enzymatic reactions. Natural products are highly diverse in their structures, enabling a wide range of biological and chemical activities. Recent advances in bioinformatic filtering algorithms have revealed previously unannotated small open reading frames (sORFs). sORFs often colocalize with maturases that further process peptides post-translationally. These ribosomally synthesized and post-translationally modified peptides (RiPPs) vary significantly in peptide length, structure, and biological function. RiPP maturases include members of the radical S-adenosylmethionine (rSAM) superfamily. This superfamily is responsible for a variety of RiPP modifications, including CC, CN, CO, and CS bond formation at unactivated carbons via radical mechanisms. These molecular mechanisms have attracted great interest as they provide access to a unique semisynthetic chemical space for the generation of biomimetic peptide therapeutics. RiPP maturases have the potential to provide biotechnological applications in peptide modifications such as thioether introduction or peptide stapling. rSAM enzymes use radical intermediates to complete chemical transformations involved in natural product biosynthesis and primary metabolism. These enzymes contain one or more iron-sulfur [Fe-S] clusters that are essential for function. The [4Fe-4S]rSAM (RS) cluster is coordinated by a canonical CxxxCxxC motif in the enzyme. In the [4Fe-4S]RS cluster, one iron coordinates the α-amino and α-carboxylate moieties of the SAM. When the RS cluster is catalytically active, it transfers an electron to the bound SAM. Because the RS cluster is catalytically inactive in the +2 state, either chemical or biological reduction systems are useful for product turnover. Homolytic cleavage of the SAM forms a reactive 5'-deoxyadenosyl radical (5'-dAdo, Figure 1). 5'-dAdo' functions as a radical initiator by abstracting a hydrogen atom from a specific site on the substrate, thereby forming 5'-deoxyadenosine (5'-dAdoH, Figure 1) and the theoretical RiPP radical intermediate. The substrate radical formed is useful for substrate maturation.Although only one [4Fe-4S] cluster is required for reductive SAM cleavage, many rSAM enzymes also employ one or more auxiliary iron-sulfur clusters (ACs) for substrate turnover (Fig. 4c). These ACs are coordinated to the enzyme by a cysteine-rich C-terminal extension from the RS canonical motif (Fig. 2). Recent studies have characterized rSAM maturases with multiple [Fe-S] clusters that form intrapeptide bonds between Cα, Cβ, or Cγ on specific residues and cysteine ​​thiols in peptide substrates. Many of these thioether-assembling maturases form only a single thioether in the mature peptide and have relatively slow substrate turnover. In addition to at least one AC cluster, an RS cluster is required for thioether formation. rSAM RiPP maturases also employ a critical RiPP recognition element (RRE) involved in binding of the immature peptide to the leader sequence (Fig. 2, left).

[0006] PapB is a RiPP maturase that catalyzes the insertion of six thioether bridges in the PapA polypeptide. PapB catalyzes the insertion of a bond between the Cys thiol and the b-carbon of Asp, the binding residue being in the CX3D motif. Previous studies have shown that the enzyme can also accept Glu at the modification site, and that PapB introduces a crosslink to the chemically similar γ-carbon. In addition, PapB has also been shown to accept a shorter minimal substrate (msPapA) that has only a single pair of crosslinking amino acids in the CX3D motif. PapB can catalyze both Cβ and Cγ thioether bonds, forming six thioether bonds in wild-type PapA. PapB contains an RS cluster and two ACs (Figure 2). Replacing the Asp residue(s) in WT-PapA with Glu residue(s) still results in successful crosslinking. Both Cβ and Cy thioether bonds were confirmed by 2D NMR.

[0007] Despite the advent of various techniques in peptide-based therapeutics, there remains a need in the art for enzymatic systems to rapidly and highly specifically modify a wide range of peptide entities to yield natural products that are not achievable by traditional synthetic chemistry methods. These and other needs are addressed herein. Summary of the Invention

[0008] In accordance with the object(s) of the invention as embodied and broadly described herein, the invention in one aspect relates to a method for chemically modifying a peptide sequence to introduce one or more thioether bonds. Additionally, compounds formed using the method for chemically modifying a peptide sequence are disclosed. Methods for chemically modifying a modified PapA sequence, and compounds formed using the method for chemically modifying a modified PapA sequence are also disclosed.

[0009] A method for chemically modifying a compound to introduce a thioether bond is disclosed, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, t is an integer from 0 to 500, v is 1, 2, 3, 4, or 5, A is S or Se, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl; R 8 is selected from hydrogen and methyl, with the proviso that the compound is not PapA.

[0010] Also disclosed is a method for chemically modifying a compound to introduce a thioether bond, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, t is an integer of 0 to 500, v is 1, 2, 3, 4, or 5, A is S or Se, and Q 1 is the leader sequence, and Q 2 is the cleavable moiety, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R5 Or R 5’ and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl; R 8 is selected from hydrogen and methyl, with the proviso that the compound is not PapA.

[0011] Also disclosed is a method for chemically modifying a compound to introduce a thioether bond, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , wherein m is 0, 1, 2, 3, or 4, n is 0 or 1, each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, A is S or Se, and L, if present, is C2-C4 alkyl, -(C1-C4 alkyl)(OCH2CH2) q ,and [ka] wherein q is 1, 2, 3, or 4; and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 2 is a residue of the side chain of an amino acid, with the proviso that the amino acid is not isoleucine or threonine; R 3a and R 3b each, when present, is independently selected from C2-C5 alkynyl, C1-C5 azide, and a residue of the side chain of an amino acid; R 4 is selected from hydrogen and methyl; R5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ covalently bonded to and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl, with the proviso that the compound is not PapA.

[0012] Also disclosed is a method for chemically modifying a compound to introduce a thioether bond, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , wherein m is 0, 1, 2, 3, or 4, n is 0 or 1, each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, A is S or Se, and L, if present, is C2-C4 alkyl, -(C1-C4 alkyl)(OCH2CH2) q ,and [ka] where q is 1, 2, 3, or 4; and Q 1 is the leader sequence, and Q 2 is the cleavable moiety, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 2 is a residue of the side chain of an amino acid, with the proviso that the amino acid is not isoleucine or threonine; R 3a and R 3b each, when present, is independently selected from C2-C5 alkynyl, C1-C5 azide, and a residue of the side chain of an amino acid; R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ covalently bonded to and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl, with the proviso that the compound is not PapA.

[0013] Also disclosed is a method for chemically modifying a peptide sequence to introduce a thioether bond, the method comprising reacting the peptide sequence with PapB, the peptide sequence being XY n -Z, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, X is an amino acid residue containing an -SH or -SeH group, each occurrence of Y, if present, is independently an amino acid residue, and Z is a carboxyl- or tetrazolyl-functionalized amino acid residue, with the proviso that the peptide sequence is not PapA.

[0014] Also disclosed is a method for chemically modifying a peptide sequence to introduce a thioether bond, the method comprising reacting the peptide sequence with PapB, the peptide sequence being XY n-Z, where X is penicillamine, or an amino acid residue containing an -SH group, or an amino acid residue containing an -SeH group, Y is a series of amino acid residues where n=0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, and Z is an aspartic acid residue, a glutamic acid residue, a hydroxy-glutamic acid residue, 2-amino-3-(2H-tetrazol-5-yl)propanoic acid, or a carboxyl-functionalized amino acid residue, and the peptide sequence is not PapA.

[0015] Also disclosed is a method for chemically modifying a modified PapA sequence to introduce a thioether bond, the method comprising reacting the modified PapA sequence with PapB, the modified PapA sequence comprising a Cys-Y n -Asp, and Y is a series of amino acid residues where n=0, 1, 2, 4, 5, 6, or 7.

[0016] Also disclosed are thioether compounds produced by the disclosed methods.

[0017] Also disclosed is a method for chemically modifying a modified PapA sequence to introduce a thioether bond, the method comprising reacting the modified PapA sequence with PapB, the modified PapA sequence comprising a Cys-Y n -Asp, and Y is a series of amino acid residues where n=0, 1, 2, 4, 5, 6, or 7.

[0018] Also disclosed are compounds produced by the disclosed methods.

[0019] Also, [ka] JPEG2025512475000013.jpg62170, or a pharma- ceutically acceptable salt thereof.

[0020] Also, [ka] or a pharma- ceutically acceptable salt thereof.

[0021] Also disclosed are pharmaceutical compositions comprising an effective amount of the disclosed compounds, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0022] Although aspects of the present invention may be described and claimed in a particular statutory class, such as a system statutory class, this is for convenience only, and one of ordinary skill in the art will understand that each aspect of the present invention may be described and claimed in any statutory class. Unless otherwise expressly stated, any method or aspect set forth herein is never intended to be interpreted as requiring its steps to be performed in a particular order. Thus, method claims are never intended to infer order in any respect unless specifically stated in the claims or specification that the steps are to be limited to a particular order. This is to be maintained for any possible non-express basis for interpretation, including obvious meanings derived from the arrangement of steps or operational flow, grammatical construction or punctuation, or logical matters regarding the number or type of aspects described in the specification.

[0023] The above and other objects, features and advantages of the present invention will become more apparent to those skilled in the art from the detailed description of illustrative embodiments thereof taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic showing the proposed mechanism for beta-thioether cross-linking. [Diagram 2] 1 is a scheme showing the predicted structure of PapB. [Diagram 3] Representative images showing SDS-PAGE analysis of reconstituted and purified PapB on a 12% cross-linked gel. [Figure 4A]Representative cross-linking data for minimal substrate PapA (msPapA) with PapB is shown. Specifically, a representative TIC of msPapA chromatographed on a C18 HPLC column (spectrum at top left) is shown. The peptide elutes at 8.1 min. A representative mass spectrum corresponding to the peak eluting at 8.1 min is shown at the bottom. The z=3 charge state was chosen for most peptide mass envelope comparisons. A representative mass spectral comparison of the z=3 charge state envelopes of unreacted and reacted msPapA±PapB is shown at the top right. [Figure 4B] Representative cross-linking data for minimal substrate PapA (msPapA) with PapB is shown, specifically the sequence of cross-linked PapB showing all of the b- and y-ions observed from tandem mass spectrometry. [Diagram 5] Representative plots showing a comparison of activity of PapB processing Y17W msPapA with dithionite or FldA / FPR / NADPH. [Figure 6] Representative mass spectra demonstrating the effect of 2- and 4-fold enzyme concentration are shown. [Figure 7] Representative mass spectra demonstrating the effect of 2- and 4-fold peptide concentration are shown. [Figure 8A] Representative data for leader-C(X0-X6)D(Xm) cross-link formation is shown, specifically a scheme showing that unmodified and modified peptide sequences exhibit a thioether cross-link based on the msPapA modification reported by Prorecord et al. [Figure 8B] Representative data for leader-C(X0-X6)D(Xm) crosslink formation is shown. Specifically, a representative mass spectrum for the CX0D-CX2D PapB modification is shown. [Figure 8C] Representative data for leader-C(X0-X6)D(Xm) crosslink formation is shown. Specifically, a representative mass spectrum for the CX4D-CX6D PapB modification is shown. [Figure 8D]Representative data for leader-C(X0-X6)D(Xm) bridge formation is shown, specifically a schematic showing the expected 2 Da loss seen in each b and y fragment in tandem mass spectrometry. [Figure 9A] Representative data for iodoacetic acid treatment of CXOD is shown, specifically representative mass spectral data for CXOD without PapB. [Figure 9B] Representative data for iodoacetic acid treatment of CX0D is shown. Specifically, representative mass spectral data for CX0D containing PapB is shown. [Figure 10A] Representative data for iodoacetic acid treatment for CX1D is shown. Representative mass spectral data for CX0D, specifically, which does not contain PapB, is shown. [Figure 10B] Representative data for iodoacetic acid treatment of CX1D is shown. Specifically, representative mass spectral data for CX1D containing PapB is shown. [Figure 11A] Representative data for iodoacetic acid treatment for CX2D is shown. Representative mass spectral data for CX0D, specifically, which does not contain PapB, is shown. [Figure 11B] Representative data for iodoacetic acid treatment of CX2D is shown. Specifically, representative mass spectral data for CX2D containing PapB is shown. [Figure 12A] Representative data for iodoacetic acid treatment for CX4D is shown. Representative mass spectral data for CX0D, specifically, which does not contain PapB, is shown. [Figure 12B] Representative data for iodoacetic acid treatment of CX4D is shown. Specifically, representative mass spectral data for CX4D containing PapB is shown. [Figure 13A] Representative data for iodoacetic acid treatment for CX5D is shown. Representative mass spectral data for CX0D, which does not contain PapB, is shown. [Figure 13B] Representative data for iodoacetic acid treatment of CX5D is shown. Specifically, representative mass spectral data for CX5D containing PapB is shown. [Figure 14A] Representative data for iodoacetic acid treatment for CX6D is shown. Representative mass spectral data for CX0D, which does not contain PapB, is shown. [Figure 14B] Representative data for iodoacetic acid treatment of CX6D is shown. Specifically, representative mass spectral data for CX6D containing PapB is shown. [Figure 15A] Representative data is shown for a leader extension with a single nested linear bridge, specifically a peptide scheme showing the apparent bridge location that remains consistent after distancing the thioether motif from the leader peptide. [Figure 15B] Representative data is shown for leader extensions with a single nested linear bridge, specifically representative mass spectra showing the isotopic distribution of the peptide, a shift of 2 Da in the case of a single thioether motif, or a shift of 4 Da with a double thioether motif upon addition of PapB. [Figure 15C] Representative data for leader extension with a single nested linear bridge is shown. Specifically, FIG. 1 is a schematic showing the display of tandem mass spectrometry results. [Figure 16A] Representative data for iodoacetic acid treatment for leader-AAACSANDA is shown. Representative mass spectral data for leader-AAACSANDA without PapB is shown. [Figure 16B] Representative data for iodoacetic acid treatment for leader-AAACSANDA is shown. Representative mass spectral data for leader-AAACSANDA containing PapB is shown. [Figure 17A]Representative data for iodoacetic acid treatment for leader-AAACSANDACSANDA is shown. Representative mass spectral data for leader-AAACSANDACSANDA without PapB is shown. [Figure 17B] Representative data for iodoacetic acid treatment for leader-AAACSANDACSANDA is shown. Representative mass spectral data for leader-AAACSANDACSANDA containing PapB is shown. [Figure 18A] Representative data for iodoacetic acid treatment for leader-AAACSACDAADA is shown. Representative mass spectral data for leader-AAACSACDAADA without PapB is shown. [Figure 18B] Representative data for iodoacetic acid treatment for leader-AAACSACDAADA is shown. Representative mass spectral data for leader-AAACSACDAADA containing PapB is shown. [Figure 19A] Representative data for iodoacetic acid treatment for leader-AAAASACDAADA is shown. Representative mass spectral data for leader-AAAASACDAADA without PapB is shown. [Figure 19B] Representative data for iodoacetic acid treatment for leader-AAAASACDAADA is shown. Representative mass spectral data for leader-AAAASACDAADA containing PapB is shown. [Figure 20A] Representative data for iodoacetic acid treatment for leader-AAACSAADAADA is shown. Representative mass spectral data for leader-AAACSAADAADA without PapB is shown. [Figure 20B] Representative data for iodoacetic acid treatment for leader-AAACSAADAADA is shown. Representative mass spectral data for leader-AAACSAADAADA containing PapB is shown. [Figure 21A]1 shows representative data demonstrating that PapB generates two thioether cross-links in the AMK-1057 precursor peptide in vitro. FIG. 2 is a scheme showing that the AMK-1057 precursor peptide contains a leader peptide sequence, a TEV protease recognition sequence, and two CX3E motifs. [Figure 21B] Representative data showing that PapB generates two thioether bridges in the AMK-1057 precursor peptide in vitro. Representative mass spectra demonstrating the formation of two bridges upon reaction with PapB in an in vitro assay. Further treatment with TEV protease produces the expected bicyclized peptide. [Figure 21C] 1 shows representative data demonstrating that PapB generates two thioether bridges in the AMK-1057 precursor peptide in vitro. A scheme demonstrating the topology of the bonds as confirmed by tandem mass spectrometry. [Figure 22A] Representative data for PapB cross-linking DC and DD msPapA peptides are shown. Scheme showing thioether cross-linking. [Figure 22B] Representative data for PapB cross-linking DC and DD msPapA peptides are shown. Representative mass spectra showing the formation of thioether cross-links. [Figure 22C] Representative data for PapB cross-linking DC and DD msPapA peptides are shown. Scheme demonstrating the topology of binding as confirmed by mass spectrometry. [Figure 23A] Representative data for iodoacetic acid treatment for leader-DCSANDA is shown. Representative mass spectral data for leader-DCSANDA without PapB is shown. [Figure 23B] Representative data for iodoacetic acid treatment for leader-DCSANDA is shown. Representative mass spectral data for leader-DCSANDA containing PapB is shown. [Figure 24A]Representative data for iodoacetic acid treatment for leader-CSANDDA is shown. Representative mass spectral data for leader-CSANDDA without PapB is shown. [Figure 24B] Representative data for iodoacetic acid treatment for leader-CSANDDA is shown. Representative mass spectral data for leader-CSANDDA containing PapB is shown. [Figure 25A] Representative data for iodoacetic acid treatment for leader-DCSANDDA is shown. Representative mass spectral data for leader-DCSANDDA without PapB is shown. [Figure 25B] Representative data for iodoacetic acid treatment for leader-DCSANDDA is shown. Representative mass spectral data for leader-DCSANDDA containing PapB is shown. [Figure 26A] Representative data for the msPapA "DSANCA" peptide is shown. Representative mass spectral data for leader-DSANCA and leader-DDSANCA with and without PapB is shown. [Figure 26B] Representative data for the msPapA "DSANCA" peptide is shown. Representative mass spectral data for leader-DSANDCA and leader-DDSANDCA with and without PapB is shown. [Figure 27A] Representative data for the synthesis of octreotide analogs is shown. The structure of FDA approved therapeutic octreotide. [Figure 27B] Representative data for the synthesis of octreotide analogs are shown. Schematic representation of the designed peptides and the predicted modification sites upon modification by PapB. A TEV cleavage site is included in the second peptide to allow release of the modified peptide sequence by PapB. [Figure 27C]1 shows representative data for the synthesis of octreotide analogs. Representative mass spectral data showing the isotopic envelope of these peptides, indicating that a mixed population of processed and unprocessed peptides exists following modification with PapB. [Figure 27D] 1 shows representative data for the synthesis of octreotide analogs. 2 shows representative mass spectral data demonstrating that the TEV cleavage peptide isotopic envelope reveals the expected 2 Da mass shift. [Figure 27E] 1 shows representative data for the synthesis of octreotide analogs. A scheme showing the expected loss of 2 Da in each y fragment after C and each b fragment after the C-terminal E, confirmed by tandem mass spectrometry. [Figure 28] 1 is a structure of a synthesized thioether-linked octreotide analogue. [Figure 29] FIG. 1 is a scheme providing a brief summary of successful PapB-mediated thioether cross-linking in the peptide sequences tested. [Diagram 30] Representative data are shown demonstrating that a leader peptide sequence is not required for PapB-mediated modification. [Diagram 31] Representative mass spectrometry data for one-to-one interpeptide cross-linking and polymeric-like addition of X-mer subunits are shown. [Diagram 32] Representative mass spectrometry results for a typical pre- and post-PapB assay peptide are shown, demonstrating the presence of interpeptide products. [Diagram 33] Representative mass spectral data showing evidence of simple and complex mass envelopes are shown. [Diagram 34] FIG. 1 is a schematic showing the experimental approach for generating modified insulin analogs using PapB. [Diagram 35] Representative mass spectral data for the synthesized insulin analogues are shown. [Diagram 36] Representative mass spectral data for cross-linking in a peptide containing EneA is shown. [Figure 37] Representative tandem mass spectrometry data for the dAdo+D24EneA msPapA adduct is shown. [Figure 38] Representative data including mass spectrometry and EXAFS are shown for crosslinks in selenopeptides. [Figure 39] Representative tandem mass spectrometry data for C19U msPapA is shown. [Diagram 40] Representative mass spectrometry data is shown demonstrating that aspartic acid can be replaced by glutamic acid and cysteine ​​can be replaced by homocysteine. Crosslinks are observed. [Diagram 41]

[0023] Figure 1 shows representative mass spectrometry data demonstrating that β-amino acids can be incorporated into peptides. Cross-links are observed. [Diagram 42] Representative mass spectrometry data is shown demonstrating that no cross-linking was observed when the positions of C and D residues were changed. [Diagram 43] Representative data is presented demonstrating the effect of components in the reduction system used. [Diagram 44] FIG. 1 is a schematic diagram summarizing the findings of experiments performed with pre-reduced PapB. [Diagram 45] FIG. 1 is a scatter plot showing representative data of % product as a function of time for PapB experiments before reduction. [Figure 46] Representative data are shown, including photodiode array chromatography, UV-Vis, and extracted ion chromatography, for PapB with and without reducing agent, and for PapB before reduction. [Figure 47] FIG. 1 is a conceptual schematic of the bioreactor set-up for PapB-mediated peptide modification. [Figure 48A] Representative data for a C-terminal glycine sequence is shown. Scheme showing thioether cross-linking. [Figure 48B] Representative data for a C-terminal glycine sequence is shown. Representative mass spectra showing the formation of a thioether bridge. [Figure 49A]Representative data for deuterium-labeled C-terminal glycine analogs are shown. Scheme showing thioether cross-linking. [Figure 49B] Representative data for a deuterium-labeled C-terminal glycine analog is shown. Representative mass spectra showing the formation of a thioether bridge. [Figure 50A] Representative data for a C-terminal glycine carboxamide sequence is shown. [Figure 50B] Representative data for a C-terminal glycine carboxamide sequence is shown. Representative mass spectra showing the lack of formation of a thioether bridge. [Figure 51A] Representative data for cross-linking with a C-terminal β-amino acid is shown. Scheme showing general thioether cross-linking reactions for C-terminal β-amino acids. [Figure 51B] Representative data for cross-linking with a C-terminal β-amino acid is shown. Scheme showing thioether cross-linking reaction with a C-terminal β-alanine. [Figure 51C] Representative data for cross-linking to the C-terminal β-amino acid is shown, along with corresponding mass spectral data showing the formation of a thioether cross-link. [Figure 52A] 1 shows representative data for cross-linking with various C-terminal β-amino acids. FIG. 2 is a scheme showing the absence of cross-linking reaction with C-terminal 2,2-dimethyl-beta-alanine. [Figure 52B] Representative data for cross-linking with various C-terminal β-amino acids is shown. Scheme showing the absence of cross-linking reaction with C-terminal (R)-3-amino-2-methylpropanoic acid. [Figure 52C] Representative data for cross-linking with various C-terminal β-amino acids are shown. Scheme showing cross-linking reaction with C-terminal (S)-3-amino-2-methylpropanoic acid. [Fig. 52D] Representative data for cross-linking with various C-terminal β-amino acids is shown, along with corresponding mass spectral data showing the formation of thioether cross-links. [Figure 53]Representative data for cross-linking with a common C-terminal β-amino acid is shown. [Figure 54A] FIG. 1 shows a schematic diagram of a thioether bridge with a D-tryptophan β-amino acid. [Figure 54B] 13 is the corresponding mass spectral data showing the formation of thioether crosslinks. [Figure 55A] Representative structures of thioether bridges of N-methyl amino acids are shown. Unsubstituted N-methylated thioether bridge products are shown. [Figure 55B] Representative structures of thioether bridges of N-methyl amino acids are shown. Substituted N-methylated thioether bridge products are shown. [Figure 55C] Representative structures of thioether bridges of N-methyl amino acids are shown. A schematic diagram of thioether bridges with substituted N-methylated substrates is shown. [Fig. 55D] 1 shows a representative structure of a thioether bridge of an N-methyl amino acid and corresponding mass spectral data showing the formation of the thioether bridge. [Figure 56A] Representative data for thioether cross-linking with C-terminal L-alanine or D-alanine are shown. A schematic diagram of C-terminal L-alanine with no thioether cross-linking product is shown. [Figure 56B] Representative data for a thioether bridge with a C-terminal L-alanine or D-alanine are shown, along with corresponding mass spectrometry data showing the lack of formation of the thioether bridge. [Figure 56C] Representative data for thioether cross-linking with C-terminal L-alanine or D-alanine are shown. A schematic diagram of the C-terminal D-alanine with thioether cross-linking product is shown. [Figure 56D] 1 shows representative data for a thioether bridge with a C-terminal L-alanine or D-alanine and corresponding mass spectral data showing the formation of the thioether bridge. [Figure 57A] 1 shows representative data for thioether cross-linking with deuterium-labeled C-terminal D-alanine. 2 shows a schematic diagram of deuterium-labeled C-terminal D-alanine with thioether cross-linking product. [Figure 57B] Representative data for a thioether bridge with a deuterium-labeled C-terminal D-alanine are shown, along with corresponding mass spectral data showing the formation of the thioether bridge and loss of labeled deuterium, confirmed by a mass shift and loss of 3 Da. [Figure 58A] Representative data for thioether cross-linking with deuterium-labeled C-terminal D-methionine is shown. A schematic diagram of deuterium-labeled C-terminal D-methionine with thioether cross-linking product is shown. [Figure 58B] Representative data for a thioether bridge with deuterium-labeled C-terminal D-methionine are shown, along with corresponding mass spectral data showing the formation of the thioether bridge and loss of labeled deuterium, confirmed by a mass shift and loss of 3 Da. [Figure 59A] Representative data for thioether cross-linking with d2-labeled D-valine is shown. The structure of deuterium-labeled C-terminal D-valine is shown. [Figure 59B] Representative data for a thioether bridge with d2-labeled D-valine is shown, along with corresponding mass spectral data showing the formation of a thioether bridge, but the mass shift does not indicate loss of deuterium. [Figure 60A] Representative data for thioether cross-linking with d3-labeled D-valine is shown. A schematic diagram of deuterium-labeled side chain C-terminal D-valine with thioether cross-linking product is shown. [Figure 60B] Representative data for a thioether bridge with d3-labeled D-valine is shown, along with corresponding mass spectral data showing the formation of a thioether bridge and loss of labeled deuterium, confirmed by a mass shift and loss of 3 Da. [Figure 61A] Representative data for thioether cross-linking with deuterium-labeled C-terminal D-phenylalanine is shown. The structure of deuterium-labeled Cα C-terminal D-phenylalanine is shown. [Figure 61B]Representative data for a thioether bridge with a deuterium-labeled C-terminal D-phenylalanine are shown, along with corresponding mass spectral data showing the formation of a thioether bridge, but the mass shift does not indicate loss of deuterium. [Figure 61C] Representative data for thioether cross-linking with deuterium-labeled C-terminal D-phenylalanine is shown. The structure of deuterium-labeled aryl C-terminal D-phenylalanine is shown. [Fig.61D] Representative data for a thioether bridge with a deuterium-labeled C-terminal D-phenylalanine are shown, along with corresponding mass spectral data showing the formation of a thioether bridge, but the mass shift does not indicate loss of deuterium. [Figure 62A] Representative data for thioether crosslinking with deuterium-labeled d8-C-terminal D-phenylalanine is shown. A schematic diagram of deuterium-labeled d8-C-terminal D-methionine with thioether crosslinking product is shown. [Figure 62B] Representative data for a thioether bridge with deuterium-labeled d8-C-terminal D-phenylalanine is shown, along with corresponding mass spectral data showing the formation of the thioether bridge as confirmed by mass shift and loss of labeled deuterium. [Figure 63] The structures of the corresponding D-amino acid sactipeptide thioether bridges are shown. [Figure 64] The structures of the corresponding D-amino acid lanthipeptide thioether bridges are shown. [Figure 65A] Representative data for a six-membered non-peptide thioether bridge is shown. A scheme for the leader-Cys-Gly reaction is shown. [Figure 65B] Representative data for a six-membered non-peptide thioether bridge is shown, along with corresponding mass spectral data showing the lack of formation of the six-membered ring thioether bridge. [Figure 66A] Representative data for a seven-membered non-peptide thioether bridge is shown. A scheme for the leader-hCys-Gly reaction is shown. [Figure 66B]1 shows representative data for a seven-membered non-peptide thioether bridge and corresponding mass spectrometry data showing the formation of a seven-membered ring thioether bridge. [Figure 67A] Representative data for a seven-membered non-peptide thioether bridge is shown. A scheme for the leader-Cys-βAla reaction is shown. [Figure 67B] Representative data for a seven-membered non-peptide thioether bridge is shown, along with corresponding mass spectral data showing the formation of a seven-membered ring thioether bridge. [Figure 68A] Representative data for an 8-membered non-peptide thioether bridge is shown. A scheme for the leader-hCys-βAla reaction is shown. [Figure 68B] Representative data for an 8-membered non-peptide thioether bridge is shown, along with corresponding mass spectral data showing the formation of an 8-membered ring thioether bridge. [Figure 69A] Representative data for an eight-membered non-peptide thioether bridge is shown. A scheme for the leader-Cys-GABA reaction is shown. [Figure 69B] Representative data for an 8-membered non-peptide thioether bridge is shown, along with corresponding mass spectral data showing the formation of an 8-membered ring thioether bridge. [Figure 70A] Representative data for a nine-membered non-peptide thioether bridge is shown. A scheme for the leader-hCys-GABA reaction is shown. [Figure 70B] Representative data for a 9-membered non-peptide thioether bridge is shown, along with corresponding mass spectral data showing the formation of a 9-membered ring thioether bridge. [Figure 71A] Representative data for a 16-membered non-peptide thioether bridge is shown. A scheme for the leader-hCys-NH-PEG3-CO2H reaction is shown. [Figure 71B] 1 shows representative data for a 16-membered non-peptide thioether bridge and corresponding mass spectral data showing the formation of a 16-membered ring thioether bridge. [Figure 72A]Representative data for a 20-membered non-peptide thioether bridge is shown. A scheme for the leader-hCys-NH-PEG4-CO2H reaction is shown. [Fig. 72B] 1 shows representative data for a 20-membered non-peptide thioether bridge and corresponding mass spectral data showing the formation of a 20-membered ring thioether bridge. [Figure 73A] Representative data for unusual non-peptide thioether crosslinks are shown. A scheme for the leader-Cys-Ser-Ala-Asn-2-(2-aminophenyl)acetic acid reaction is shown. [Figure 73B] Representative data for an unusual non-peptide thioether bridge are shown, along with corresponding mass spectrometry data showing the formation of a 17-membered ring thioether bridge. [Fig. 74A] Representative data for unusual non-peptide thioether crosslinks are shown. A scheme for the leader-Cys-Ser-Ala-Asn-2-(2-(aminomethyl)phenyl)acetic acid reaction is shown. [Fig. 74B] Representative data for an unusual non-peptide thioether bridge are shown, along with corresponding mass spectral data showing the formation of an 18-membered ring thioether bridge. [Fig. 75A] Representative data for coumarin thioether crosslinks are shown. A scheme for the leader-Cys-coumarin reaction is shown. [Fig. 75B] Representative data for a coumarin thioether bridge is shown with corresponding mass spectral data indicating the formation of a 12-membered ring thioether bridge. [Figure 76A] Representative data for synthetic thioether peptidomimetics are shown: Structure of the FDA-approved drug, cetomaranotide. [Figure 76B] Representative data for synthetic thioether peptidomimetics are shown. Schematic thioether cross-linking with modified peptide structures (e.g., analogs of cetomaranotide) is shown. [Figure 76C]1 shows representative data for synthetic thioether peptidomimetics with corresponding mass spectral data showing the formation of thioether crosslinks. [Figure 77A] Representative data for synthetic thioether peptidomimetics are shown. Structures of orally available peptides from Novartis. [Fig. 77B] Representative data for synthetic thioether peptidomimetics are shown: Structure of the designed peptide (an analog of the therapeutic peptide of Figure 77A) and the predicted product upon modification with PapB. [Fig. 77C] Representative data for synthetic thioether peptidomimetics are shown. A schematic thioether cross-link with modified peptide structure is shown. [Fig. 77D] 1 shows representative data for synthetic thioether peptidomimetics with corresponding mass spectral data showing the formation of thioether crosslinks. [Fig. 78A] 1 shows representative therapeutic cyclic peptides that can be mimicked by thioether bridged peptides. FIG. 2 shows the structure of a representative cyclic peptide, bremeianotide. [Fig. 78B] 1 shows a representative therapeutic cyclic peptide that can be mimicked by a thioether cross-linked peptide. 2 shows a representative structure of a thioether cross-linked product that is an analog of bremeianotide, containing the amino acid sequences norleucine, cysteine, D-phenylalanine, arginine, tryptophan, and epsilon-aminohexanoic acid (ACP). [Fig. 78C] 1 shows a representative therapeutic cyclic peptide that can be mimicked by a thioether bridged peptide. 2 shows a representative scheme of the leader-XCDFRWZ XXX reaction. [Fig. 78D] 1 shows representative therapeutic cyclic peptides that can be mimicked by thioether bridged peptides and corresponding mass spectral data showing the formation of thioether bridges in therapeutic analogs. [Figure 79A]Representative data showing that PapB forms cross-links at extended side chains containing thiols and carboxylates are shown, specifically, a generalized linear scenario of C19hCys msPapA where n = CH2(Asp), (CH2)2(Glu), or (CH2)3(hGlu). [Fig. 79B] Representative data showing that PapB forms cross-links at extended side chains containing thiols and carboxylates are shown, specifically showing a 2 Da shift in MS for carboxylate-containing residues as Asp. [Figure 79C] Representative data showing that PapB forms cross-links at extended side chains containing thiols and carboxylates are shown, specifically showing a 2 Da shift in MS for carboxylate-containing residues such as Glu. [Fig. 79D] Representative data showing that PapB forms cross-links at extended side chains containing thiols and carboxylates are shown, specifically showing a 2 Da shift in MS for carboxylate-containing residues as homoGlu. [Figure 79E] Representative data showing that PapB forms cross-links at extended side chains containing thiols and carboxylates are shown, specifically, MS of the large cyclized peptide core released from the leader sequence after cleavage of the TEV protease recognition sequence by TEV protease. [Figure 80] A representative proton NMR spectrum of the linear G(hC)SAN(hE)A peptide is shown. [Figure 81] A representative proton NMR spectrum of the cyclized G(hC)SAN(hE)A peptide is shown. [Figure 82] A representative ROESY spectrum of the linear G(hC)SAN(hE)A peptide is shown. [Figure 83] A representative ROESY spectrum of the cyclized G(hC)SAN(hE)A peptide is shown. [Fig. 84A]Representative data for carboxylate isosteres (tetrazole moieties) cross-linked by PapB are shown, specifically, schematics of linear and cyclized peptides showing the putative cross-linking positions are shown. [Fig. 84B] Representative data are shown for a carboxylate isostere (tetrazole moiety) cross-linked by PapB, specifically MS results showing a clear 2 Da loss between assays without PapB (darker grey) and assays with the addition of PapB (lighter grey). [Fig. 84C] Representative data is shown for a carboxylate isostere (tetrazole moiety) cross-linked by PapB, specifically showing the expected tandem mass spectrometry with no fragmentation between Cys and T4Az. [Figure 85] Representative fragmentation of the reacted D23T4Az msPapA variant is shown. [Figure 86] Representative fragments of tetrazole loss in the D23T4Az msPapA variant are shown.

[0025] Additional advantages of the invention will be set forth in part in the description which follows and in part will be obvious from the description or may be learned by the practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0027] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods, unless otherwise specified, or to specific reagents, unless otherwise specified, as they may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein.

[0028] Although aspects of the present invention may be described and claimed in a particular statutory class, such as a system statutory class, this is for convenience only, and one of ordinary skill in the art will understand that each aspect of the present invention may be described and claimed in any statutory class. Unless otherwise expressly stated, any method or aspect set forth herein is never intended to be interpreted as requiring its steps to be performed in a particular order. Thus, method claims are never intended to infer order in any respect unless specifically stated in the claims or specification that the steps are to be limited to a particular order. This is to be maintained for any possible non-express basis for interpretation, including obvious meanings derived from the arrangement of steps or operational flow, grammatical construction or punctuation, or logical matters regarding the number or type of aspects described in the specification.

[0029] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this application pertains. The references disclosed are also discussed in the sentence in which they are relied upon and are individually and specifically incorporated by reference herein for the material contained therein. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publications provided herein may be different from the actual publication dates and may be independently confirmed.

[0030] A.Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "functional group," an "alkyl," or a "residue" includes a mixture of two or more such functional groups, alkyls, or residues, etc.

[0031] As used in this specification and claims, the term "comprising" can include the embodiments "consisting of" and "consisting essentially of."

[0032] As used herein, ranges can be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each range are significant in relation to the other endpoint, and independently of the other endpoint. Additionally, certain values ​​are disclosed herein, and each value is understood to be disclosed herein as "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0033] As used herein, the terms "about" and "approximately" mean that the quantity or value in question may be a value that indicates approximately or roughly the same some other value. As used herein, it is generally understood to be a nominal value that indicates a variation of ±10% unless otherwise indicated or inferred. The term is intended to convey that similar values ​​promote the same results or effects as described in the claims. That is, it is understood that the amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those skilled in the art. In general, the amounts, sizes, formulations, parameters, or other quantities or characteristics are "about" or "approximate", whether or not they are expressly stated as such. When "about" is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0034] References in the specification and concluding claims to parts by weight of a particular element or component in a composition indicate the weight relationship between the element or component and any other element or component in the composition or product for which the parts by weight are expressed. Thus, in a composition comprising 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are included in the composition.

[0035] Weight percentage (wt %) of a component is based on the total weight of the formulation or composition in which the component is included, unless specifically stated to the contrary.

[0036] As used herein, "IC 50 " is intended to refer to the concentration of a substance (e.g., a compound or drug) required for 50% inhibition of a biological process or component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. In one embodiment, the IC 50 can refer to the concentration of a substance required for 50% inhibition in vivo, as further defined elsewhere herein. In a further embodiment, IC 50 refers to the half-maximal (50%) inhibitory concentration (IC) of a substance.

[0037] As used herein, "EC 50 " is intended to refer to the concentration of a substance (e.g., a compound or drug) required for 50% agonism of a biological process or component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. 50 can refer to the concentration of a substance required for 50% agonism in vivo, as further defined elsewhere herein. In a further embodiment, EC 50 refers to the concentration of agonist that elicits a response halfway between the baseline and maximum response.

[0038] As used herein, the term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0039] As used herein, the term "subject" may be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Thus, the subject of the methods disclosed herein may be a human, a non-human primate, a horse, a pig, a rabbit, a dog, a sheep, a goat, a cow, a cat, a guinea pig, or a rodent. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, as well as fetuses of either sex. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease, disorder, or condition. The term "patient" includes human and veterinary subjects.

[0040] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorate a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed to remove the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to reduce symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy directed to ameliorate the associated disease, condition, or disorder. In various embodiments, the term covers any treatment of a subject, including a mammal (e.g., a human), including (i) preventing the onset of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (ii) inhibiting the disease, i.e., arresting its onset, or (iii) relieving the disease, i.e., causing regression of the disease. In one embodiment, the subject is a mammal such as a primate, and in a further embodiment, the subject is a human. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), farm animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.).

[0041] As used herein, the term "prevent" or "preventing" refers to preventing, avoiding, eliminating, forestalling, hindering, or impeding something from happening, especially by prior action. Where reduce, inhibit, or prevent are used herein, it is understood that the use of the other two words is also expressly disclosed unless specifically indicated otherwise.

[0042] As used herein, the term "diagnosed" means having undergone a physical examination by a person skilled in the art, e.g., a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

[0043] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectables such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the preparation can be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation can be administered prophylactically, i.e., administered for the prevention of a disease or condition.

[0044] As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired outcome or to affect an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic outcome or to have an effect on an undesired symptom, but insufficient to generally cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition, the particular composition used; the age, weight, general health, sex, and diet of the patient; the timing of administration; the route of administration; the excretion rate of the particular compound used; the duration of treatment; drugs used in combination with or simultaneously with the particular compound used, and similar factors well known in the medical arts. For example, it is well within the skill of the art to start a dose of a compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. If desired, an effective daily dose can be divided into multiple doses for administration. Consequently, a single dose composition can contain such amounts or submultiples thereof to make up the daily dose. In the event of any contraindications, the dosage can be adjusted by the individual physician. Dosages can vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical product. In further various embodiments, the preparations can be administered in a "prophylactically effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0045] As used herein, a "dosage form" refers to a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. The dosage form can include the disclosed compound of the present invention, the product of the disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharma- ceutically acceptable excipient, such as a preservative, a buffer, saline, or phosphate buffered saline. The dosage form can be made using conventional pharmaceutical manufacturing and compounding techniques. The dosage form can contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphates, carbonates, acetates, or citrates) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrates or acetates, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium desoxycholate), solution and / or cryo / freeze stabilizers (e.g., Injectables may include, for example, sucrose, lactose, mannitol, trehalose), osmolality adjusters (e.g., salts or sugars), antimicrobial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity adjusters (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). Dosage forms formulated for injectable use may have the disclosed compounds, products of the disclosed methods of making, or salts, solvates, or polymorphs thereof suspended in sterile saline for injection together with a preservative.

[0046] As used herein, a "kit" refers to an assemblage of at least two components that make up the kit. Together, the components constitute a functional unit for a given purpose. The individual member components may be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include instructions with the other individual member components. Alternatively, the instructions may be provided as separate member components, either in paper form, or in electronic form, such as provided on a computer-readable memory device, or downloaded from an internet website, or as a recorded presentation.

[0047] As used herein, "instructions"(s) refers to documents describing the relevant materials or methodologies associated with the kit. These materials may include any combination of the following: background information, a list of components and their availability information (such as purchasing information), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and any other relevant documentation. The instructions may be provided in either paper form, or electronic form, either provided on a computer readable memory device, or downloaded from an internet website, or as a recorded presentation, with the kit or as a separate member component. The instructions may include one or more documents and are meant to include future updates.

[0048] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition of matter that, when administered to an organism (human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action. Thus, the term encompasses compounds or chemicals traditionally considered to be drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, and the like. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th Edition), Physicians' Desk Reference (64th Edition), and The Pharmacological Basis of Therapeutics (12th Edition), and include, without limitation, pharmaceuticals, vitamins, mineral supplements, substances used in the treatment, prevention, diagnosis, cure, or mitigation of disease or illness, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions for use in all of the major therapeutic areas, including, but not limited to, adjuvants, anti-infective agents such as antibiotics and antivirals, anti-cancer and anti-tumor agents such as kinase inhibitors, poly ADP-ribose polymerase (PARP) inhibitors and other DNA damage response modifiers, epigenetic agents such as bromodomain and extraterminal (BET) inhibitors, histone deacetylase (HDAc) inhibitors, iron activators and other ribonucleotide reductase inhibitors, proteasome inhibitors and Nedd8 activating enzyme (NAE) inhibitors, laminin inhibitors, and other anti-cancer and anti-tumor agents such as ribonucleotide inhibitors. conventional cytotoxic agents such as mammalian target of mycobacterium (mTOR) inhibitors, paclitaxel, doxorubicin, irinotecan, and platinum compounds; immune checkpoint blockade agents such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibodies (mABs), programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1) mABs, cluster of differentiation 47 (CD47) mABs, toll-like receptor (TLR) agonists and other immune modifiers; cellular therapeutic agents such as chimeric antigen receptor T cells (CAR-T) / chimeric antigen receptor natural killer (CAR-NK);Proteins such as interferons (IFNs), interleukins (ILs), and mAbs, anti-ALS agents such as entry inhibitors, fusion inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors, NCP7 inhibitors, protease inhibitors, and integrase inhibitors, analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptic agents, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuronal blockers, anticholinergics and cholinoergic agents, antimuscarinic and muscarinic agonists, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, anti-arthritic agents, anti-asthmatic agents, anticonvulsants, antihistamines, antiemetics, antitumor agents, antipruritics, antipyretics, sedatives, Included are spasmodics, cardiovascular agents (including calcium channel blockers, beta blockers, beta agonists and antiarrhythmic agents), antihypertensives, diuretics, vasodilators, central nervous system stimulants, cough and cold remedies, decongestants, diagnostic agents, hormones, bone growth stimulants and bone resorption inhibitors, immunosuppressants, muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced), as well as nucleic acid molecules (polymeric forms of two or more nucleotides, ribonucleotides (RNA) or deoxyribonucleotides (DNA), including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other biologically active macromolecules, such as proteins and enzymes. The drug may be a biologically active agent used in medical applications, including veterinary medicine, and in agriculture, such as plants, and other areas. The term "therapeutic agent" also includes, but is not limited to, a pharmaceutical agent; a vitamin; a mineral supplement; a substance used to treat, prevent, diagnose, cure, or mitigate a disease or illness; or a substance that affects the structure or function of the body; or a prodrug that becomes biologically active or more active after being placed in a given physiological environment.

[0049] The term "pharmacologically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., does not cause unacceptable levels of undesirable biological effects or interact in a deleterious manner.

[0050] As used herein, the term "sactipeptide" refers to a sulfur-alpha carbon thioether bridged peptide belonging to the ribosomally synthesized post-translationally modified peptide (RiPP) superfamily. As shown by the following structure, a sactipeptide contains an intramolecular thioether bond bridging the sulfur atom of a cysteine ​​residue to the α-carbon of an acceptor amino acid. [ka]

[0051] As used herein, the term "lanthipeptide" refers to a radical non-α-thioether-containing peptide that, like the sactipeptides described above, is also a member of the RiPP superfamily. For example, as shown below, a lanthipeptide can contain an intramolecular thioether bond bridging the sulfur atom of a cysteine ​​residue to any carbon other than the α-carbon of the acceptor amino acid. [ka]

[0052] Exemplary lantipeptide residues that contain a β- or γ-carbon are shown below. [ka]

[0053] As used herein, the term "derivative" refers to a compound having a structure derived from that of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to that disclosed herein that it would be expected by one of skill in the art to exhibit the same or similar activity and utility as the claimed compound or to induce the same or similar activity and utility as the compound claimed as a precursor, based on that similarity. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound.

[0054] As used herein, the term "pharmaceutical acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents such as aluminum monostearate and gelatin which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer employed. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0055] The term "substituted" as used herein is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. The permissible substituents can be one or more of the same or different suitable organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted with" also include the implicit proviso that such substitution is subject to the permissible valences of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. It is also contemplated that in certain embodiments, unless expressly indicated to the contrary, individual substituents may be further optionally substituted (ie, may be further substituted or unsubstituted).

[0056] In defining various terms, 1 ","A 2 ","A 3 " and "A 4 " is used herein as a generic symbol to represent various specific substituents. These symbols are not limited to those disclosed herein and may be any substituent, and if they are defined as a certain substituent in one instance, they may be defined as some other substituent in another instance.

[0057] The terms "aliphatic" or "aliphatic group," as used herein, refer to a hydrocarbon moiety that may be straight-chained (i.e., unbranched), branched-chained, or cyclic (including fused, bridged, and spiro-fused polycyclic), and may be fully saturated or may contain one or more units of unsaturation, but is not aromatic. Unless otherwise specified, an aliphatic group contains 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, and alkynyl groups, as well as hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0058] The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms. The term alkyl group can also be C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, etc., including up to C1-C24 alkyl.

[0059] Throughout this specification, "alkyl" is used generally to refer to both unsubstituted and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the particular substituent(s) on the alkyl group. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl group that is substituted with one or more halides, such as fluorine, chlorine, bromine, or iodine. Alternatively, the term "monohaloalkyl" specifically refers to an alkyl group that is substituted with a single halide, such as fluorine, chlorine, bromine, or iodine. The term "polyhaloalkyl" specifically refers to an alkyl group that is independently substituted with two or more halides, i.e., each halide substituent need not be the same halide as another halide substituent, nor do multiple instances of a halide substituent have to be on the same carbon. The term "alkoxyalkyl" specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term "aminoalkyl" specifically refers to an alkyl group that is substituted with one or more amino groups. The term "hydroxyalkyl" specifically refers to an alkyl group substituted with one or more hydroxy groups. Where "alkyl" is used in one instance and a specific term such as "hydroxyalkyl" is used in another instance, the term "alkyl" is not meant to imply that the specific term also includes "hydroxyalkyl."

[0060] This convention is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, but substituted moieties may additionally be specifically identified herein, e.g., a particular substituted cycloalkyl may be specifically referred to as, e.g., an "alkylcycloalkyl". Similarly, a substituted alkoxy may be specifically referred to as, e.g., a "halogenated alkoxy", a particular substituted alkenyl may be, e.g., an "alkenylalcohol", etc. Again, the convention of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" is not meant to imply that the general term does not include the specific term.

[0061] The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, etc. The term "heterocycloalkyl" is a cycloalkyl group of the type defined above, included within the meaning of the term "cycloalkyl", in which at least one of the carbon atoms of the ring is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0062] As used herein, the term "polyalkylene group" refers to a group having two or more CH groups linked together. A polyalkylene group has the formula -(CH) a -, where "a" is an integer from 2 to 500.

[0063] The terms "alkoxy" and "alkoxyl" are used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage, i.e., an "alkoxy" group is -OA 1 A 1 is alkyl or cycloalkyl as defined above. "Alkoxy" also includes polymeric alkoxy groups as described above, i.e., alkoxy includes -OA, -O, -O-C, -O-H ... 1 -OA 2 OR-OA 1 -(OA 2 ) a -OA 3 and the like, where "a" is an integer from 1 to 200, and A 1 , A 2 , and A 3 is an alkyl and / or cycloalkyl group.

[0064] The term "alkenyl" as used herein refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon double bond. 1 A 2 )C=C(A 3 A 4 Asymmetric structures such as aryl, aryl, aryl, aryls ...

[0065] The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bond, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term "heterocycloalkenyl" is a cycloalkenyl group of the type defined above and included within the meaning of the term "cycloalkenyl", in which at least one of the carbon atoms of the ring is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl and heterocycloalkenyl groups can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0066] The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0067] The term "cycloalkynyl" as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bond. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, etc. The term "heterocycloalkynyl" is a type of cycloalkenyl group defined above and included within the meaning of the term "cycloalkynyl", in which at least one of the carbon atoms of the ring is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkynyl and heterocycloalkynyl groups can be substituted or unsubstituted. The cycloalkynyl and heterocycloalkynyl groups may be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0068] The term "aromatic group" as used herein refers to a ring structure having a cyclic cloud of delocalized π-electrons above and below the plane of the molecule, the π-cloud containing (4n+2) π-electrons. Further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled "Aromaticity," pages 477-497, which is incorporated herein by reference. The term "aromatic group" includes both aryl and heteroaryl groups.

[0069] The term "aryl" as used herein is a group that includes any carbon-based aromatic group, including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, -NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of "aryl". In addition, aryl groups can include multiple ring structures that can be a single ring structure or that are either fused ring structures or are linked through one or more bridging groups, such as carbon-carbon bonds. For example, biaryl can be two aryl groups that are linked together through a fused ring structure, as in naphthalene, or are linked through one or more carbon-carbon bonds, as in biphenyl.

[0070] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout this specification, "C(O)" is a shorthand notation for a carbonyl group, i.e., C=O.

[0071] As used herein, the term "amine" or "amino" refers to a group of the formula -NA 1 A 2 In the formula, A 1 and A 2 may be independently hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A particular example of an amino is -NH2.

[0072] The term "alkylamino" as used herein is represented by the formula -NH(-alkyl), where alkyl is as described herein. Representative examples include, but are not limited to, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, (sec-butyl)amino, (tert-butyl)amino, pentylamino, isopentylamino, (tert-pentyl)amino, hexylamino, and the like.

[0073] The term "dialkylamino" as used herein is represented by the formula -N(-alkyl)2, where alkyl is as described herein. Representative examples include, but are not limited to, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, (sec-butyl)amino, di(tert-butyl)amino, dipentylamino, diisopentylamino, di(tert-pentyl)amino, dihexylamino, N-ethyl-N-methylamino, N-methyl-N-propylamino, N-ethyl-N-propylamino, and the like.

[0074] The term "carboxylic acid" as used herein is represented by the formula --C(O)OH.

[0075] As used herein, the term "ester" refers to an ester of the formula -OC(O)A 1 or -C(O)OA 1 In the formula, A 1 may be an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As used herein, the term "polyester" refers to a group of compounds of the formula -(A 1 O(O)CA 2 -C(O)O) a -or-(A 1 O(O)CA 2 -OC(O) a In the formula, A 1and A 2 can be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, and "a" is an integer from 1 to 500. "Polyester" is intended as a term used to describe a group produced by the reaction between a compound having at least two carboxylic acid groups and a compound having at least two hydroxyl groups.

[0076] As used herein, the term "ether" refers to a compound of formula A 1 Office Automation 2 In the formula, A 1 and A 2 may independently be an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As used herein, the term "polyether" refers to a group having the formula -(A 1 Office Automation 2 O) a In the formula, A 1 and A 2 may independently be an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, and "a" is an integer from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0077] As used herein, the terms “halo,” “halogen,” or “halide,” which may be used interchangeably, refer to F, Cl, Br, or I.

[0078] As used herein, the terms "pseudohalide," "pseudohalogen," or "pseudohalo" may be used interchangeably and refer to functional groups that behave substantially similarly to halides. Such functional groups include, by way of example, cyano, thiocyanato, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0079] The term "heteroalkyl" as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, with the nitrogen, phosphorus, and sulfur atoms being optionally oxidized, and the nitrogen heteroatom being optionally quaternized. Heteroalkyl can be substituted as defined above for alkyl groups.

[0080] The term "heteroaryl" as used herein refers to an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, with N-oxides, sulfur oxides, and dioxides being permissible heteroatom substitutions. Heteroaryl groups can be substituted or unsubstituted. Heteroaryl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. Heteroaryl groups can be monocyclic or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further non-limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0081] As used herein, the terms "heterocycle" or "heterocyclyl" may be used interchangeably and refer to monocyclic and polycyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, this term includes, but is not limited to, "heterocycloalkyl," "heteroaryl," "bicyclic heterocycle," and "polycyclic heterocycle." Examples of the heterocycle include pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazoles including 1,2,3-oxadiazole, 1,2,5-oxadiazole, and 1,3,4-oxadiazole, thiadiazoles including 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazoles including 1,2,3-triazole and 1,3,4-triazole, tetrazoles including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazines including 1,2,4-triazine and 1,3,5-triazine, tetrazines including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, and dioxane. The term heterocyclyl group can also be C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, etc., including up to C2-C18 heterocyclyl. For example, C2 heterocyclyl includes groups having two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, etc. Alternatively, for example, C5 heterocyclyl includes groups having five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, etc.It is understood that heterocyclyl groups may be attached, if chemically possible, either through a heteroatom within the ring or through one of the carbons comprising the heterocyclyl ring.

[0082] The term "bicyclic heterocycle" or "bicyclic heterocyclyl" as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl includes ring systems in which an aromatic ring is fused to another aromatic ring or an aromatic ring is fused to a non-aromatic ring. Bicyclic heterocyclyl includes ring systems in which a benzene ring is fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms, or a pyridine ring is fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[1,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; 1H-pyrrolo[3,2-b]pyridin-3-yl; and 1H-pyrazolo[3,2-b]pyridin-3-yl.

[0083] The term "heterocycloalkyl" as used herein refers to an aliphatic, partially unsaturated or fully saturated 3-14 membered ring system, including monocyclic rings of 3-8 atoms, as well as bicyclic and tricyclic ring systems. Heterocycloalkyl ring systems contain 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur, where the nitrogen and sulfur heteroatoms can be optionally oxidized and the nitrogen heteroatom can be optionally substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0084] The term "hydroxyl" or "hydroxyl" as used herein is represented by the formula --OH.

[0085] As used herein, the term "ketone" refers to a compound of formula A 1 C(O)A 2 In the formula, A 1 and A 2 can be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0086] The term "azide" or "azide" as used herein is represented by the formula --N3.

[0087] The term "nitro" as used herein is represented by the formula --NO.sub.2.

[0088] The term "nitrile" or "cyano" as used herein is represented by the formula --CN.

[0089] As used herein, the term "silyl" refers to a group of the formula -SiA 1 A 2 A 3 In the formula, A 1 , A 2 , and A 3 may be independently hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0090] As used herein, the term "sulfo-oxo" refers to a group of the formula -S(O)A 1 , -S(O)2A 1 , -OS(O)2A 1 , or -OS(O)2OA 1 In the formula, A 1may be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification, "S(O)" is a shorthand notation for S=O. The term "sulfonyl" is used herein to refer to a group of the formula -S(O)A 1 is used to refer to a sulfo-oxo group represented by the formula: 1 may be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As used herein, the term "sulfone" refers to a group of formula A 1 S(O)2A 2 In the formula, A 1 and A 2 may be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As used herein, the term "sulfoxide" refers to a group of formula A 1 S(O)A 2 In the formula, A 1 and A 2 can be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0091] The term "thiol" as used herein is represented by the formula --SH.

[0092] "R 1 ", "R 2 ", "R 3 ", "R n ", where n is an integer, and as used herein, can independently have one or more of the groups listed above. For example, R 1When is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can be optionally replaced with a hydroxyl group, an alkoxy group, an alkyl group, a halide, etc. Depending on the group selected, the first group can be embedded within the second group, or alternatively, the first group can be pendant (i.e., attached) to the second group. For example, in the phrase "an alkyl group comprising an amino group," the amino group can be embedded within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) selected will determine whether the first group is embedded or attached to the second group.

[0093] As described herein, the compounds of the present invention may include "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally", means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. The combination of substituents envisioned by the present invention is preferably one that results in the formation of a stable or chemically feasible compound. In certain embodiments, it is also contemplated that individual substituents may be further optionally substituted (i.e., further substituted or not substituted), unless explicitly indicated to the contrary.

[0094] The term "stable" as used herein refers to compounds that are substantially unchanged when subjected to conditions that enable their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0095] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently: halogen; -(CH) 0~4 R°;-(CH2) 0~4OR°;-O(CH2) 0~4 R°, -O-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 CH(OR°)2;-(CH2) 0~4 SR°; may be substituted with R°, -(CH2) 0~4 Ph; may be substituted with R°, -(CH2) 0~4 O(CH2) 0~1 Ph; may be substituted with R°, -CH=CHPh; may be substituted with R°, -(CH2) 0~4 O(CH2) 0~1 -Pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0~4 N(R°)2;-(CH2) 0~4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0~4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0~4 N(R°)C(O)OR°;-N(R°)N(R°)C(O)R°;-N(R°)N(R°)C(O)NR°2;-N(R°)N(R°)C(O)OR°;-(CH2) 0~4 C(O)R°;-C(S)R°;-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 C(O)SR°;-(CH2) 0~4 C(O)OSiR°3;-(CH2) 0~4 OC(O)R°;-OC(O)(CH2) 0~4 SR-;SC(S)SR°;-(CH2) 0~4 SC(O)R°;-(CH2) 0~4 C(O)NR°2;-C(S)NR°2;-C(S)SR°;-(CH2) 0~4 OC(O)NR°2;-C(O)N(OR°)R°;-C(O)C(O)R°;-C(O)CH2C(O)R°;-C(NOR°)R°;-(CH2) 0~4 SSR°;-(CH2) 0~4 S(O)2R°;-(CH2) 0~4 S(O)2OR°;-(CH2) 0~4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0~4S(O)R°;-N(R°)S(O)2NR°2;-N(R°)S(O)2R°;-N(OR°)R°;-C(NH)NR°2;-P(O)2R°;-P(O)R°2;-OP(O)R°2;-OP(O)(OR°)2;SiR°3;-(C 1~4 Linear or branched alkylene)ON(R°)2; or -(C 1~4 linear or branched alkylene)C(O)ON(R°)2, where each R° may be substituted as defined below and is independently hydrogen, C 1~6 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2- (a 5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definitions, two independent occurrences of R° taken together with their intervening atom(s) form a 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be a substituent as defined below.

[0096] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with their intervening atoms) are independently halogen, —(CH2), 0~2 R ● , -(Halo R ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2;-O(HaloR ● ), -CN, -N3, -(CH2) 0~2 C(O)R ● , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR ● , -(CH2) 0~2 S.R. ● , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR● , -(CH2) 0~2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● , -(C 1~4 Linear or branched alkylene)C(O)OR ● , or -SSR ● where each R ● is unsubstituted or, if preceded by "halo", is substituted with only one or more halogens, and independently represents C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0097] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2~3 O- or -S(C(R * 2)) 2~3 S-, wherein R * Each independent occurrence of 1~6 Aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to adjacent substitutable carbons of the "optionally substituted" group include -O(CR * 2) 2~3 O-, wherein R * Each independent occurrence of 1~6It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0098] R * Suitable substituents on the aliphatic group include halogen, -R ● , -(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, wherein each R ● is unsubstituted or, if preceded by "halo", is substituted with only one or more halogens, and independently represents C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0099] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH2C(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † In the formula, each R † are independently hydrogen, C which may be substituted as defined below 1~6 aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, regardless of the above definitions, R† two independent occurrences of together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0100] R † Suitable substituents on the aliphatic groups are independently halogen, -R ● , -(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", is substituted with only one or more halogens, and independently represents C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0101] The term "leaving group" refers to an atom (or group of atoms) having electron-withdrawing capability that can be displaced as a stable species with the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflates, mesylates, tosylates, and brosylates.

[0102] The terms "hydrolyzable group" and "hydrolyzable moiety" refer to functional groups that can undergo hydrolysis, for example, under basic or acidic conditions. Examples of hydrolyzable residues include, but are not limited to, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, "Protective Groups in Organic Synthesis," TW Greene, PGM Huts, Wiley-Interscience, 1999).

[0103] The term "organic residue" defines a carbon-containing residue, i.e., a residue that contains at least one carbon atom, including, but not limited to, carbon-containing groups, residues, or radicals as defined above. The organic residue may contain various heteroatoms or may be bonded to another molecule via heteroatoms including oxygen, nitrogen, sulfur, phosphorus, and the like. Examples of organic residues include, but are not limited to, alkyl or substituted alkyl, alkoxy or substituted alkoxy, mono- or di-substituted amino, amido groups, and the like. The organic residue may preferably contain 1-18 carbon atoms, 1-15 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In further embodiments, the organic residue may contain 2-18 carbon atoms, 2-15 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-4 carbon atoms, or 2-4 carbon atoms.

[0104] A close synonym of the term "residue" is the term "radical," which, as used herein and in the concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, the 2,4-thiazolidinedione radical in certain compounds has the structure: [ka] , Regardless of which thiazolidinedione is used to prepare the compound. In some embodiments, the radical (e.g., alkyl) can be further modified by having one or more "substituent radicals" attached thereto (i.e., substituted alkyl). The number of atoms in a given radical is not critical to the invention, unless otherwise indicated to the contrary elsewhere in this specification.

[0105] An "organic radical," as that term is defined and used herein, includes one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In further aspects, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bonded to at least some of the carbon atoms of the organic radical. An example of an organic radical that does not include inorganic atoms is the 5,6,7,8-tetrahydro-2-naphthyl radical. In some embodiments, an organic radical can include 1-10 inorganic heteroatoms bonded thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include, but are not limited to, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, monosubstituted amino, disubstituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamido, substituted alkylcarboxamido, dialkylcarboxamido, substituted dialkylcarboxamido, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, as these terms are defined elsewhere herein. Some non-limiting examples of organic radicals that contain heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals, and the like.

[0106] The compounds described herein contain one or more double bonds and may thus potentially occur as cis / trans (E / Z) isomers, as well as other conformational isomers, and unless stated to the contrary, the present invention includes all such possible isomers, as well as mixtures of such isomers.

[0107] Unless stated to the contrary, formulas with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplate each possible isomer, e.g., each enantiomer and diastereomer, as well as mixtures of isomers, e.g., racemic or scalemic mixtures. The compounds described herein may contain one or more asymmetric centers, thus potentially giving rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers, as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and their pharma- ceutically acceptable salts. Mixtures of stereoisomers, and isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or when using racemization or epimerization procedures known to those skilled in the art, the products of such procedures may be mixtures of stereoisomers.

[0108] Many organic compounds exist in optically active forms that have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be indicated with an asterisk (*). When a bond to a chiral carbon is shown as a straight line in a disclosed formula, it is understood that both the (R) and (S) configurations of the chiral carbon, and thus both enantiomers and mixtures thereof, are encompassed within the formula. As used in the art, when it is desired to specify the absolute configuration for a chiral carbon, one of the bonds to the chiral carbon can be shown as a wedge (a bond to an atom above the plane) and the other can be shown as a row of short parallel lines or a wedge (a bond to an atom below the plane). The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0109] When the disclosed compounds contain one chiral center, the compounds exist in two enantiomeric forms.Unless specifically stated to the contrary, the disclosed compounds include mixtures of enantiomers, such as both enantiomers and a particular 50:50 mixture referred to as a racemic mixture.Enantiomers can be resolved by methods known to those skilled in the art, such as, for example, the formation of diastereomeric salts that can be separated by crystallization (see CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)), the formation of diastereomeric derivatives or complexes that can be separated by, for example, crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiospecific reagent, for example, enzymatic esterification, or, for example, a chiral support, such as silica with a bound chiral ligand, or gas-liquid or liquid chromatography in a chiral environment in the presence of a chiral solvent. It will be appreciated that if the desired enantiomer is converted to another chemical entity by one of the separation procedures described above, a further step can be performed to liberate the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by converting one stereoisomer into the other by asymmetric transformation.

[0110] It is understood that the designation of a particular absolute configuration at a chiral carbon in a disclosed compound means that the designated enantiomeric form of the compound can be obtained in enantiomeric excess (ee). Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%, for example, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. In one embodiment, the designated enantiomer is substantially free of the other enantiomer. For example, the "R" form of the compound can be substantially free of the "S" form of the compound and is thus in enantiomeric excess of the "S" form. Conversely, the "S" form of the compound can be substantially free of the "R" form of the compound and is thus in enantiomeric excess of the "R" form.

[0111] When a disclosed compound has two or more chiral carbons, it may have more than two optical isomers and may exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound may have up to four optical isomers, as well as two pairs of enantiomers ((S,S) / (R,R) and (R,S) / (S,R)). Enantiomer pairs (e.g., (S,S) / (R,R)) are mirror image stereoisomers of each other. Stereoisomers that are not mirror images (e.g., (S,S) and (R,S)) are diastereomers. Diastereoisomeric pairs can be separated by methods known to those skilled in the art, such as, for example, chromatography or crystallization, and individual enantiomers within each pair can be separated as described above. Unless specifically excluded otherwise, the disclosed compounds include each diastereoisomer of such compounds and mixtures thereof.

[0112] The compounds according to the present disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc. as prodrug-forming moieties. For example, hydroxymethyl positions may form monophosphates, diphosphates, or triphosphates, and these phosphates may form prodrugs as well. The preparation of such prodrug derivatives is discussed in various literature sources (e.g., Alexander et al., J.Med.Chem.1988,31,318; Aligas-Martin et al., PCT WO2000 / 041531, p.30). The nitrogen function that is converted in preparing these derivatives is one (or more) of the nitrogen atoms of the compounds of the present disclosure.

[0113] The "derivatives" of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radioactively labeled forms, isomers, solvates, and combinations thereof. In this context, the term "combination" refers to derivatives that fall into at least two of the following groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radioactively labeled forms, isomers, and solvates. Examples of radioactively labeled forms include compounds labeled with tritium, phosphorus-32, iodine-129, carbon-11, fluorine-18, and the like.

[0114] The compounds described herein can contain atoms in both their natural isotopic abundance and non-natural abundance.The disclosed compounds can be isotopically labeled or isotopically substituted compounds identical to those described, but due to the fact that one or more atoms are replaced by atoms with atomic mass or mass number different from the atomic mass or mass number typically found in nature.Exemplary isotopes that can be incorporated into the compounds of the present invention include, respectively: 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, and36 Included within the scope of the invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as Cl. The compounds further include prodrugs thereof, and pharma- ceutically acceptable salts of the compounds or of the prodrugs that contain the isotopes and / or other isotopes of other atoms. Certain isotopically labeled compounds of the invention, such as 3 H and 14 Those in which a radioactive isotope such as C is incorporated are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e. 14 C isotopes are particularly preferred for their ease of preparation and detectability. Additionally, deuterium, i.e., 2 Substitution with heavier isotopes such as H can confer certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. The isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the following procedure by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0115] The compounds described in the present invention may exist as solvates.In some cases, the solvent used to prepare solvates is an aqueous solution, and solvates are often called hydrates.The compounds can exist as hydrates, which can be obtained, for example, by crystallization from a solvent or from an aqueous solution.In this regard, one, two, three, or any number of solvents or water molecules can be combined with the compounds according to the present invention to form solvates and hydrates.Unless otherwise stated, the present invention includes all such possible solvates.

[0116] The term "co-crystal" refers to a physical association of two or more molecules that are responsible for their stability through non-covalent interactions. One or more components of this molecular complex provide a stable framework in the crystal lattice. In certain instances, the guest molecule is incorporated into the crystal lattice as an anhydride or solvate, see, for example, "Crystal Engineering of the Composition Pharmaceutical Phase. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?" Almarasson, O., et.al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.

[0117] It is also understood that certain compounds described herein may exist in equilibrium between tautomers, for example, a ketone having an α-hydrogen may exist in equilibrium between the keto and enol forms. [ka]

[0118] Similarly, amides with an N-hydrogen can exist in equilibrium between the amide and imidic acid forms. As another example, pyrazole can exist in two tautomeric forms, N 1 -unsubstituted, 3-A 3 and N 1 -unsubstituted, 5-A 3 It can exist in. [ka] Unless stated to the contrary, the present invention includes all such possible tautomers.

[0119] Chemical substances are known to form solids that exist in different ordered states, called polymorphic forms or modifications. Different modifications of polymorphic substances can vary greatly in their physical properties. Compounds according to the present invention can exist in different polymorphic forms, and it is possible that certain modifications are metastable. Unless otherwise stated, the present invention includes all such possible polymorphic forms.

[0120] In some embodiments, the structure of the compound may be represented by the formula: [ka] , is understood to be equivalent to the formula: [ka] , In the formula, n is typically an integer. That is, R n is a group consisting of five independent substituents R n(a) , R n(b) , R n(c) , R n(d) , R n(e) By "independent substituents" it is meant that each R substituent can be defined independently. For example, in one example, R n(a) is a halogen, then R n(b) does not necessarily have to be a halogen.

[0121] Certain of the materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, starting materials and reagents used in preparing the disclosed compounds and compositions may be obtained from commercial suppliers, such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, NJ), Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.), or may be generally described in such publications as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers, 1997). They can be prepared according to procedures described in references such as (E. G., Inc., 1989) and by methods known to those skilled in the art.

[0122] Unless otherwise expressly stated, any method set forth herein is never intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed or the claims or specification specifically state that the steps are to be limited to a particular order, no order is ever intended to be inferred in any respect. This holds true for any possible implicit basis for interpretation, including the obvious meaning derived from the arrangement of steps or operational flow, grammatical construction or punctuation, and logical matters regarding the number or type of embodiments described in the specification.

[0123] Disclosed are the components used to prepare the compositions of the invention, as well as the compositions themselves used within the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific references to the various individual and collective combinations and permutations of each of these compounds cannot be explicitly disclosed, but each is specifically contemplated and described herein. For example, where a particular compound is disclosed and discussed, and a number of modifications that can be made to some molecules that include the compound are discussed, any and all combinations and permutations of the compounds and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C, as well as a class of molecules D, E, and F, are disclosed, and one example of a combination molecule, A-D, is disclosed, each is considered to be disclosed individually and collectively, even if each is not individually described, as are combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups A through E, B through F, and C through E are considered disclosed. This concept applies to all aspects of this application including, without limitation, steps in methods of making and using the compositions of the invention. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the methods of the invention.

[0124] It is understood that the compounds and compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it will be understood that there are various structures that can perform the same functions associated with the disclosed structures, and that these structures will generally achieve the same results.

[0125] B. Compound In one aspect, disclosed is a compound having a structure represented by the formula: [ka] , In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, t is an integer from 0 to 500, v is 1, 2, 3, 4, or 5, A is S or Se, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl; R 8 is selected from hydrogen and methyl, with the proviso that the compound is not PapA.

[0126] Also disclosed is a compound having a structure represented by the formula: [ka] , In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, t is an integer of 0 to 500, v is 1, 2, 3, 4, or 5, A is S or Se, and Q 1 is the leader sequence, and Q2 is the cleavable moiety, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl; R 8 is selected from hydrogen and methyl, with the proviso that the compound is not PapA.

[0127] Also disclosed is a compound having a structure represented by the formula: [ka] , wherein m is 0, 1, 2, 3, or 4, n is 0 or 1, each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, A is S or Se, and L, if present, is C2-C4 alkyl, -(C1-C4 alkyl)(OCH2CH2) q ,and [ka] wherein q is 1, 2, 3, or 4; and R1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 2 is a residue of the side chain of an amino acid, with the proviso that the amino acid is not isoleucine or threonine; R 3a and R 3b each, when present, is independently selected from C2-C5 alkynyl, C1-C5 azide, and a residue of the side chain of an amino acid; R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ covalently bonded to and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl, with the proviso that the compound is not PapA.

[0128] Also disclosed is a compound having a structure represented by the formula: [ka] , wherein m is 0, 1, 2, 3, or 4, n is 0 or 1, each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, A is S or Se, and L, if present, is C2-C4 alkyl, -(C1-C4 alkyl)(OCH2CH2) q ,and [ka] where q is 1, 2, 3, or 4; and Q 1 is the leader sequence, and Q 2 is the cleavable moiety, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 2 is a residue of the side chain of an amino acid, with the proviso that the amino acid is not isoleucine or threonine; R 3a and R 3b each, when present, is independently selected from C2-C5 alkynyl, C1-C5 azide, and a residue of the side chain of an amino acid; R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ covalently bonded to and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl, with the proviso that the compound is not PapA.

[0129] In various embodiments, o is independently 0, 1, 2, 3, 4, 5, 6, or 7.

[0130] In various embodiments, t is 0.

[0131] In various embodiments, v is 1 or 2.

[0132] In various embodiments, R 1 is -CO2H or the following structure: [ka]

[0133] In various embodiments, R 1 is -COH.

[0134] In various embodiments, the cleavable moiety is -CO2-(C4-C8 alkylene)-OC(O)-. In further embodiments, the cleavable moiety is -CO2CH2CH=CHCH2OC(O)-.

[0135] In various embodiments, the cleavable portion is a protease recognition sequence. In further embodiments, the protease recognition sequence is a TEV recognition sequence.

[0136] In various embodiments, the compounds include one or more D-amino acid residues. In further embodiments, the compounds include one or more β-amino acid residues. In yet further embodiments, the compounds include one or more N-methylated amino acid residues.

[0137] In various embodiments, PapB introduces a single thioether bond into the compound. In further embodiments, PapB introduces two or more thioether bonds into the compound.

[0138] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0139] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0140] In various embodiments, m is 0. In further embodiments, m is 1.

[0141] In various embodiments, n is 0. In further embodiments, n is 1.

[0142] In various embodiments, o is 0, 1, 2, 3, 4, 5, 6, or 7. In further embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, or 9. In yet further embodiments, o is 1, 2, 3, or 4.

[0143] In various embodiments, p is 1. In further embodiments, p is 2.

[0144] In various embodiments, A is S. In further embodiments, A is Se.

[0145] In various embodiments, L is C2-C4 alkyl. In further embodiments, L is -(C1-C4 alkyl)(OCH2CH2). q In still further embodiments, L is [ka] The structure is selected from:

[0146] In various embodiments, the cleavable portion is a protease recognition sequence. In further embodiments, the protease recognition sequence is a TEV protease recognition sequence. In yet further embodiments, the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), where X is any amino acid and Z is any amino acid that contains a hydrophobic residue. In another further embodiment, the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).

[0147] In various embodiments, the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3). In further embodiments, the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2).

[0148] In various embodiments, R 1 is selected from -CO2H and the following structures: [ka]

[0149] In various embodiments, R 1 is -COH.

[0150] In various embodiments, R 2 is a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan. 2 is a side chain residue of an amino acid selected from alanine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, and glycine.

[0151] In various embodiments, R 3a and R 3b One of, if present, is hydrogen and R 3a and R 3b When present, one of is a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.

[0152] In various embodiments, R 4 is hydrogen. In a further embodiment, R 4 is methyl.

[0153] In various embodiments, R5 Each occurrence of, if present, is independently a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.

[0154] In various embodiments, R 6 Each occurrence of, when present, is hydrogen. In a further embodiment, R 6 Each occurrence, when present, is methyl.

[0155] In various embodiments, R 7a and R 7b Each, when present, is hydrogen. In a further embodiment, R 7a and R 7b Each of, when present, is methyl.

[0156] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0157] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0158] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0159] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0160] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0161] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0162] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0163] In various embodiments, the compound has a structure represented by the following formula: [ka] In further embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0164] In various embodiments, R 3a and R 3b One of, if present, is hydrogen and R 3a and R 3b When present, one of is a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.

[0165] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0166] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0167] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0168] In various embodiments, the compound has a structure represented by the following formula: [ka] In further embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0169] In various embodiments, the compound has a structure represented by the formula: [ka] , In the formula, r is 2, 3, or 4.

[0170] In various embodiments, the compound has a structure represented by the formula: [ka] , In the formula, s is 1 or 2.

[0171] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0172] C. Thioether Compounds In one aspect, a thioether compound produced by the disclosed method is disclosed. Thus, in various aspects, the method produces a thioether compound having a structure represented by the following formula: [ka] , In the formula, v′ is 0, 1, 2, or 3.

[0173] In various embodiments, the method further comprises the addition of a reducing agent, hi further embodiments, the method further comprises the addition of a protease.

[0174] In various embodiments, the method produces a thioether compound having a structure represented by the formula: [ka] , In the formula, v′ is 0, 1, 2, or 3.

[0175] In various embodiments, the thioether compound is selected from the following: [ka]

[0176] In various embodiments, the method produces a thioether compound having a structure represented by the following formula: [ka]

[0177] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0178] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0179] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0180] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0181] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0182] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0183] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0184] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0185] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0186] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka]

[0187] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka] JPEG2025512475000068.jpg145170

[0188] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka] JPEG2025512475000070.jpg145170

[0189] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka]

[0190] In various embodiments, the thioether compound is a sactipeptide. In further embodiments, the sactipeptide has a structure represented by a formula selected from the following: [ka]

[0191] In various embodiments, the thioether compound is a lanthipeptide. In further embodiments, the lanthipeptide has a structure represented by a formula selected from the following: [ka]

[0192] In various embodiments, the method produces a thioether compound having a structure represented by the following formula: [ka]

[0193] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0194] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0195] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0196] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0197] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0198] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0199] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0200] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0201] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0202] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka]

[0203] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka] JPEG2025512475000086.jpg146170

[0204] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka] JPEG2025512475000088.jpg145170

[0205] In various embodiments, the thioether compound has a structure represented by a formula selected from the following: [ka]

[0206] In various embodiments, the thioether compound is a sactipeptide. In further embodiments, the sactipeptide has a structure represented by a formula selected from the following: [ka]

[0207] In various embodiments, the thioether compound is a lanthipeptide. In further embodiments, the lanthipeptide has a structure represented by a formula selected from the following: [ka]

[0208] In various embodiments, the thioether compound is selected from the following: [ka]

[0209] In various embodiments, the thioether compound is selected from the following: [ka]

[0210] D. Analogues of Peptide Therapeutics In one aspect, a thioether compound prepared by the disclosed method is disclosed, wherein the thioether compound is an analog of a peptide therapeutic. Exemplary peptide therapeutics include, but are not limited to, octreotide, cetomalanotide, romidepsin, bremelanotide, pramlintide, oxytocin, cetomalanotide, or cyclosporine.

[0211] Thus, in one aspect, [ka] or a pharma- ceutically acceptable salt thereof.

[0212] Also, [ka] or a pharma- ceutically acceptable salt thereof.

[0213] E. Methods of Chemically Modifying Compounds In one aspect, a method for chemically modifying a compound to introduce a thioether bond is disclosed, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, t is an integer from 0 to 500, v is 1, 2, 3, 4, or 5, A is S or Se, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl; R 8 is selected from hydrogen and methyl, with the proviso that the compound is not PapA.

[0214] Also disclosed is a method for chemically modifying a compound to introduce a thioether bond, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, t is an integer of 0 to 500, v is 1, 2, 3, 4, or 5, A is S or Se, and Q 1 is the leader sequence, and Q 2 is the cleavable moiety, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 4is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl; R 8 is selected from hydrogen and methyl, with the proviso that the compound is not PapA.

[0215] Also disclosed is a method for chemically modifying a compound to introduce a thioether bond, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , wherein m is 0, 1, 2, 3, or 4, n is 0 or 1, each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, A is S or Se, and L, if present, is C2-C4 alkyl, -(C1-C4 alkyl)(OCH2CH2) q ,and [ka] wherein q is 1, 2, 3, or 4; and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 2 is a residue of the side chain of an amino acid, with the proviso that the amino acid is not isoleucine or threonine; R 3a and R 3b each, when present, is independently selected from C2-C5 alkynyl, C1-C5 azide, and a residue of the side chain of an amino acid; R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ covalently bonded to and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl, with the proviso that the compound is not PapA.

[0216] Also disclosed is a method for chemically modifying a compound to introduce a thioether bond, the method comprising reacting the compound with PapB, wherein the compound has a structure represented by the formula: [ka] , wherein m is 0, 1, 2, 3, or 4, n is 0 or 1, each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, p is 1 or 2, A is S or Se, and L, if present, is C2-C4 alkyl, -(C1-C4 alkyl)(OCH2CH2) q ,and [ka] where q is 1, 2, 3, or 4; and Q 1is the leader sequence, and Q 2 is the cleavable moiety, and R 1 -CO2H, -C(O)NHOH, -SO2NH2, -SO2NHC(O)CH3, -SO3H, -NHC(O)NHSO2CH3, -P(O)(OH)2, and [ka] and R 2 is a residue of the side chain of an amino acid, with the proviso that the amino acid is not isoleucine or threonine; R 3a and R 3b each, when present, is independently selected from C2-C5 alkynyl, C1-C5 azide, and a residue of the side chain of an amino acid; R 4 is selected from hydrogen and methyl; R 5 and R 5’ each occurrence, when present, is independently a residue of a side chain of an amino acid, R 6 and R 6’ each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ are respectively, R 5 Or R 5’ covalently bonded to and together with the intermediate atom comprises an unsubstituted 5-membered heterocyclic ring, R 7a and R 7b each, when present, is independently selected from hydrogen and C1-C4 alkyl, with the proviso that the compound is not PapA.

[0217] In various embodiments, o is independently 0, 1, 2, 3, 4, 5, 6, or 7.

[0218] In various embodiments, t is 0.

[0219] In various embodiments, v is 1 or 2.

[0220] In various embodiments, R 1 is -CO2H or the following structure: [ka]

[0221] In various embodiments, R 1 is -COH.

[0222] In various embodiments, the cleavable moiety is a chemically cleavable moiety. Exemplary chemically cleavable moieties include, but are not limited to, -CO2-(C4-C8 alkylene)-OC(O)-. In further embodiments, the chemically cleavable moiety is -CO2CH2CH=CHCH2OC(O)-.

[0223] In various embodiments, the cleavable moiety is an enzymatically cleavable moiety, such as, for example, a protease recognition sequence. In further embodiments, the protease recognition sequence is a TEV recognition sequence.

[0224] In various embodiments, the compounds include one or more D-amino acid residues. In further embodiments, the compounds include one or more β-amino acid residues. In yet further embodiments, the compounds include one or more N-methylated amino acid residues.

[0225] In various embodiments, PapB introduces a single thioether bond into the compound. In further embodiments, PapB introduces two or more thioether bonds into the compound.

[0226] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0227] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0228] In various embodiments, the method produces a thioether compound having a structure represented by the formula: [ka] , In the formula, v′ is 0, 1, 2, or 3.

[0229] In various embodiments, the method further comprises the addition of a reducing agent, hi further embodiments, the method further comprises the addition of a protease.

[0230] In various embodiments, the method produces a thioether compound having a structure represented by the formula: [ka] , In the formula, v′ is 0, 1, 2, or 3.

[0231] In various embodiments, the thioether compound is [ka] is selected from.

[0232] In various embodiments, m is 0. In further embodiments, m is 1.

[0233] In various embodiments, n is 0. In further embodiments, n is 1.

[0234] In various embodiments, o is 0, 1, 2, 3, 4, 5, 6, or 7. In further embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, or 9. In yet further embodiments, o is 1, 2, 3, or 4.

[0235] In various embodiments, p is 1. In further embodiments, p is 2.

[0236] In various embodiments, A is S. In further embodiments, A is Se.

[0237] In various embodiments, L is C2-C4 alkyl. In further embodiments, L is -(C1-C4 alkyl)(OCH2CH2). q In still further embodiments, L is [ka] is selected from.

[0238] In various embodiments, the cleavable portion is a protease recognition sequence. In further embodiments, the protease recognition sequence is a TEV protease recognition sequence. In yet further embodiments, the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), where X is any amino acid and Z is any amino acid that contains a hydrophobic residue. In another further embodiment, the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).

[0239] In various embodiments, the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3). In further embodiments, the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2).

[0240] In various embodiments, R 1 is selected from -CO2H and the following structures: [ka]

[0241] In various embodiments, R 1 is -COH.

[0242] In various embodiments, R 2is a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan. 2 is a side chain residue of an amino acid selected from alanine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, and glycine.

[0243] In various embodiments, R 3a and R 3b One of, if present, is hydrogen and R 3a and R 3b When present, one of is a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.

[0244] In various embodiments, R 4 is hydrogen. In a further embodiment, R 4 is methyl.

[0245] In various embodiments, R 5 Each occurrence of, if present, is independently a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.

[0246] In various embodiments, R 6 Each occurrence of, when present, is hydrogen. In a further embodiment, R 6 Each occurrence, when present, is methyl.

[0247] In various embodiments, R 7a and R 7b Each, when present, is hydrogen. In a further embodiment, R 7a and R 7b Each of, when present, is methyl.

[0248] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0249] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0250] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0251] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0252] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0253] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0254] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0255] In various embodiments, the compound has a structure represented by the following formula: [ka] In further embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0256] In various embodiments, R 3a and R 3b One of, if present, is hydrogen and R 3a and R 3b When present, one of is a residue of the side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.

[0257] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0258] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0259] In various embodiments, the compound has a structure represented by the following formula: [ka]

[0260] In various embodiments, the compound has a structure represented by the following formula: [ka] In further embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0261] In various embodiments, the compound has a structure represented by the formula: [ka] , In the formula, r is 2, 3, or 4.

[0262] In various embodiments, the compound has a structure represented by the formula: [ka] , In the formula, s is 1 or 2.

[0263] In various embodiments, the compound has the following formula: [ka] The compound has a structure represented by:

[0264] In various embodiments, PapB introduces a single thioether bond into the compound. In further embodiments, PapB introduces two or more thioether bonds into the compound.

[0265] In various embodiments, the method produces a thioether compound having a structure represented by the following formula: [ka]

[0266] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0267] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0268] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0269] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0270] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0271] In various embodiments, the thioether compound has the following formula: [ka] The compound has a structure represented by:

[0272] In various embodiments, the thioether compound has the following formula: [ka] The compound has a structure represented by:

[0273] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0274] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0275] In various embodiments, the thioether compound is [ka] The compound has a structure represented by a formula selected from:

[0276] In various embodiments, the thioether compound is [ka] JPEG2025512475000141.jpg145170.

[0277] In various embodiments, the thioether compound is [ka] JPEG2025512475000143.jpg145170.

[0278] In various embodiments, the thioether compound is [ka] The compound has a structure represented by a formula selected from:

[0279] In various embodiments, the thioether compound is a sactipeptide. In further embodiments, the sactipeptide is: [ka] The compound has a structure represented by a formula selected from:

[0280] In various embodiments, the thioether compound is a lanthipeptide. In further embodiments, the lanthipeptide is [ka] The compound has a structure represented by a formula selected from:

[0281] In various embodiments, the method further comprises the addition of a reducing agent, hi further embodiments, the reducing agent comprises dithionite, flavodoxin, flavodoxin reductase, titanium citrate, reduced nicotinamide adenine dinucleotide phosphate, or any combination thereof.

[0282] In various embodiments, the method further comprises the addition of a protease. In further embodiments, the protease is a TEV protease.

[0283] In various embodiments, the method produces a thioether compound having a structure represented by the following formula: [ka]

[0284] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0285] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0286] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0287] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0288] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0289] In various embodiments, the thioether compound has the following formula: [ka] The compound has a structure represented by:

[0290] In various embodiments, the thioether compound has the following formula: [ka] The compound has a structure represented by:

[0291] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0292] In various embodiments, the thioether compound has a structure represented by the following formula: [ka]

[0293] In various embodiments, the thioether compound is [ka] The compound has a structure represented by a formula selected from:

[0294] In various embodiments, the thioether compound is [ka] JPEG2025512475000159.jpg145143 The compound has a structure represented by a formula selected from:

[0295] In various embodiments, the thioether compound is [ka] JPEG2025512475000161.jpg145170.

[0296] In various embodiments, the thioether compound is [ka] The compound has a structure represented by a formula selected from:

[0297] In various embodiments, the thioether compound is a sactipeptide. In further embodiments, the sactipeptide is: [ka] The compound has a structure represented by a formula selected from:

[0298] In various embodiments, the thioether compound is a lanthipeptide. In further embodiments, the lanthipeptide is [ka] The compound has a structure represented by a formula selected from:

[0299] In various embodiments, the thioether compound is [ka] is selected from.

[0300] In various embodiments, the thioether compound is [ka] is selected from.

[0301] F. Peptides 1. Peptide Substrate In one aspect, the present invention relates to chemically modifying a peptide sequence, the peptide sequence being XY n -Z, where X is penicillamine or an amino acid residue containing an -SH group or an amino acid residue containing an -SeH group, Y is a series of amino acid residues where n=0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, and Z is an aspartic acid residue, a glutamic acid residue, a hydroxy-glutamic acid residue, 2-amino-3-(2H-tetrazol-5-yl)propanoic acid, or a carboxyl-functionalized amino acid residue, and the peptide sequence is not PapA.

[0302] In a further embodiment, the peptide sequence is the sequence CY a -CDY b -D, where C is a cysteine ​​residue, D is an aspartic acid residue, and Y is a series of amino acid residues, a=1, 2, 3, 4, 5, 6, or 7, and b=0, 1, 2, 3, 4, 5, 6, or 7.

[0303] In a further embodiment, the peptide sequence is the sequence CY x -DY y -CY z-D, where C is a cysteine ​​residue, D is an aspartic acid residue, Y is a series of amino acid residues, x=0, 1, 2, 3, 4, 5, 6, or 7, y=1, 2, 3, 4, 5, 6, 7, or 8, and z=0, 1, 2, 3, 4, 5, 6, or 7.

[0304] In a further embodiment, the peptide sequence comprises octreotide or vapreotide. In another further embodiment, the peptide sequence comprises octreotide. In another further embodiment, the peptide sequence comprises vapreotide.

[0305] In a further embodiment, the peptide sequence is D FCF D WKTET (SEQ ID NO:3), where the first and fourth positions are D-amino acids.

[0306] In a further embodiment, the peptide sequence comprises FCFAKTETA.

[0307] In various embodiments, the peptide sequence further comprises a leader sequence of LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3). In further embodiments, the peptide sequence further comprises a leader sequence of LKQINVIAGVKEPIRAYG (SEQ ID NO: 3).

[0308] In various embodiments, the peptide sequence further comprises a TEV protease recognition sequence. In a further embodiment, the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), where X is any amino acid and Z is any amino acid that contains a hydrophobic residue. In another further embodiment, the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).

[0309] In further embodiments, the peptide sequence includes one or more D-amino acid residues.

[0310] In a further embodiment, the peptide sequence comprises one or more β-amino acid residues.

[0311] In a further embodiment, the peptide sequence comprises one or more N-methylated amino acids.

[0312] In one embodiment, the peptide sequence is a modified PapA sequence, the modified PapA sequence being Cys-Y n -Asp, and Y is a series of amino acid residues where n=0, 1, 2, 4, 5, 6, or 7.

[0313] In a further embodiment, the modified PapA sequence comprises a minimal substrate PapA.

[0314] In a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCDSNNAANA (SEQ ID NO:6), LKQINVIAGVKEPIRAYGCSDNNAAA (SEQ ID NO:7), LKQINVIAGVKEPIRAYGCSNDAAA (SEQ ID NO:8), LKQINVIAGVKEPIRAYGCSAANDA (SEQ ID NO:9), LKQINVIAGVKEPIRAYGCSAAANDA (SEQ ID NO:10), or LKQINVIAGVKEPIRAYGCSAAAANDA (SEQ ID NO:11). In a still further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCDSNNAANA (SEQ ID NO:6). In a still further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSDNNAAA (SEQ ID NO:7). In a still further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSNDAAA (SEQ ID NO:8). In yet a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSAANDA (SEQ ID NO: 9). In yet a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSAAANDA (SEQ ID NO: 10). In yet a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSAAAANDA (SEQ ID NO: 11).

[0315] In a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSANDA (SEQ ID NO: 12), LKQINVIAGVKEPIRAYGAAACSANDACSANDA (SEQ ID NO: 13), LKQINVIAGVKEPIRAYGAAACSACDAADA (SEQ ID NO: 14), LKQINVIAGVKEPIRAYGAAAASACDAADA (SEQ ID NO: 15), or LKQINVIAGVKEPIRAYGAAACSAADAAADA (SEQ ID NO: 16). In a still further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSANDA (SEQ ID NO: 12). In a still further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSANDACSANDA (SEQ ID NO: 13). In a still further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSACDAADA (SEQ ID NO: 14). In yet a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAAASACDAADA (SEQ ID NO: 15). In yet a further embodiment, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSAADAAADA (SEQ ID NO: 16).

[0316] In a further embodiment, the modified PapA sequence includes one or more D-amino acid residues.

[0317] In a further embodiment, the modified PapA sequence includes one or more β-amino acid residues.

[0318] In a further embodiment, the modified PapA sequence comprises one or more N-methylated amino acid residues.

[0319] aX group In various embodiments, X is penicillamine, or an amino acid residue containing an -SH group, or an amino acid residue containing an -SeH group.

[0320] In a further embodiment, X is penicillamine.

[0321] In a further embodiment, X is an amino acid residue containing an -SH group. In a still further embodiment, X is cysteine, homocysteine, D-cysteine, or D-homocysteine. In a still further embodiment, X is homocysteine. In a still further embodiment, X is D-cysteine. In a still further embodiment, X is D-homocysteine. In another further embodiment, X is cysteine.

[0322] In a further embodiment, X is an amino acid residue comprising a -SeH group. In yet a further embodiment, X is selenocysteine ​​or homoselenocysteine. In yet a further embodiment, X is selenocysteine. In another further embodiment, X is homoselenocysteine.

[0323] bY N base In various embodiments, Y n is a series of amino acid residues where n=0, 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0324] Examples of amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, β-amino acid residues, and N-methylated amino acid residues.

[0325] Natural amino acid residues can include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0326] Non-naturally occurring amino acid residues can include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine. Also, p-propargyloxyphenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), 3,4,6-trihydroxy-L-phenylalanine, 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-tyrosine, 3-thiol-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0327] In a further embodiment, Y n contains one or more D-amino acids.

[0328] In a further embodiment, Y n contains one or more β-amino acids.

[0329] In a further embodiment, Y ncontains one or more N-methylated amino acids.

[0330] In various embodiments, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. In further embodiments, n is 0, 1, 2, 3, 4, 5, 6, 7, or 8. In still further embodiments, n is 0, 1, 2, 3, 4, 5, 6, or 7. In still further embodiments, n is 0, 1, 2, 3, 4, 5, or 6. In still further embodiments, n is 0, 1, 2, 3, 4, or 5. In still further embodiments, n is 0, 1, 2, 3, or 4. In still further embodiments, n is 0, 1, 2, or 3. In still further embodiments, n is 0, 1, or 2. In another further embodiment, n is 0 or 1. In another further embodiment, n is 0. In another further embodiment, n is 1. In another further embodiment, n is 2. In another further embodiment, n is 3. In another further embodiment, n is 4. In another further embodiment, n is 5. In another further embodiment, n is 6. In another further embodiment, n is 7. In another further embodiment, n is 8. In another further embodiment, n is 9.

[0331] cZ group In various embodiments, Z is an aspartic acid residue, a glutamic acid residue, a hydroxy-glutamic acid residue, 2-amino-3-(2H-tetrazol-5-yl)propanoic acid, or a carboxyl-functionalized amino acid residue.

[0332] In a further embodiment, Z is aspartic acid or glutamic acid. In another further embodiment, Z is aspartic acid. In another further embodiment, Z is glutamic acid.

[0333] In a further embodiment, Z is a hydroxy-glutamic acid residue.

[0334] In a further embodiment, Z is 2-amino-3-(2H-tetrazol-5-yl)propanoic acid.

[0335] In further embodiments, Z is a carboxyl functionalized amino acid residue. Examples of carboxyl functionalized amino acid residues include, but are not limited to, (2S,3S)-2-amino-3-methylsuccinic acid, (2S,3R)-2-amino-3-methylsuccinic acid, (2S,3S)-2-amino-3-methylpentanedioic acid, (2S,3R)-2-amino-3-methylpentanedioic acid, (2S,4S)-2-amino-4-methylpentanedioic acid, (2S,4R)-2-amino-4-methylpentanedioic acid, and homoglutamic acid.

[0336] dY A base In a further embodiment, Y a is a series of amino acid residues where a=0, 1, 2, 3, 4, 5, 6, or 7.

[0337] Examples of amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, β-amino acid residues, and N-methylated amino acid residues.

[0338] Natural amino acid residues can include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0339] Non-naturally occurring amino acid residues can include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine. Also, p-propargyloxyphenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), 3,4,6-trihydroxy-L-phenylalanine, 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-tyrosine, 3-thiol-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0340] In a further embodiment, Y a contains one or more D-amino acids.

[0341] In a further embodiment, Y a contains one or more β-amino acids.

[0342] In a further embodiment, Y acontains one or more N-methylated amino acids.

[0343] In a further embodiment, a is 0, 1, 2, 3, 4, 5, 6, or 7. In a further embodiment, a is 0, 1, 2, 3, 4, 5, or 6. In a still further embodiment, a is 0, 1, 2, 3, 4, or 5. In a still further embodiment, a is 0, 1, 2, 3, or 4. In a still further embodiment, a is 0, 1, 2, or 3. In a still further embodiment, a is 0, 1, or 2. In another further embodiment, a is 0 or 1. In another further embodiment, a is 0. In another further embodiment, a is 1. In another further embodiment, a is 2. In another further embodiment, a is 3. In another further embodiment, a is 4. In another further embodiment, a is 5. In another further embodiment, a is 6. In another further embodiment, a is 7.

[0344] eY B base In a further embodiment, Y b is a series of amino acid residues where b=0, 1, 2, 3, 4, 5, 6, or 7.

[0345] Examples of amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, β-amino acid residues, and N-methylated amino acid residues.

[0346] Natural amino acid residues can include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0347] Non-naturally occurring amino acid residues can include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine. Also, p-propargyloxyphenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), 3,4,6-trihydroxy-L-phenylalanine, 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-tyrosine, 3-thiol-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0348] In a further embodiment, Y b contains one or more D-amino acids.

[0349] In a further embodiment, Y b contains one or more β-amino acids.

[0350] In a further embodiment, Y bcontains one or more N-methylated amino acids.

[0351] In a further embodiment, b is 0, 1, 2, 3, 4, 5, 6, or 7. In a further embodiment, b is 0, 1, 2, 3, 4, 5, or 6. In a still further embodiment, b is 0, 1, 2, 3, 4, or 5. In a still further embodiment, b is 0, 1, 2, 3, or 4. In a still further embodiment, b is 0, 1, 2, or 3. In a still further embodiment, b is 0, 1, or 2. In another further embodiment, b is 0 or 1. In another further embodiment, b is 0. In another further embodiment, b is 1. In another further embodiment, b is 2. In another further embodiment, b is 3. In another further embodiment, b is 4. In another further embodiment, b is 5. In another further embodiment, b is 6. In another further embodiment, b is 7.

[0352] fY X base In a further embodiment, Y x is a series of amino acid residues where x=0, 1, 2, 3, 4, 5, or 6.

[0353] Examples of amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, β-amino acid residues, and N-methylated amino acid residues.

[0354] Natural amino acid residues can include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0355] Non-naturally occurring amino acid residues can include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine. Also, p-propargyloxyphenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), 3,4,6-trihydroxy-L-phenylalanine, 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-tyrosine, 3-thiol-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0356] In a further embodiment, Y x contains one or more D-amino acids.

[0357] In a further embodiment, Y x contains one or more β-amino acids.

[0358] In a further embodiment, Y xcontains one or more N-methylated amino acids.

[0359] In further embodiments, x is 0, 1, 2, 3, 4, 5, or 6. In still further embodiments, x is 0, 1, 2, 3, 4, or 5. In still further embodiments, x is 0, 1, 2, 3, or 4. In still further embodiments, x is 0, 1, 2, or 3. In still further embodiments, x is 0, 1, or 2. In another further embodiment, x is 0 or 1. In another further embodiment, x is 0. In another further embodiment, x is 1. In another further embodiment, x is 2. In another further embodiment, x is 3. In another further embodiment, x is 4. In another further embodiment, x is 5. In another further embodiment, x is 6.

[0360] gY Y base In a further embodiment, Y y is a series of amino acid residues where y=0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0361] Examples of amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, β-amino acid residues, and N-methylated amino acid residues.

[0362] Natural amino acid residues can include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0363] Non-naturally occurring amino acid residues can include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine. Also, p-propargyloxyphenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), 3,4,6-trihydroxy-L-phenylalanine, 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-tyrosine, 3-thiol-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0364] In a further embodiment, Y y contains one or more D-amino acids.

[0365] In a further embodiment, Y y contains one or more β-amino acids.

[0366] In a further embodiment, Y ycontains one or more N-methylated amino acids.

[0367] In a further embodiment, y is 0, 1, 2, 3, 4, 5, 6, 7, or 8. In a still further embodiment, y is 0, 1, 2, 3, 4, 5, 6, or 7. In a still further embodiment, y is 0, 1, 2, 3, 4, 5, or 6. In a still further embodiment, y is 0, 1, 2, 3, 4, or 5. In a still further embodiment, y is 0, 1, 2, 3, or 4. In a still further embodiment, y is 0, 1, 2, or 3. In a still further embodiment, y is 0, 1, or 2. In another further embodiment, y is 0 or 1. In another further embodiment, y is 0. In another further embodiment, y is 1. In another further embodiment, y is 2. In another further embodiment, y is 3. In another further embodiment, y is 4. In another further embodiment, y is 5. In another further embodiment, y is 6. In another further embodiment, y is 7. In another further embodiment, y is 8.

[0368] hY Z base In a further embodiment, Y z is a series of amino acid residues, where z=0, 1, 2, 3, 4, 5, 6, or 7.

[0369] Examples of amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, β-amino acid residues, and N-methylated amino acid residues.

[0370] Natural amino acid residues can include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0371] Non-naturally occurring amino acid residues can include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine. Also, p-propargyloxyphenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), 3,4,6-trihydroxy-L-phenylalanine, 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-tyrosine, 3-thiol-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0372] In a further embodiment, Y z contains one or more D-amino acids.

[0373] In a further embodiment, Y z contains one or more β-amino acids.

[0374] In a further embodiment, Y zcontains one or more N-methylated amino acids.

[0375] In further embodiments, z is 0, 1, 2, 3, 4, 5, 6, or 7. In further embodiments, z is 0, 1, 2, 3, 4, 5, or 6. In still further embodiments, z is 0, 1, 2, 3, 4, or 5. In still further embodiments, z is 0, 1, 2, 3, or 4. In still further embodiments, z is 0, 1, 2, or 3. In still further embodiments, z is 0, 1, or 2. In another further embodiment, z is 0 or 1. In another further embodiment, z is 0. In another further embodiment, z is 1. In another further embodiment, z is 2. In another further embodiment, z is 3. In another further embodiment, z is 4. In another further embodiment, z is 5. In another further embodiment, z is 6. In another further embodiment, z is 7.

[0376] It is contemplated that each of the disclosed derivatives can be optionally further substituted. It is also contemplated that any one or more of the derivatives can be optionally omitted from the present invention. It is understood that the disclosed compounds can be provided by the disclosed methods.

[0377] 2. Exemplary Peptide Products In one aspect, the present invention provides a method for producing [ka] The present invention relates to a product compound having a structure selected from:

[0378] In a further embodiment, the compound is: [ka]

[0379] In a further embodiment, the compound is: [ka]

[0380] G. Methods for Chemically Modifying Peptide Sequences The compounds of the present invention can be prepared by using reactions as shown in the following schemes, in addition to other standard manipulations known in the literature, illustrated in the experimental section, or apparent to one skilled in the art. For clarity, examples with a single substituent are shown, although multiple substituents are possible under the definitions disclosed herein.

[0381] Preferred methods include, but are not limited to, those described below. During any of the following synthetic sequences, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved. This can be accomplished by conventional protecting groups, such as those described in TW Greene, Protective Groups in Organic Chemistry, John Wiley & Sons, 1981, and TW Greene and PGM Huts, Protective Groups in Organic Chemistry, John Wiley & Sons, 1991, which are incorporated herein by reference.

[0382] The reactions used to produce the compounds of the present invention are prepared as described and exemplified below by using reactions as shown in the following reaction schemes. The following examples are provided so that the invention may be more fully understood and are merely illustrative and should not be construed as limiting.

[0383] In one aspect, the present invention relates to a method for chemically modifying a peptide sequence to introduce a thioether bond, the method comprising reacting a peptide substrate with PapB.

[0384] In a further embodiment, the peptide sequence further comprises a leader sequence of LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3). The leader sequence facilitates recognition of the entire peptide sequence by PapB. However, the leader sequence is not required.

[0385] In a further embodiment, the method further comprises the addition of a protease. In a further embodiment, the peptide sequence comprises a protease recognition sequence. A protease in combination with a peptide sequence comprising a protease recognition sequence allows for cleavage of a desired product from a leader sequence. In a further embodiment, the protease is a TEV protease. A TEV protease in combination with a peptide sequence comprising a TEV protease recognition sequence allows for cleavage of a desired product from a leader sequence. In a further embodiment, the peptide sequence comprises a TEV protease recognition sequence. In yet a further embodiment, the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), where X is any amino acid and Z is any amino acid comprising a hydrophobic residue. In another further embodiment, the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).

[0386] In a further embodiment, the method further comprises the addition of a reducing agent. Examples of reducing agents include, but are not limited to, dithionite, flavodoxin, flavodoxin reductase, titanium citrate, reduced nicotinamide adenine dinucleotide phosphate, and Hantzsch ester. In still further embodiments, the reducing agent comprises dithionite, flavodoxin, flavodoxin reductase, titanium citrate, reduced nicotinamide adenine dinucleotide phosphate, or any combination thereof. In still further embodiments, the reducing agent comprises dithionite, flavodoxin, flavodoxin reductase, and titanium citrate. In still further embodiments, the reducing agent comprises dithionite, flavodoxin, and flavodoxin reductase. In still further embodiments, the reducing agent comprises dithionite and flavodoxin. In still further embodiments, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate, titanium citrate, flavodoxin reductase, and flavodoxin. In still further aspects, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate, titanium citrate, and flavodoxin reductase. In still further aspects, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate and titanium citrate. In still further aspects, the reducing agent comprises dithionite. In still further aspects, the reducing agent comprises flavodoxin. In still further aspects, the reducing agent comprises flavodoxin reductase. In still further aspects, the reducing agent comprises titanium citrate. In still further aspects, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate.

[0387] In further embodiments, PapB introduces two or more thioether bonds into the peptide sequence. For example, the peptide sequence may include the sequence CY a -CDY b-D, where C is a cysteine ​​residue, D is an aspartic acid residue, and Y is a series of amino acid residues, a=1, 2, 3, 4, 5, 6, or 7, and b=0, 1, 2, 3, 4, 5, 6, or 7. In this example, thioether bonds are introduced between the first cysteine ​​residue and the first aspartic acid residue, and between the second cysteine ​​residue and the second aspartic acid residue, resulting in nested crosslinks. By way of example, the peptide sequence may also include the sequence CY x -DY y -CY z -D, where C is a cysteine ​​residue, D is an aspartic acid residue, Y is a series of amino acid residues, x=0, 1, 2, 3, 4, 5, 6, or 7, y=1, 2, 3, 4, 5, 6, 7, or 8, and z=0, 1, 2, 3, 4, 5, 6, or 7. In this example, thioether bonds are introduced between the first cysteine ​​residue and the first aspartic acid residue, and between the second cysteine ​​residue and the second aspartic acid residue, resulting in a linear crosslink.

[0388] H. Methods for Chemically Modifying Modified PapA Sequences In one aspect, the invention relates to a method for chemically modifying a modified PapA sequence to introduce a thioether bond, the method comprising reacting the modified PapA sequence with PapB.

[0389] In a further embodiment, the modified PapA sequence comprises a minimal substrate PapA.

[0390] In a further embodiment, the modified PapA sequence further comprises a leader sequence of LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3). The leader sequence facilitates recognition of the entire peptide sequence by PapB. However, the leader sequence is not required.

[0391] In a further embodiment, the method further comprises the addition of a protease. In a further embodiment, the modified PapA sequence comprises a protease recognition sequence. A protease in combination with a modified PapA sequence comprising a protease recognition sequence allows for cleavage of a desired product from the leader sequence. In a further embodiment, the protease is a TEV protease. A TEV protease in combination with a modified PapA sequence comprising a TEV protease recognition sequence allows for cleavage of a desired product from the leader sequence. In a further embodiment, the modified PapA sequence comprises a TEV protease recognition sequence. In yet a further embodiment, the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), where X is any amino acid and Z is any amino acid comprising a hydrophobic residue. In another further embodiment, the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).

[0392] In a further embodiment, the method further comprises the addition of a reducing agent. Examples of reducing agents include, but are not limited to, dithionite, flavodoxin, flavodoxin reductase, titanium citrate, reduced nicotinamide adenine dinucleotide phosphate, and Hantzsch ester. In still further embodiments, the reducing agent comprises dithionite, flavodoxin, flavodoxin reductase, titanium citrate, reduced nicotinamide adenine dinucleotide phosphate, or any combination thereof. In still further embodiments, the reducing agent comprises dithionite, flavodoxin, flavodoxin reductase, and titanium citrate. In still further embodiments, the reducing agent comprises dithionite, flavodoxin, and flavodoxin reductase. In still further embodiments, the reducing agent comprises dithionite and flavodoxin. In still further embodiments, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate, titanium citrate, flavodoxin reductase, and flavodoxin. In still further aspects, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate, titanium citrate, and flavodoxin reductase. In still further aspects, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate and titanium citrate. In still further aspects, the reducing agent comprises dithionite. In still further aspects, the reducing agent comprises flavodoxin. In still further aspects, the reducing agent comprises flavodoxin reductase. In still further aspects, the reducing agent comprises titanium citrate. In still further aspects, the reducing agent comprises reduced nicotinamide adenine dinucleotide phosphate.

[0393] In further embodiments, PapB introduces two or more thioether bonds into the peptide sequence. For example, the peptide sequence may include the sequence CY a -CDY b-D, where C is a cysteine ​​residue, D is an aspartic acid residue, and Y is a series of amino acid residues, a=1, 2, 3, 4, 5, 6, or 7, and b=0, 1, 2, 3, 4, 5, 6, or 7. In this example, thioether bonds are introduced between the first cysteine ​​residue and the first aspartic acid residue, and between the second cysteine ​​residue and the second aspartic acid residue, resulting in nested crosslinks. By way of example, the peptide sequence may also include the sequence CY x -DY y -CY z -D, where C is a cysteine ​​residue, D is an aspartic acid residue, Y is a series of amino acid residues, x=0, 1, 2, 3, 4, 5, 6, or 7, y=1, 2, 3, 4, 5, 6, 7, or 8, and z=0, 1, 2, 3, 4, 5, 6, or 7. In this example, thioether bonds are introduced between the first cysteine ​​residue and the first aspartic acid residue, and between the second cysteine ​​residue and the second aspartic acid residue, resulting in a linear crosslink. EXAMPLES

[0394] I. Working Example The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, are intended to be purely exemplary of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is ° C. or is at ambient temperature, and pressure is at or near atmospheric pressure.

[0395] The examples are provided herein to illustrate the invention and are not to be construed as limiting the invention in any manner.The examples are provided herein to illustrate the invention and are not to be construed as limiting the invention in any manner.

[0396] 1. Method Cloning and expression of PapB Plasmids PapB and pPH151 were co-transformed into Escherichia coli BL21(DE3)T1 resistant cells (NEB C2527). Plasmid pPH151 contains the suf operon encoding the sufABCDE proteins that assist in the release of sulfur, function as an Fe-S scaffold, and donate the Fe-S cluster to the apo protein. The suf operon is frequently included with the radical SAM enzyme to aid in the assembly of iron-sulfur clusters in heterologously expressed proteins. The transformation mixture was suspended in SOC recovery medium and shaken at 200 rpm at 37°C for 1 hour. The mixture was plated on agar Lennox broth (LB) plates containing 34 μg / mL chloramphenicol and 34 μg / mL kanamycin and placed in an oven set at 37°C for 16 hours. An overnight culture (0.15 L) of LB containing 34 μg / mL chloramphenicol and 34 μg / mL kanamycin was inoculated with a single colony from the plate. Twelve aliquots (12 mL each) of the overnight culture were used to inoculate twelve 2.8 L Fernbach flasks each containing 1 L of LB supplemented with 34 μg / mL chloramphenicol and 34 μg / mL kanamycin. The culture was grown at 37° C. and 180 rpm to an OD600nm of approximately 0.35, at which point 0.1 mM iron(III) chloride (0.1 mM) and L-cysteine ​​hydrochloride monohydrate (0.1 mM) were added. At an OD600nm of approximately 0.5, the flask was immersed in an ice bath and cooled for 20 minutes before induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cultures were grown overnight (approximately 16 hours) and cells were harvested by centrifugation at 6500×g. Typical yield is approximately 45 g wet cell paste per 12 L of growth. Cell pellets were flash frozen in liquid N2 and stored at −80° C. until use.

[0397] B. Purification of PapB PapB was purified in a Coy Laboratories anaerobic chamber maintained at 98% N2 / 2% H2 atmosphere. Cell paste (15 g) was resuspended in a metal beaker containing 0.1 L of 0.05 M KPi (pH 7.4) buffer containing 0.5 M KCl, 0.05 M imidazole, 20% glycerol (v / v), 0.1 mg / mL lysozyme, 10 μg / mL DNAse, and two cOmplete EDTA-free protease inhibitor cocktail tablets (Fisher Scientific NC0939481). The suspension was stirred on ice for 30 min, after which the cells were lysed with a Branson digital sonicator operated at 50% amplitude for a total of 17 min (25 sec on / 35 sec off) while stirring on ice. The resulting liquid was centrifuged at 18,442 × g for 45 min at 4 °C. Three 5 mL HisTrap HP columns (GE healthcare) packed with nickel sulfate were connected in series and equilibrated with loading buffer containing 0.05 M KPi (pH 7.4), 0.5 M KCl, 20% glycerol (v / v), and 0.05 M imidazole. The clarified lysate was loaded onto the column at 3 mL / min. The column was washed with 8 column volumes (CV) of loading buffer, and PapB was eluted with a linear gradient spanning 8 CV to 0.5 M imidazole in loading buffer. Fractions containing PapB were identified by brown and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels.

[0398] The pooled fractions were further purified by an amylose resin (NEB E8022S) column equilibrated in loading buffer containing 0.05 M KPi (pH 7.4), 0.5 M KCl, and 0.05 M imidazole. The column was washed with 0.15 L of loading buffer and eluted with loading buffer containing 10 mM maltose. The resulting dark brown fractions were pooled and solid dithiothreitol (DTT) powder was added to the combined fractions to a final concentration of 2 mM. An aliquot (1 mL) of 90 μM TEV protease was added and the mixture was stirred at room temperature for 14 h. Cleaved MBP was removed from PapB through three consecutively connected 5 mL HisTraps equilibrated in loading buffer. The flow-through from this column contained cleaved PapB. The resulting PapB protein was desalted in a buffer containing 0.05 M PIPES·NaOH (pH 7.4), 0.3 M NaCl, 2 mM DTT, and 20% glycerol (v / v). The concentration of PapB was determined by the Bradford method using bovine serum albumin (BSA) as a standard. PapB was reconstituted by mixing 12 molar equivalents of 0.1 M FeCl3 hexahydrate and Na2S nonahydrate as follows: Aliquots (5 μL) of FeCl3 hexahydrate and Na2S were added individually with a 15 s interval between additions to ensure complete mixing. FeCl3 was added first until complete, followed by Na2S. The reconstituted mixture was stirred at room temperature for 4 h. The resulting solution was centrifuged at 16,000 x g for 10 min to remove any debris and desalted on BioGel P6 DG desalting gel 100-200 mesh (wet) (Bio-Rad) in a buffer containing 0.05 M PIPES·NaOH (pH 7.4), 0.3 M NaCl, 2 mM DTT, and 20% glycerol (v / v). Protein was concentrated to approximately 3 mL using an Amicon concentrator with a YM-10 membrane (Millipore) under N2.

[0399] Reconstituted PapB was further purified by a Cytiva XK26 (1000 mm) S-300 column equilibrated with a buffer containing 0.05 M PIPES·NaOH (pH 7.4), 0.3 M KCl, 2 mM DTT, and 10% glycerol (v / v). Protein was eluted isocratically at 2.7 mL / min, and fractions containing PapB were identified by visual inspection of dark brown and Coomassie stained SDS-PAGE gels. Pooled fractions were concentrated to approximately 0.5 mL. Aliquots were flash frozen in liquid N2 and stored at -80°C. PapB was quantified by Bradford assay using BSA as a standard. A typical yield from the purification outlined above is 16.5 mg of pure protein for 15 g of wet cell paste.

[0400] c. Amino acid analysis and iron concentration determination Correction factors for the Bradford assay were determined by direct amino acid analysis on three independent preparations of protein. Amino acid analysis was performed by the Molecular Structure Facility at the University of California-Davis as follows: A 0.1 mL aliquot of concentrated PapB was desalted in a solution containing 10 mM NaOH using an Illustra NICK column (GE Healthcare). Protein samples were hydrolyzed in a solution containing 6 M HCl and 1% phenol at 110° C. in vacuum and resuspended in a norleucine solution as an internal standard. PapB samples were analyzed by a Hitachi 8800 amino acid analyzer calibrated with amino acid standards for protein hydrolysates on a Na-based Hitachi 8800 (Sigma, A-9906). These standards were verified by the National Institute of Standards and Technology (NIST) Standard Reference 2389a. PapB samples were run through a brief ion exchange column (AminoSep Beckman Style Na+, part number AAA-99-6312) with a secondary ninhydrin reaction for detection using Pickering Na buffer. The correction factor for the Bradford assay was determined to be 0.60 based on the results of three independent purifications, and this factor was used in all subsequent protein concentration determinations to correct values ​​obtained from the less laborious Bradford determinations.

[0401] The iron content of reconstituted PapB was determined by inductively coupled plasma mass spectrometry (ICP-MS) on the same three separate enzyme preparations. This was performed at the Center for Water, Ecosystems and Climate Science, Department of Geology and Geophysics, University of Utah, as follows. PapB preparations were diluted to concentrations of 2–5 μM with 10% trace metal grade nitric acid before submission. Iron concentration was performed using a triple quadrupole inductively coupled plasma mass spectrometer (ICP-MS, Agilent 8900, Santa Clara, CA). 10 nM In / mL was added as an internal standard. An external calibration curve was prepared from 1000 mg / L single element standards (Inorganic Ventures, Christiansburg, VA). The Fe concentrations in the six calibration solutions were 0, 8.3, 20.7, 66.2, 165.5, and 331.1 ng Fe / mL, and all solutions contained 10 ng In / mL. Blanks, calibration solutions, and diluted samples were run by ICP-MS in a quartz platinum shielded torch using a double-pass quartz spray chamber, PTFE nebulizer, and dual syringe introduction system (Teledyne, AVX72000), platinum cone, and sapphire injector. In and Fe were detected at masses of 115 and 56 with a flow rate of 8 mL He / min in the collision cell. Certified reference manual CRM 1643f (National Institute of Standards and Technology, Gaithersburg, MD) was diluted 1:20 and run with the samples and calibration curves as a quality control for the calibration. Fe in CRM 1643f was measured to be 10% within the certified value.

[0402] d. TEV protease purification SG1200008 pRARE chemically competent cells were transformed with pNB512. The transformation was suspended in SOC recovery medium and shaken at 200 rpm for 45 minutes at 37°C. The mixture was plated on an agar Lennox broth (LB) plate containing 34 ug / mL chloramphenicol and 100 mg / mL ampicillin and placed in an oven set at 37°C for 16 hours. An overnight culture (0.15 L) of LB containing 34 mg / mL chloramphenicol and 34 mg / mL ampicillin was inoculated with a single colony from the plate. Twelve aliquots (0.010 L each) of the overnight culture were used to inoculate twelve 2.8 L Fernbach flasks each containing 1 L of LB supplemented with 34 μg / mL chloramphenicol and 100 μg / mL ampicillin. The culture was grown at 37°C and 175 rpm to an OD of approximately 0.49. 600nm The cultures were grown to 30° C. for 1 h at which point 1 mM IPTG was added to each flask and the temperature was reduced to 16° C. Cultures were grown overnight (approximately 16 hours) and cells were harvested by centrifugation at 6500×g. Cell pellets were flash frozen in liquid N2 and stored at −80° C. until use.

[0403] The cell paste (15 g) was resuspended in a metal beaker containing 0.1 L of 0.05 M KPi (pH 7.4) buffer containing 0.5 M KCl, 0.05 M imidazole, 100 mg / mL lysozyme, 10 mg / mL PMSF, and 20% (v / v) glycerol. The suspension was stirred for 2 h at 4 °C. The cells were lysed with a Branson digital sonicator operated at 50% amplitude for a total of 15 min (10 s on / 20 s off) with stirring on ice. The resulting liquid was centrifuged at 18,442 x g for 50 min at 4 °C. Two 5 mL HisTrap HP columns (GE healthcare) packed with nickel sulfate were connected in series and equilibrated with loading buffer containing 0.05 M KPi (pH 7.4), 0.5 M KCl, and 0.05 M imidazole. The clarified lysate was loaded onto the column at 3 mL / min. The column was washed with 8 column volumes (CV) of loading buffer and the TEV protease was eluted with a linear gradient spanning 8 CV to 0.5 M imidazole in loading buffer. Fractions containing TEV protease were identified by SDS-PAGE, pooled and dialyzed three times against 4 L of 0.05 M KPi (pH 7.4), 0.5 M KCl, 0.05 M imidazole and 20% glycerol. The dialyzed protein was concentrated to a minimum volume and subsequently glycerol was added to a final concentration of 50% glycerol (v / v). Aliquots were flash frozen in liquid nitrogen and stored at -80 °C until use.

[0404] e. Synthesis of minimal substrate PapA (MSPapA) and variants PapA peptides were synthesized on either a PS3 peptide synthesizer (Protein Technologies Inc.) or a Prelude peptide synthesizer (Protein Technologies Inc.). In comparison to the previously reported msPapA peptide (Van der Donk, WA; Bindman, NAnat. Prod.: Discourse, Delivery, and Design, John Wiley & Sons: Oxford, 2014; pp 197-218), the N-terminal methionine was removed in all peptide syntheses. Synthesis was performed on a 0.025 mmol scale using standard Fmoc procedures from the manufacturer. All natural Fmoc-amino acids were purchased from Protein Technologies Inc. N-alpha-Fmoc-S-trityl-D-cysteine ​​and Fmoc-D-aspartic acid α-tert-butyl ester were purchased from Chem Impex (04314). For the synthesis, 150 mg of 2-chlorotrityl chloride resin 100-200 mesh (ChemPep) was loaded with 9.3 mg of Fmoc-Ala-OH (approximately 0.2 mmol / g of resin). The resin was washed three times with 5 mL of DMF and three times with 5 mL of dichloromethane (DCM). 9.3 mg of Fmoc-Ala-OH was dissolved in 1 mL of 1:1 dichloromethane (DCM):N,N-dimethylformamide (DMF) containing 0.15 mmol of diisopropylethylamine (DIPEA). This solution was added to the resin and gently shaken for 1 h. The Fmoc-Ala / DIPEA solution was then removed and the resin was washed three times with 5 mL of DCM. Uncapped sites on the resin were capped by washing the resin with 20 mL of 17:2:1 DCM:methanol:DIPEA. The resin was then washed 3 times with 5 mL of DCM and 3 times with 5 mL of DMF. The resin was then transferred to a reaction vessel.

[0405] All Fmoc-amino acids (0.15 mmol, 6 equiv.) were coupled by in situ activation with N-[(dimethylamino)-1H-1,2,3-triazo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate-N-oxide (HATU) (0.15 mmol, 6 equiv.; ChemPep) in 0.6 M N-methylmorpholine. The peptides were deprotected and cleaved from the resin by adding 5 mL of cleavage solution (87.5% (v / v) TFA, 5% (v / v) thioanisole, 3% (v / v) ethanedithiol, 2.5% (v / v) triisopropylsilane, and 2% (v / v) anisole) followed by stirring at room temperature for 2 h. The cleavage reaction was filtered into 30 mL of ice-cold diethyl ether to precipitate the peptide. The solution was poured onto a Buchner funnel filter and vacuumed to collect the peptide precipitate. The peptide was dried on a vacuum for 15 minutes and then washed with 80 mL of ice-cold diethyl ether. After drying for an additional hour, the peptide was resuspended in 20 mL of water and sonicated for 15 minutes to aid in dissolving the peptide. The solution was then flash frozen in liquid nitrogen and lyophilized.

[0406] Peptides were purified using high performance liquid chromatography (HPLC) using a Phenomenex Jupiter C18 preparative column (21.2 mm × 250 mm, 5 μm particle size, 300 Å pore size) with buffer A as 0.1% trifluoroacetic acid (TFA, HPLC grade) in nanopure water and buffer B as 0.1% TFA (HPLC grade) in acetonitrile (ACN, HPLC grade). Separation was performed at a flow rate of 5 mL / min with a linear gradient of 88 to 60% buffer A over 65 min. Fractions were analyzed by LC-MS using one of two HPLC-MS / MS setups (Vanquish UHPLC with a diode array detector interfaced to a Q-Exactive or Ultimate 3000 HPLC with a diode array detector interfaced to an LTQ OrbiTrap XL mass spectrometer) fitted with a Hypersil GOLD C18 column (2.1 mm × 150 mm, 1.9 μm particle size) for separation at 0.2 mL / min. The LC-MS program for peptide fraction identification was set as follows: Buffer A was LC-MS Optima water (Fisher) / 0.1% (v / v) LC-MS Optima TFA (Fisher) and Buffer B was LC-MS Optima acetonitrile (Fisher) / 0.1% (v / v) LC-MS Optima TFA (Fisher). The 12 min separation consisted of washing the column with 100% A for 3 min, followed by a linear gradient to 100% B for 3–6 min, followed by washing the column with 100% B for 6–9 min, and finally re-equilibrating with 100% A for 9–12 min. The MS detector was operated in positive ion mode and the FT analyzer settings were as follows: 70,000 resolution for the Q-Exactive and 100,000 resolution for the LTQ OrbiTrap, 1 microscan, and a maximum injection time of 200 ms. MS data analysis was performed using Xcalibur software (Thermo Fisher).

[0407] f. Enzymatic reaction of MSPapA peptide with PapB Assays were performed in a Coy Laboratories anaerobic chamber in a 98% N2 / 2% H2 atmosphere at room temperature. All reactions contained 0.05 M PIPES·NaOH (pH 7.4), 2 mM DTT, 2.4 mM SAM (enzymatically synthesized and purified as previously described (deGruyter, JN, et al. Biochem. 2017, 56(30), 3863-3873)), approximately 100-400 μM msPapA variant (concentration determined by peptide dry weight or spectroscopic analysis in the case of Y19W), and 430 nm-10 μM PapB. PapB was reduced using either dithionite (dT) or flavodoxin (FldA), flavodoxin reductase (FPR), and NADPH. For assays using chemical reductants, the total concentration was 2 mM dT. For assays using the biological reduction system, the mixture contained 25 μM FldA, 2 μM FPR, and 2 mM NADPH. The total reaction volume ranged from 0.1 mL to 0.5 mL for the initial screen for MS / MS collision-induced dissociation (CID) fragmentation experiments described below. Control reactions in the absence of dT, SAM, and PapB were also performed. Reactions were initiated by the addition of PapB and quenched for times ranging from 15 s to 2 h by adding 10% of the reaction volume of 30% (w / v) trichloroacetic acid (TCA, ACS grade). Samples were centrifuged at 16,000 x g for 10 min in a microcentrifuge to pellet precipitated PapB.

[0408] g. Alkylation of MSPapA peptides and variants After the initial incubation, half of the enzymatic reaction and half of the control reaction were aliquoted for alkylation with iodoacetic acid. A 500 mM stock of iodoacetic acid (IAC) was prepared in the dark and added to the enzymatic reaction to a final concentration of 10 mM (5x higher than the DTT concentration). These reactions were incubated in the dark for an additional 6 hours before being quenched by the addition of 10% of the reaction volume of 30% (w / v) TCA. The samples were then centrifuged at 16,000xg for 10 minutes in a microcentrifuge to pellet precipitated PapB.

[0409] h. TEV protease cleavage of peptide If TEV cleavage was required, 90 μM TEV protease was added directly to the complete PapB assay at a 1:1 volume ratio after the initial incubation. The TEV assay combination was incubated for 4 hours and then quenched by adding 10% of the reaction volume of 30% (w / v) TCA. The samples were then centrifuged at 16,000 x g for 10 minutes in a microcentrifuge to pellet precipitated PapB and TEV proteases.

[0410] i. U / HPLC-MS analysis of enzyme reactions and controls Assays were analyzed using either a Vanquish UHPLC equipped with a diode array detector coupled to a Q-Exactive mass spectrometer, or an Ultimate 3000 HPLC equipped with a diode array detector coupled to an LTQ OrbiTrap XL mass spectrometer. Each was operated in positive ion mode, with the FT analyzer set to 100,000 resolution, 1 microscan, and a maximum injection time of 200 ms. Data were analyzed using Xcalibur software. Aliquots of 20 μL were injected onto a Hypersil GOLD C18 column (2.1 mm × 150 mm, 1.9 μm particle size) (Thermo Fisher) pre-equilibrated in LC-MS Optima TFA (Fisher) with 0.1% (v / v) in LC-MS Optima water (Fisher). Chromatographic steps were performed with buffer A containing 0.1% (v / v) TFA in Optima water, and buffer B containing Optima grade acetonitrile with 0.1% (v / v) TFA at 0.2 mL / min. Separation consisted of a wash with 100% A for 0–3 min, followed by a linear gradient of 100%–0% A for 3–6 min, a wash with 0% A for 6–10 min, and re-equilibration at 100% A for 10–14 min.

[0411] j. Collision-induced dissociation (CID) fragmentation of unmodified and modified PapB The enzymatic reaction was carried out on a 0.5 mL scale as described above to obtain sufficient material. After quenching the reaction with TCA and centrifuging to remove precipitated proteins, the reaction mixture was desalted using C18 ZipTips (Millipore) according to the manufacturer's protocol. The analyzer was first calibrated to the mass of each msPapA peptide. The 3 msPapA peptide corresponding to each msPapA peptide was then added to the elution buffer. + Charge states were isolated in a CID cell using an isolation width of 1.7–2.4 m / z (depending on complete or incomplete peptide turnover), an activation time of 0.1 ms, a resolution of 70,000, and fragmented using a normalized collision energy (NCE) of 25. Fragmentation analysis was performed using mMass software.

[0412] 2. Characterization of Purified PapB PapB was obtained to homogeneity using His6 affinity chromatography for the initial separation, followed by TEV cleavage and amylose chromatography to remove MBP, and reconstituted with Fe / S. Gel filtration was used to remove higher molecular weight complexes (Figure 3). Because previous sequence analysis and ferrozine assays have shown that PapB likely has three [4Fe-4S] clusters, a 12-fold molar excess of iron and sulfide was added to the maturase for reconstitution. Amino acid and ICP-MS analysis of the protein from multiple independent purifications indicates that the purified protein obtained by this procedure contains 13.5 ± 0.3 moles of iron per mole of PapB. This is consistent with three [4Fe-4S] clusters per polypeptide chain. The enzymatic activity of PapB was established with HPLC-purified msPapA (Figures 4A and 4B). The peptide elutes at 8 min under the conditions used for separation (Figure 4A, top left) and HR-MS / MS reveals two clearly visible charge states (Figure 4A, bottom). Zooming in on the +3 charge state (Figure 4A, top right) reveals an isotopic envelope with a monoisotopic peak at m / z of 844.1201, which is within 0.5 ppm of the calculated unmodified peptide (calc: m / z 844.1197). In the presence of PapB, dithionite (dT), and SAM, the monoisotopic peak of the +3 charge state shifts by 0.6716, which corresponds to a loss of 2 Da from the peptide. This is within 0.8 ppm of the expected mass for a single cross-linked peptide. To verify that the thioether cross-link bond was formed, the modification reaction was performed in bulk and the resulting sample was desalted and subjected to HR-MS / MS analysis. As expected for a cross-linked lanthipeptide (Precord, TW, et al. ACS Chem. Biol. 2019, 14(9), 1981-89), no fragmentation is seen between C19 and D23 in the reacted peptide. In addition, b 23A 2 Da loss was observed in the y ion and all y series ions above y7 (Figure 4B). Fragmentation data is shown in Table 1 below. Note that under these conditions, complete conversion of msPapA to a single cross-linked peptide is routinely observed in 5 minutes using 0.1 nmol PapB and 20 mmol msPapA. [Table 1-1] JPEG2025512475000171.jpg46170 [Table 1-2] Unmodified indicates a charge state of z=1. The underline indicates the z=2 charge state. The italics indicate the z=3 charge state. *Indicates fragments derived from modified peptides.

[0413] Next, the kinetics of the modification reaction catalyzed by PapB in the presence of dT or a biological reduction system (FldA / FPR / NADPH) were evaluated (Figure 5). In these experiments, the enzyme concentration was kept low (430 nM) compared to the peptide (191 μM; established by tryptophan absorbance). Under these conditions, both show robust turnover, with dT showing kinetics about three times faster than those observed with the biological reduction system. Three replicate runs were performed using either the dT or FldA / FPR / NADPH biological reduction systems. At each time point, an aliquot was removed from the first assay batch quenched with TCA. Both the biological reduction system and the chemical reduction system had 100% substrate conversion after 300 seconds. At 15 seconds, a two-fold and four-fold increase in the concentration of PapB resulted in the conversion of unmodified msPapA to modified msPapA, which was about two- and four-fold greater than the initial conditions, suggesting that activity is proportional to the PapB concentration. However, increasing the msPapA concentration by 2- and 4-fold did not change the distribution of responses, suggesting that the peptide concentration was saturated. Thus, the rates measured in these experiments were k catUsing the linear portion of the curve, we obtain a dT of 7.4 ± 0.1 s. -1 , and 2.6±0.2s -1 The turnover number of is estimated in a biological reduction system.

[0414] 3. Exploiting the Substrate Promiscuity of Radical SAM RIPP Maturase for Intramolecular Peptide Cross-Linking Applications a. PapB modifies the expanded and contracted C(X3)D motif To assess the sequence dependence of the modification, minimal substrates containing 0–6 amino acids between the bridged Cys and Asp were synthesized and incubated with PapB (Figure 8A). In each case, a loss of 2 Da is observed upon addition of PapB (compare Figure 8B and Figure 8C). While reactions with 1–5 intervening residues appear to go to completion, CX0D (Figure 8B) and CX6D (Figure 8C) do not react completely, suggesting that PapB does not process these motifs efficiently. The observed monoisotopic masses for each treated and untreated species of the peptide are consistent with the expected monoisotopic masses (within less than 4 ppm error) (Table 2). [Table 2-1]

[0415] Treatment with iodoacetic acid (IAC) indicated that no free thiols were present in the treated samples, other than C at the unmodified portions of CX0D and CX6D (Figures 9-14), indicating that PapB had introduced thioether bridges into each peptide.

[0416] The location of the modifications in each msPapA peptide variant was investigated by collision-induced dissociation (CID) MS / MS. The modified msPapA peptides were analyzed and compared to unmodified control peptides. In each case, after quenching with TCA and removing excess salt, the samples were introduced into the mass spectrometer by direct infusion. The +3 charge state envelope was isolated and fragmented in the CID cell of the instrument. Fragmentation data showing all b and y ions that could be identified are shown in Tables 3-8 below. In general, all unmodified peptides showed fragmentation between the Cys and Asp residues. Upon modification, no fragmentation peak is observed between these two residues with the addition of PapB. In the case of the b fragment, no change in mass is observed until after the Asp residue, after which a loss of -2 Da is seen for each fragment. In contrast, a loss of -2 Da is observed after the Cys residue for each y fragment. [Table 3-1] JPEG2025512475000175.jpg50170 [Table 3-2] JPEG2025512475000177.jpg93170 [Table 3-3] The underline indicates the z=2 charge state. The italics indicate the z=3 charge state. *Indicates fragments derived from modified peptides. [Table 4-1] JPEG2025512475000180.jpg49170 [Table 4-2] JPEG2025512475000182.jpg91170 [Table 4-3]

Table 5-1

Table 5-2

Table 5-3

Table 6-1

Table 6-2

Table 6-3

Table 7-1

Table 7-2

Table 7-3

Table 8-1

Table 8-2

Table 8-3

[0417] The MS / MS data are consistent with the formation of thioether bridges at non-α positions. Under mild CID conditions, sactipeptide (sulfur-α carbon thioether bridged peptide) MS / MS spectra generally generate fragments at each residue position but contain a 2 Da loss at the acceptor (non-Cys) residue (Rea, MC, et al. Proc. Natl. Acad. Sci. USA 2010, 107(20), 9352-9357 and Lohans, CTJ Antibiot. 2014, 67, 23-30). Conversely, Cβ- and Cγ-thioether bridged peptides do not generate fragments within the macrocycle under mild CID conditions (Hudson, GA, et al. J. Am. Chem. Soc. 2019, 141, 8228-8238). Previous work by the Mitchell laboratory calculated the zero-point energies between Cα- and Cβ-thioethers and found that the electronic energy of the Cβ-bond is 12 kcal / mol more stable than that of the Cα-bond. This energy difference provides an explanation for the differences seen in the MS / MS spectra for these classes of RiPPs. All MS / MS spectra of the expansion and contraction of the msPapA thioether motif show that no fragments are found in the stable macrocycle, i.e., between the Cys and Asp residues.

[0418] Reactions with the CX0D and CX6D peptides did not go to completion, therefore, unmodified peptide fragments are also seen in these reactions revealing cleavage between C and D residues in the unmodified part of the isolated envelope, which served as an internal control (Figure 4D).

[0419] b. PapB allows extension from the leader peptide and processes linear and nested cross-links independently Next, we investigated whether the sequence context of the CX3D sequence in the native peptide and specific amino acids within the motif are essential for recognition and cross-linking (Figure 15A). We did not test an exhaustive number of modifications, since adding three or four Ala residues directly adjacent to the recognition motif did not clearly impair cross-linking activity. As Figure 15B shows, all peptides tested were efficiently cross-linked by the enzyme. Figure 15C demonstrates that cross-linking occurs within the CX3D sequence, even if alternative D residues are available downstream.

[0420] The naturally occurring PapA peptide is treated with PapB to introduce six lanthionine bonds that are either linear with Cys and Asp residues in the CX3D motifs that are crosslinked, or nested with a C residue present in one CX3D motif that bridges it with an Asp residue located at the C-terminus (Precord, TW, et al. ACS Chem. Biol. 2019, 14(9), 1981-89). As Figure 15 shows, both nested and linear variants of the peptide could be crosslinked by simply rearranging the CX3D element within the peptide. In the case of linear and nested crosslinks, treatment with PapB results in the loss of 4 Da from the peptide (Figure 15B). The observed monoisotopic masses for these species are less than 5 ppm of the expected monoisotopic mass (Table 9). [Table 9-1]

[0421] Tandem mass spectrometry reveals similar patterns in the b and y fragments. A mass loss of 2 Da is seen in each b fragment after D and in each y fragment after C. Fragmentation data for all discernible peaks are shown in Tables 10-14, and stable macrocycles are seen in each peptide. In all these cases, treatment with IAA did not result in carboxymethylation of the modified peptides (Figures 16-20). [Table 10-1] JPEG2025512475000208.jpg51170

Table 10-2

Table 10-3

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Table 12-1

Table 12-2

Table 12-3

Table 12-4

[0422] CX in the previous section nResults with the extension of the D motif demonstrate a lack of defined specificity in the recognition sequence beyond a preference for Cys and Asp. The data with nested crosslinks above extend this to include the leader peptide recognition sequence and the distance from individual amino acids in the processed peptide. These observations suggest that the only elements directing binding and crosslinking activity are the presence of proximal Cys and Asp residues, as well as the leader sequence presumed to be required for RiPP maturase (Precord, TW, et al. ACS Chem. Biol. 2019, 14(9), 1981-89). These observations support the notion that PapB can be used broadly to introduce thioether crosslinks into peptides that are completely unrelated to naturally occurring PapA substrates.

[0423] Indeed, PapB has recently been used to prepare peptide products capable of binding to a single protein target, such as the SARS-CoV-2 spike receptor-binding domain (King, AM, et al. Nat. Commun. 2021, 12, 6343). The peptide of that design contained a leader sequence connected via a TEV protease recognition sequence to a minimal substrate containing two CX3E motifs. The first report on PapB demonstrated that both Asp and Glu were cross-linked by the enzyme (Precord, TW, et al. ACS Chem. Biol. 2019, 14(9), 1981-89). However, in a more recent paper, although the peptide contained two potential cross-linking motifs, only a single cross-link was observed. Given the in vitro data with highly active proteins, showing that the topology of the modification can be directed in nature, this result was revisited to determine whether the absence of a second cross-link reflects the in vivo system used rather than being intrinsic to PapB. A synthetic peptide identical to the non-native peptide used to target the SARS-CoV-2 spike receptor binding domain was synthesized and treated with the above mentioned protein (Figure 21A). As the mass spectrum of the peptide shows, the enzyme introduces two crosslinks as evidenced by the loss of 4 Da in the modified peptide (Figure 21B). TEV cleavage of the resulting product was then performed to release the mature peptide, as shown by MS, which also showed a loss of 4 Da, localizing the modification to the peptide. The monoisotopic masses observed for both the full-length and TEV-cleaved peptides are less than 3 ppm of the expected values ​​(Table 15). [Table 15]

[0424] Tandem mass spectrometry showed a fragmentation pattern indicating that two thioether events occur, one between Cys3 and Glu7 and the other between Cys9 and Glu13 (Figure 21C, Table 16). Thus, the presence of a single crosslink in the reported peptide was likely due to the in vivo conditions used. [Table 16-1] JPEG2025512475000244.jpg45170 [Table 16-2]

[0425] cD-amino acids are processed by PapB In the initial experiments with PapA / PapB, the CX3D spacing was intriguing, suggesting that the enzyme recognizes Cys and Asp residues as part of a helical fragment, since the Cys and Asp side chains are expected to be located on the same face of the alpha helix. However, expansion and contraction of the motif clearly demonstrated that the spacing is not important. The expansion and contraction results suggest that only the identity of the amino acid or specific chemical moiety is important. Therefore, we investigated whether PapB could process msPapA when Cys and Asp were replaced with their dextrorotatory enantiomers (Figure 22A). Leader- D For the CSANDA peptide, there is complete conversion to the cross-linked peptide, as evidenced by the loss of 2 Da (Figure 22B). D For the DA peptide, significant substrate turnover is also observed, although the conversion is not complete (Figure 22B). D CSAN DDA is inefficiently processed under these conditions, although some products are clearly observed in the MS. It is possible to suggest that the small amount of product observed with this peptide is due to contaminating L-amino acids from a commercial source, but impurities would only account for 1-2% of product turnover. Based on the MS data, at least approximately 15% of the substrate is converted to product, suggesting that modification is a true D From Cys D Evidence is provided that this represents an Asp thioether bridge. Finally, CID MS / MS spectroscopy shows the loss of 2 Da in each y fragment after the C residue and in a single b fragment after the D residue in all three D-peptide scenarios (Figure 22C, Tables 17-19), indicating a stable macrocycle. Control experiments show that treatment with IAA reduces the C19 D It is shown that this does not result in carboxymethylation in the C-peptide (Figure 23). D D and C19 D C / D23 D In the case of the D-peptides, carboxymethylation is present upon IAA treatment due to incomplete turnover (Figures 23-25). However, the carboxymethylated species showed no evidence of 2 Da loss, providing evidence that the Cys thiol is involved in the newly introduced bond in these non-natural peptides. [Table 17-1] JPEG2025512475000247.jpg49170 [Table 17-2] JPEG2025512475000249.jpg86170 [Table 17-3] [Table 18-1] JPEG2025512475000252.jpg51170 [Table 18-2] JPEG2025512475000254.jpg87170 [Table 18-3] [Table 19-1] JPEG2025512475000257.jpg49170 [Table 19-2] JPEG2025512475000259.jpg87170 [Table 19-3]

[0426] We next attempted to transpose Cys and Asp residues by using a leader-bound DSANCA motif. However, we were unable to observe any cross-linking products with the transposed peptides, whether with L- or D-amino acids (Figure 26). These data support the notion that the active site has substantial flexibility with respect to Cys, but that interactions with Asp limit the range of available productive conformations. Previous studies have shown that mutation of a conserved PapB Arg residue to Ala, which may be near the PapA peptide Asp-binding site, prevents activity. Inversion of the side chain similarly eliminates interactions that do not result in cross-linking.

[0427] d. PapB processes sequences unrelated to the wild-type peptide sequence The results presented in the previous section highlight the remarkable lack of sequence specificity in PapB and suggest that the enzyme can crosslink virtually any sequence linked to a leader sequence, so long as Cys and downstream Asp / Glu residues are present in the peptide. As a proof of concept, we investigated the use of PapB to generate analogs of octreotide. Octreotide is an FDA-approved drug used to treat excess human growth hormone production, control symptoms of several types of cancer, and treat gastrointestinal bleeding (Lamberts, S.W.J., et al. Eur. J. Endocrinol. 2019, 181, R173-R183). Octreotide has two D-amino acids, reducing its susceptibility to protease degradation in vivo (Muttenthaler, M., et al. Nat. Rev. Drug Discov. 2021, 20, 309-325).

[0428] Octreotide is D FCF D It is an octameric peptide with WKTCT, with D-amino acids at the first and fourth positions. The two C residues form a disulfide bond macrocycle. Note that WT-PapA contains positively charged, non-polar, polar non-charged, and bulky side chain residues between the six donor and acceptor residue motifs (Precord, TW, et al. ACS Chem. Biol. 2019, 14(9), 1981-89). This observation, combined with the successful cross-linking in the extended motif, suggested that PapB may be able to introduce disulfide mimetic bonds via thioethers in various peptide substrates, as long as thiol and carboxylate moieties are present. As a proof of concept, two octreotide analogs were synthesized. Both designs omitted the C-terminal Cys, choosing Glu, which was used to cross-link with PapB to Cys. In the first design attempt, DThe sequence was further simplified by replacing W4 with Ala (Figure 27A). The octreotide analog sequence was covalently attached to the PapA leader peptide by solid-phase peptide synthesis (SPPS). The second design contained only the C7E substitution, but incorporated an ENLYFQ sequence between the leader and peptide to provide a convenient site for TEV cleavage to facilitate removal of the leader peptide. Incubation of any of the designed octreotide analogs with PapB leads to the formation of new products. In each case, the products are 2 Da lighter than the starting material, consistent with the formation of crosslinks (Figure 27B). Disulfides within the peptide can be excluded as a source of this loss, as the peptide contains only one Cys residue. Note that the reaction is approximately 75% complete with this analog, as assessed from the isotopic envelope. However, the observed monoisotopic masses for each peptide product species are in good agreement with the expected monoisotopic mass for a single bridge (<3 ppm, Figures 27B and 27C, Table 20). The structures of the synthesized octreotide analogs can be found in Figure 28. Subsequent MS / MS analysis confirmed the initial mass spectrometry data, with the fragmentation patterns of the peptides showing small fragments between the bridge Cys and Glu due to incomplete bridges. There is a clear 2 Da loss pattern in all y fragments after Cys, and a 2 Da loss in all b fragments after the C-terminal Glu with modified peptides (Tables 21-22). [Table 20] [Table 21-1] JPEG2025512475000263.jpg44170 [Table 21-2] [Table 22-1] JPEG2025512475000266.jpg50170 [Table 22-2]

[0429] We next attempted to use TEV protease to release modified peptides to demonstrate the feasibility of using this method to generate novel octreotide analogs. TEV protease can accommodate other amino acids at the P1' position other than Pro (Kapust, RB, et al. Biochem. Biophy. Res. Commun. 2002, 294(5), 949-955), but G or S is preferred. Residues other than G or S are tolerated at the P1' position, but they result in reduced enzyme efficacy. The crystal structure of a catalytically inactive form of TEV protease co-crystallized with an oligopeptide substrate revealed that the side chains of the residues at the P1' position are partially exposed to solvent (Phan, J., et al. J. Biol. Chem. 2002, 277(52), 50564-60672). D-amino acids have likely not been tested at the P1' position. Treatment with TEV protease cleaves the peptide containing the TEV cleavage site, releasing the C-terminal fragment (Figure 27C). D It is emphasized that F is tolerated at the P1' position. This proof-of-concept experiment demonstrates that PapB and TEV proteases can be used together to generate therapeutic analogs from synthetic peptide substrates containing both Cys and Asp / Glu residues, with PapB introducing thioether bond(s) between Cys and Asp / Glu to replace disulfide bridges.

[0430] These findings support the notion that PapB can modify peptides with large spacing between the thiol and carboxylate moieties, as well as sequences unrelated to PapA. These initial results demonstrate the utility of PapB as a bipartite-dependent thioether introduction tool: (1) it tolerates a variety of side chains across the peptide between the donor and acceptor Cys and Asp / Glu residues, (2) the orientation and spacing of the carboxylate and thiol moieties is flexible, and (3) a TEV recognition sequence can be introduced to allow the isolation of modified peptides from the leader sequence.

[0431] e. Consideration In the two decades since Sofia and colleagues established the RS superfamily (Sofia, HJ, et al. Nucleic Acids Res. 2001, 29(5), 1097-1106), there has been a proliferation of complex transformations attributed to RS enzymes. RS enzymes greatly expand their biochemical reaction repertoire due to their ability to activate C-H bonds for a variety of transformations that can range from epimerization to attachment to other carbon or heteroatoms. PapB catalyzes one such transformation, which involves activation of the carbon adjacent to a carboxylate moiety to crosslink to the thiol side chain of Cys (Precord, TW, et al. ACS Chem. Biol. 2019, 14(9), 1981-89). The mechanistic details of thioether crosslink formation remain to be elucidated. However, these results highlight a previously unknown PapA / B promiscuity, which will have implications for the mechanism of substrate recognition.

[0432] Based on all available structural and biochemical data on the RiPP maturase proteins, it would be expected that the leader sequence would bind to the RiPP recognition element (RRE) domain and direct the peptide to the active site of the protein to be modified. Implicit in this is the assumption that specificity in substrate selection is governed by the binding energy of the interaction with the leader sequence to the RRE domain. The conserved Asn side chain in the leader sequence has been previously proposed as necessary for the peptide-RRE interaction. The results showing that PapB can accept substrates with Cys to Asp separations ranging from 0 to 6 amino acids are likely evidence for this, in that the binding energy for the interaction with the leader sequence is utilized for reactivity. However, when these studies were first initiated, it was assumed that a separation of three amino acids would likely mean that the peptide has a helical structure, as previously proposed (King, A. M., et al. Nat. Commun. 2021, 12, 6343), which would place the side chains of the Cys and Asp residues close to each other in three-dimensional space. However, the observation that the enzyme can accept substrates with variable Cys to Asp spacing suggests that recognition depends on specific side chains rather than secondary structure. In other words, the enzyme specifically recognizes Cys and Asp / Glu side chains. In the absence of structural data on this enzyme, it is difficult to know how this could be accomplished, but note that in thioether cross-linking enzymes, it has been proposed that the thiolate of Cys could interact with one of the auxiliary Fe-S clusters. One can imagine that Asp / Glu recognition could involve hydrophilic or positively charged patches of residues. An Arg residue (Arg372) in PapB was previously suggested by sequence alignment, and mutation of the sequence alignment abolished cross-linking activity. Thus, the model for recognition that best fits the data is one in which the modified peptide has only two, but very specific interactions with the enzyme, outside of the leader sequence.

[0433] The observation that cross-linking efficiency decreases when the separation is zero or six could be due to either restricted degrees of freedom at shorter spans or the presence of too many possible conformations at longer separations, both of which would lead to less productive interactions between the cross-linked residues and the specific positions to which they are attached. Further evidence that there is no significant sequence dependency, other than the identity of Cys and Asp / Glu, is the fact that D-amino acids are tolerated. Outside of the leader sequence, it is proposed that there are no specific interactions between the enzyme and the rest of the peptide, other than thiolate and carboxylate binding. As with other RS ​​enzymes, binding can be expected to occur placing the 5' position of the SAM within or near the van der Waals radius of the H atom to be extracted, which in turn is within close proximity of the bridging Cys sulfur.

[0434] rSAMs, including hybrid RiPPs generated using chimeric leader peptides (Burkhart, BJ, et al. ACS Cent. Sci. 2017, 3(6), 629-638), RiPPs with acceptor residue changes (Himes, PM, et al. ACS Chem. Biol. 2016, 11(6), 1737-1744), truncated (i.e., leaderless) peptides accepted by epimerases (Himes, PM, et al. ACS Chem. Biol. 2016, 11(6), 1737-1744), changes in the tolerated macrocyclic core (King, AM, et al. Nat. Commun. 2021, 12, 6343), and residue-epimerization shifts based on core sequence changes (Korneli, M., et al. ACS Synth. Biol. 2021, 10(2), 236-242). Although some examples of RiPP maturase-induced promiscuity exist, this study demonstrates a highly predictable pattern for cross-link formation.

[0435] FIG. 29 provides a brief summary of successful PapB-mediated thioether cross-linking in the peptide sequences tested.

[0436] 4. Leader peptide sequence is not required As shown in Figure 30, the leader peptide sequence is not required for PapB-mediated modification: a PapB "leaderless" sequence containing non-proteinogenic amino acids still demonstrated thioether linkages via mass spectrometry.

[0437] 5. Interpeptide cross-linking studies As shown in Figures 31-35, mass spectrometry results reveal evidence that interpeptide cross-linking can also be achieved with PapB. Figure 31 shows mass spectrometry data for one-to-one interpeptide cross-linking and polymerization-like addition of X-mer peptide subunits. Figure 32 shows results for a typical assay peptide before and after PapB, demonstrating the presence of interpeptide products. Figure 33 shows mass spectrometry results showing evidence of simple and complex mass envelopes. As a proof of concept for interpeptide cross-linking using PapB, a thioether insulin analog was synthesized. Results showing cross-linked products can be found in Figures 34 and 35.

[0438] 6. Further Research Results showing cross-linking occurring in studies in which the peptide sequence contained (S,E)-5-aminohex-2-enedioic acid are shown in Figure 36. Tandem mass spectrometry results of dADo+msPapA adducts are shown in Figure 37.

[0439] Studies demonstrating that thioether cross-linking occurs via PapB in peptides containing selenopeptide sequences were conducted and the results (mass spectrometry and EXAFS) are shown in Figure 38. The tandem mass spectrometry results of C19U msPapA are shown in Figure 39.

[0440] Substitutions of donor and acceptor were examined and successful cross-linking results are shown in Figures 40 and 41. However, swapping the positions of model system C and D residues was found to not result in cross-linking (Figure 42).

[0441] Studies were then performed to investigate the nature of the electron transfer reaction occurring during modification. The results are shown in Figure 43 and demonstrate that the system is active in both reduced systems tested, with only a loss of activity when flavodoxin mononucleotide was removed from the reduced system control series. Following these results, "pre-reduced" PapB studies were performed to characterize turnover in the absence of reducing agent. These results are shown in Figures 44-46.

[0442] A conceptual schematic of the bioreactor set-up for PapB mediated peptide modification is shown in FIG.

[0443] 7. PapB tolerates a C-terminal glycine The sequence of leader-CX3G for recognition and cross-linking was examined (Figure 48A-B). Labeling experiments with deuterated glycine showed the cross-linked peptide with a corresponding loss of 3 Da, indicating that a thioether cross-link had occurred on the carbon adjacent to the carboxylic acid (Figure 49A-B).

[0444] However, the sequence of the leader-CX3(CH2)C(O)NH2 did not demonstrate a thioether bridge (Figure 50A-B). The loading, synthesis, and cleavage of the C-terminal glycine carboxamide peptide, which differs from that previously described for all alternative C-terminal carboxylate peptides, is detailed below.

[0445] Briefly, 150 mg of Rink amide resin was swelled in 5 mL of DMF for 1 hour in a polyprep chromatography column. The DMF was then removed from the column using nitrogen gas. The Fmoc protecting group on the resin was deprotected by adding 10 mL of 20% (v / v) piperidine in DMF and rocking the resin for 1 hour. The piperidine mixture was removed from the resin using nitrogen gas. The resin was then washed three times with 5 mL of DMF. Next, 0.03 mmol of Fmoc-Gly was added to 0.15 mmol of HATU and 0.15 mmol of HOAt in a glass scintillation vial and weighed out. 10 mL of 20% N-methylmorpholine in DMF was added to the vial and gently rocked to dissolve the material. This mixture was then added to the deprotected resin and rocked at room temperature for 4 hours. The amino acid solution was then pushed off the column with nitrogen gas and washed three times with 5 mL of DMF. The resin was then capped with acetic anhydride and pyridine in a 3:2 ratio. 10 mL of the mixture was added to the column and the resin mixture was shaken at room temperature for 30 min. The capping solution was pushed off the column with nitrogen gas and washed three times with 5 mL of DMF. All Fmoc-amino acids (0.15 mmol, 6 equiv.) in the peptide synthesis were coupled by in situ activation with N-[(dimethylamino)-1H-1,2,3-triazo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate-N-oxide (HATU) (0.15 mmol, 6 equiv.; ChemPep) in 0.4 M N-methylmorpholine. After completion of the synthesis, the peptide was cleaved from the resin with 10 mL of 18:1:1 TFA:H2O:triisopropylsilane. The solution was rocked at room temperature for 1 h. The cleavage reaction was filtered into 30 mL of ice-cold diethyl ether to precipitate the peptide. The solution was poured onto a Buchner funnel filter and vacuumed to collect the peptide precipitate. The peptide was dried on a vacuum for 15 minutes and then washed with 80 mL of ice-cold diethyl ether. After drying for an additional hour, the peptide was resuspended in 20 mL of water and sonicated for 15 minutes to aid in dissolving the peptide. The solution was then flash frozen in liquid nitrogen and lyophilized.

[0446] After lyophilization, the peptides were resuspended in 0.05 M PIPES·NaOH (pH 7.4), 2 mM DTT, 300 mM KCl, and 15% glycerol buffer solution. The peptides were then assayed with PapB using the following parameters: 6.1 uM PapB, 100 uM msPapA C-terminal Gly carboxamide, 2 mM DTT, 2.1 mM SAM, and 15% glycerol in a total volume of 100 uL. The negative control (no PapB) and the overnight complete assay (+PapB) were quenched by adding 11 uL of 30% (w / v) TCA to the mixture. The quenched assays were spun at 16,000xg for 10 minutes to pellet any precipitated enzyme or PIPES. The assay was analyzed using a Vanquish UHPLC equipped with a diode array detector connected to a Q-Exactive mass spectrometer operated in positive ion mode, with the FT analyzer set to 70,000 resolution, 1 microscan, and a maximum injection time of 200 ms. Data were analyzed using Xcalibur software. Aliquots of 20 uL were injected onto a Hypersil GOLD C18 column (msPapA) (2.1 mm x 150 mm, 1.9 μm particle size) (Thermo Fisher) pre-equilibrated in LC-MS Optima TFA (Fisher) at 0.1% (v / v) in LC-MS Optima water (Fisher). Chromatographic steps were performed at 0.2 mL / min with Buffer A containing 0.1% (v / v) TFA in Optima water, and Buffer B containing Optima grade acetonitrile containing 0.1% (v / v) TFA. Separation consisted of a wash with 100% A for 0–3 min, followed by a linear gradient of 100% to 0% A for 3–6 min, a wash with 0% A for 6–10 min, and re-equilibration at 100% A for 10–14 min.

[0447] 8. PapB tolerates C-terminal β-amino acids The sequence of leader-CX3 (β-amino acid) was examined for recognition and cross-linking (Figure 51A). The sequence of leader-CX3 (L-3-aminobutyric acid) was probed and treated with PapB, resulting in a loss of 2 Da, indicative of a thioether cross-linked product (Figure 51B-C).

[0448] Alternative β-amino acids were examined to probe the sequence requirements for recognition and cross-linking. The sequences of leader-CX3 (3-amino-2,2-dimethylbutanoic acid) (Figure 52A) and leader-CX3 ((R)-3-amino-2-methylpropanoic acid) (Figure 52B) showed no evidence of the 2 Da loss, indicating that the thioether cross-linked product was not formed.

[0449] The sequence of leader-CX3 ((S)-3-amino-2-methylpropanoic acid) (Figure 52C) shows evidence of a 2 Da loss (Figure 52D), indicating that a thioether cross-linked product was formed. Without wishing to be bound by theory, the culmination of these selective methylations on the C-terminal β-amino acid demonstrates that only one position is not amenable to substitution. The position alpha to the carboxylate must contain an H atom at the pro-R position. The reference point for the described pro-R position is the singly methylated alpha to carboxylate moiety. In any scenario where a change in substituent priority may alter absolute stereochemical priority, the side chain scenario must be compared to its singly methylated case.

[0450] The sequence of leader-CX3 (β-amino acid) was further probed using analogs representative of natural amino acids (Figure 53). A representative analog with B-tryptophan (Figure 54A) showed evidence of a 2 Da loss (Figure 54B), indicating that a thioether cross-linked product was formed.

[0451] The sequence of leader-CX3 (β-amino acids) was further investigated by probing reactions with N-methylated β-amino acids (Figure 55A-B). A representative analog with the sequence of leader-CX3 (2-methyl-3-(methylamino)propanoic acid) was treated under reaction conditions with PapB (Figure 55C). The reaction showed evidence of a 2 Da loss, indicating that a thioether cross-linked product had been formed (Figure 55D).

[0452] 9. PapB tolerates C-terminal D-amino acids Leader-CSAD to assess the requirement for C-terminal carboxylate D and L variants L A and Leader-CSAD D A was prepared and incubated with PapB (Figures 56A-D). In the case of L-alanine (Figure 56A), no loss of 2 Da was observed, indicating that the thioether cross-linked product was not formed (Figure 56B). In the case of D-alanine (Figure 56C), a loss of 2 Da was observed, indicating that the thioether cross-linked product was formed (Figure 56D).

[0453] Leader - CSAD D Further investigation with deuterated D-alanine at the carbon adjacent to the carboxylic acid incorporated into the A sequence (Figure 57A) showed evidence of a 3 Da loss, indicating that a thioether crosslink product was formed adjacent to the carboxylic acid (Figure 57B). Similarly, the carboxylic acid leader-CSAD D Deuterated D-methionine at the carbon adjacent to the M sequence (Figure 58A) showed evidence of a 3 Da loss, indicating that a thioether cross-linked product was formed adjacent to the carboxylic acid (Figure 58B).

[0454] However, the carboxylic acid leader - CSAD D Deuterated D-valine at the carbon adjacent to the V sequence (Figure 59A) showed evidence of a 2 Da loss, indicating that a thioether cross-linked product had been formed (Figure 59B), but the thioether cross-linked product was not present at the carbon adjacent to the carboxylic acid. Tertiary side chain carbon leader - CSAD DDeuterated D-valine at the V sequence (Figure 60A) showed evidence of a 3 Da loss (Figure 60B), indicating that a thioether cross-linked product was formed at the tertiary carbon.

[0455] Deuterium labeling of phenylalanine at the carbon adjacent to the carboxylic acid leader -CSAD D F sequence (Figure 61A) and incubated with PapB. The product showed evidence of a 2Da loss (Figure 61B). This again indicates a successful thioether cross-linked product, but the thioether cross-linked product was not present at the carbon adjacent to the carboxylic acid. Similarly, phenylalanine d5 was incorporated into the leader-CSAD D F sequence (Figure 61C) and incubated with PapB. The product showed evidence of a 2 Da loss (Figure 61D), again indicating a successful thioether cross-linked product, however, no thioether cross-linked product was formed with an aromatic group.

[0456] Phenylalanine d8 Leader-CSAD D F sequence (Figure 62A) and incubated with PapB. The product showed evidence of a 3 Da loss (Figure 62B), indicating a successful thioether cross-linked product to the methylene of the phenylalanine side chain.

[0457] The sequence of CX3 (C-terminal D-amino acid) was further explored using analogs representative of natural amino acids to form sactipeptides (Figure 63).

[0458] The sequence of CX3 (C-terminal D-amino acids) of certain amino acids provided the lanthipeptide (Figure 64).

[0459] 10. PapB tolerates non-peptide analogs The ring size of the non-peptide sequences was examined for carbon-linked analogs (Figures 65-70). In the case of leader-CG, no crosslinks are observed (Figure 65A). This places a lower limit for proper crosslink formation (Figure 65B). In the case of leader-(hCys)-(Gly) (Figure 66A), a loss of 2 Da is observed in the mass spectrum upon reaction with PapB (Figure 67B). Similarly, for leader-(Cys)-(β-Ala) (Figure 67A), a thioether crosslink is observed upon addition of PapB (67B). The smallest ring observed is a seven-membered ring. Extension of a single CH2 group on either the thiol or carboxylate-containing moiety from the baseline leader-CG scenario resulted in crosslinks. Furthermore, the ring size can be expanded with additional CH2 groups. See Figure 68A (leader-hCys-βAla) for sequence and Figure 68B for a representative mass spectrum showing 2 Da loss, Figure 69A (leader-Cys-gamma aminobutyric acid) for sequence and Figure 69B for a representative mass spectrum showing 2 Da loss, and Figure 70A (leader-hCys-gamma aminobutyric acid) for sequence and Figure 70B for a representative mass spectrum showing 2 Da loss.

[0460] The maximum ring size of non-peptide sequences was examined using carboxylate-terminated PEG-based moieties (Figures 71-72). In the case of triple PEGylation (Figure 71A), thioether crosslinks are observed upon addition of PapB (Figure 71B). Quadruple PEGylation (Figure 72A) shows thioether formation upon addition of PapB (Figure 72B), where thioether formation is observed.

[0461] Alternative scaffolds were also explored (Figures 73-75), establishing that both aromatic and heterocyclic rings can be included in the thioether macrocycle. Figure 73A demonstrates the structure of a peptide chain containing a substituted aniline in the peptide backbone and reacted thioether macrocycle. Figure 73B shows the mass spectra of the unreacted (top, no PapB) and reacted (bottom, with PapB) aniline-containing peptide chain. Figure 74A demonstrates the structure of a peptide chain containing a substituted benzylamine in the peptide and reacted thioether macrocycle. Figure 74B shows the mass spectrum of the reacted benzylamine-containing peptide chain. Figure 75A shows the structure of a modified coumarin-containing peptide. The coumarin-like portion is the most C-terminal aspect of the peptide. Figure 75B demonstrates the mass spectra of both the unreacted (top, no PapB) and reacted (bottom, with PapB) coumarin-containing peptide.

[0462] 11. Utilizing PapB to Prepare Thioether-crosslinked Peptide Mimetics The MC4R agonist, cetomalanotide, is an FDA approved drug indicated for chronic weight management (Figure 76A). Similar peptidomimetic analogs based on the PapB reaction to form thioether crosslinked products were envisioned (Figure 76B). Leader-CD D AHD D The sequence of FRWX (Figure 76C, X = β-Ala) was examined, and the reaction showed evidence of a 2 Da loss (Figure 76D), indicating that a thioether cross-linked product had been formed.

[0463] An orally available cross-linked thioether peptidomimetic was recently disclosed (J. Med. Chem. 2021, 64, 5, 2622-2633) (Figure 77A). Based on the disclosed PapB reaction, a similar peptidomimetic analog to the thioether cross-linked product formed was envisioned (Figure 77B). The sequence of leader-CBXBXF (Figure 77C, B=norleucine, X=N-methylnorleucine) was examined. The reaction showed evidence of a 2Da loss, indicating that a thioether cross-linked product had been formed (Figure 77D).

[0464] Bremeianotide, an agonist of MC1R, MC4R, MC3R, MC5R, and MC2R, is an FDA-approved drug for treating low libido in premenopausal women. Sequence Leader-BC D A peptidomimetic analog similar to FRWZ (B=norleucine, and Z=ε-ACP) was generated (Figure 78A). A putative thioether crosslink analog is shown in Figure 78B. PapB transformation is shown in Figure 78C. Mass spectra showing the reaction in both the absence (top) and presence (bottom) of PapB are shown in Figure 78D. The proposed thioether analog is shown in Figure 78B.

[0465] 12. PapB cross-links extended side chains of thiol- and carboxylate-containing residues. Previous studies have demonstrated that PapB can tolerate extended side chains of acidic residues, as both CX3D and CX3E sequences have been crosslinked. PapB forms crosslinks with homocysteine ​​(hCys) substitutions at position 19 and Asp at position 23, C19hCys and D23E, and C19hCys and homoglutamate (hGlu) at position 23 in msPapA (Figure 79A). MS analysis confirmed that PapB catalyzes the formation of crosslinks in peptides containing hCys and Asp at position 19 (Figure 79B), hCys and Glu at position 19 (Figure 79C), or hCys at position 19 and hGlu at position 23 (Figure 79D). Previous results demonstrated that altering either the donor or acceptor residues was adaptable, but in the case of D-amino acids, little crosslinking was observed when both residues were altered simultaneously. It is rare in RiPP maturation to alter both donor and acceptor residues and observe efficient substrate conversion. MS analysis of the reaction products shows that each substrate undergoes cross-linking, as evidenced by the loss of 2 Da to the substrate due to the loss of one H from the hCys thiol at residue 23 and a second H from the side chain of the carboxylate-containing side chain. As a proof of concept for the use of PapB in generating macrocyclized peptides, in the case of the C19hCys / D23hGlu variant, the leader sequence is shown to be cleaved to generate the macrocyclized core peptide (Figure 79E).

[0466] The observation of a 2 Da shift, taken together with the MS / MS data and the loss of IAM sensitivity, clearly indicates the formation of a thioether bridge. However, it was necessary to further confirm that the bridge was formed at the carbon atom that is alpha to the carboxylate of the side chain, as in the wild-type substrate. To this end, either unmodified or cross-linked C19hCys / D23hGlu was treated with TEV protease to liberate the modified core from the leader peptide, purified by HPLC, and subjected to both 1D and 2D NMR analysis. The 1D NMR spectrum of the peptide before treatment with PapB reveals a resonance at 2.41 ppm, composed of a doublet of triplets integrated at two protons (Figure 80). This feature can be reasonably assigned to the Hε of hGlu. In the NMR spectrum of the treated peptide (Figure 81), this resonance is absent and a new triplet at 3.34 ppm that integrates to a single hydrogen is observed. This new resonance is consistent with the introduction of a thioether at position alpha to the carboxylate of the hGlu side chain. To further correlate the positions, the modified and unmodified peptides were subjected to ROESY analysis to establish through-space correlation of the protons. In the unmodified peptide, the resonance at 2.41 ppm is coupled to resonances at 1.66, 1.75, and 1.88 ppm, which correspond to through-space coupling of Hε in the hGlu side chain to Hγ and Hβ of hGlu, respectively (see FIG. 82). In the modified peptide, the new resonance at 3.25 ppm is coupled to resonances at 1.8-1.9 ppm (see FIG. 83). Comparison of the linear and cyclized peptide 1D spectra shows a 0.2 ppm shift in the resonance between 1.6-2.0, which would be consistent with through-space coupling of Hε in the hGlu side chain to Hβ and Hγ of hGlu. In addition, weak couplings between 3.25-2.10 and 2.79-2.98 are observed. These cross peaks are consistent with Hε of the hGlu side chain coupling with Hβ (2.10 ppm) and Hγ (2.79-2.98) of the hCys side chain.Without wishing to be bound by theory, these results demonstrate that PapB can crosslink extended side chains of thiol- and carboxylate-containing residues.

[0467] Although various levels of promiscuity have been reported in the rSAM RiPP field, altering the identity of both the donor and acceptor residues to form a chemically consistent product is unprecedented. These findings expand our understanding of the ability of PapB to affect the biogenesis of RiPPs containing such extended side chains.

[0468] 13. Cross-linking with tetrazole moieties: Implications for peptide-based therapies Data with hCys, hGlu, and D-amino acid-containing peptides all suggest a high level of tolerance in PapB for a variety of substrates, but the examples shown are limited by the fact that they all contain thiol- and carboxylate-containing amino acids. Although selenocysteine ​​peptides are demonstrated herein to be processed by PapB, there are currently no examples of isosteres of carboxylate moieties processed by rSAM RiPP maturase. The tetrazole moiety is commonly used as a bioisostere of carboxylic acids in small molecule drug development. Tetrazoles improve the bioavailability of drugs, increase their lipophilicity, and reduce side effects compared to carboxylate-containing compounds. This is attributed to the metabolic stability of the tetrazole moiety, where metabolic conversion of carboxylic acids is partially driven by hepatic microsomes, much of which is avoided by using tetrazole isosteres. The tetrazole pharmacophore is used in a variety of drug classes, including nonsteroidal anti-inflammatory drugs, angiotensin receptor blockers, and proton pump inhibitors.

[0469] (2H-tetrazol-5-yl)propanoic acid (T4Az) was incorporated into msPapA (D23T4Az) by SPPS and incubated with PapB to determine whether the carboxylate-containing amino acids of msPapA could be replaced with the equivalent tetrazole-containing amino acids. The structures of the linear and cyclized peptides are shown in Figure 84A. Upon reaction with PapB, a 2 Da shift is clearly observed (Figure 84B), suggesting the formation of a crosslink. The MS / MS spectrum of D23T4Az msPapA does not reveal any fragmentation between the Cys and T4Az residues (Figure 84C), although the expected 2 Da loss is observed in the b-ion after T4Az and in the y-ion after the Cys residue (see Figure 85 for MS / MS spectrum and all fragments found). Although the regiochemistry for the crosslink is unconfirmed, there are several unusual peaks in the MS / MS that are potentially informative. For example, peaks consistent with loss of the tetrazole side chain moiety are observed (Figure 84C, y' and b') (see also Figure 86 for additional y' and b' fragments). Without wishing to be bound by theory, these fragments suggest that the thioether forms alpha to the tetrazole.

[0470] "This data with a tetrazole analog is the first demonstrated ability of rSAM RiPP maturase to crosslink with tetrazole moieties and opens new avenues for the development of peptide-based therapeutics. By using a tetrazole moiety instead of a carboxylate, the metabolic stability and pharmacokinetic properties of potential peptide therapeutics can be improved. This finding significantly expands the scope of rSAM RiPP maturase." 14. References (1)Van der Donk,WA;Bindman,NANat.Prod.:Discourse,Delivery,and Design,John Wiley&Sons:Oxford,2014;pp 197-218. (2)Sberro,H.;Fremin,B.J.;Zlitni,S.;Edfors,F.;Greenfield,N.;Snyder,M.P.;Pavlopoulos,G.A.;Kyrpides,N.C.;Bhatt,A.S.Large-Scale Analyses of Human Microbiomes Reveal Thousands of Small,Novel Genes.Cell.2019,178 (5),1245-1259. (3)Hetrick,K.J.;van der Donk,W.A.Ribosomally synthesized and post-translationally modified peptide natural product discovery in the genomic era.Curr.Opin.Chem.Biol.2017,38,36-44. 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[0471] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A method for introducing a thioether bond to a compound by chemical modification, wherein the method comprises reacting the compound with PapB, and the compound has a structure represented by the following formula: 【Chemistry 1】 、 In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. p is 1 or 2, t is an integer between 0 and 500, v is 1, 2, 3, 4, or 5, A is either S or Se, Q 1 However, it is a leader array, Q 2 However, it is a part that can be cut, R 1 is -CO 2 H, -C(O)NHOH, -SO 2 NH 2 、-SO 2 NHCOCH 3 、-SO 3 H, -NHCONHSO 2 CH 3 、-P(O)(OH) 2 、and 【Chemistry 2】 Selected from the structures selected from, R 4 However, selected from hydrogen and methyl, R 5 and R 5’ Each occurrence, if present, is independently a residue of the amino acid side chain. R 6 and R 6’ Each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ However, each of them is R 5 Or R 5’ It is covalently bonded to the intermediate atom, and together they form an unsubstituted five-membered heterocycle. R 7a and R 7b If each of these is present, it is independently selected from hydrogen and C1-C4 alkyl groups. R 8 However, selected from hydrogen and methyl, A method provided that the compound is not PapA.

2. The method according to claim 1, wherein o is independently 0, 1, 2, 3, 4, 5, 6, or 7.

3. The method according to claim 1, wherein t is 0.

4. The method according to claim 1, wherein v is 1 or 2.

5. R 1 However, -CO 2 The method according to claim 1, wherein the structure is H or the following. 【Transformation 3】

6. R 1 However, -CO 2 The method according to claim 1, wherein H.

7. The aforementioned detachable portion is -CO 2 The method according to claim 1, wherein the material is -(C4-C8 alkylene)-OC(O).

8. The aforementioned detachable portion is -CO 2 CH 2 CH = CHCH 2 The method according to claim 1, wherein the result is OC(O)-.

9. The method according to claim 1, wherein the cleavable portion is a protease recognition sequence.

10. The method according to claim 1, wherein PapB introduces a single thioether bond to the compound.

11. The method according to claim 1, wherein PapB introduces two or more thioether bonds into the compound.

12. The method according to claim 1, wherein the compound has a structure represented by the following formula. 【Chemistry 4】

13. The method according to claim 1, wherein the compound has a structure represented by the following formula. 【Transformation 5】

14. The above method produces a thioether compound having a structure represented by the following formula, 【Transformation 6】 、 The method according to claim 1, wherein v' is 0, 1, 2, or 3.

15. The method according to any one of claims 1 to 14, wherein the method further comprises the addition of a reducing agent.

16. The method according to claim 15, wherein the method further comprises the addition of a protease.

17. The thioether compound is 【Transformation 8】 The method according to claim 16, selected from the following.

18. A method for introducing a thioether bond to a compound by chemical modification, wherein the method comprises reacting the compound with PapB, and the compound has a structure represented by the following formula: 【Chemistry 9】 、 In the formula, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. p is 1 or 2, t is an integer between 0 and 500, v is 1, 2, 3, 4, or 5, A is either S or Se, R 1 is -CO 2 H, -C(O)NHOH, -SO 2 NH 2 、 -SO 2 NHCOCH 3 、 -SO 3 H, -NHCONHS 2 CH 3 、 -P(O)(OH) 2 、 and 【Chemistry 10】 Selected from the structures selected from, R 4 However, selected from hydrogen and methyl, R 5 and R 5’ Each occurrence, if present, is independently a residue of the amino acid side chain. R 6 and R 6’ Each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ However, each of them is R 5 Or R 5’ It is covalently bonded to the intermediate atom, and together they form an unsubstituted five-membered heterocycle. R 7a and R 7b If each of these is present, it is independently selected from hydrogen and C1-C4 alkyl groups. R 8 However, selected from hydrogen and methyl, A method provided that the compound is not PapA.

19. A method for introducing a thioether bond to a compound by chemical modification, wherein the method comprises reacting the compound with PapB, and the compound has a structure represented by the following formula: 【Chemistry 11】 、 In the formula, m is 0, 1, 2, 3, or 4. n is 0 or 1, Each of o and o' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. p is 1 or 2, A is either S or Se, If L is present, C2-C4 alkyl, -(C1-C4 alkyl)(OCH 2 CH 2 ) q ,and 【Chemistry 12】 Selected from the structures selected from, q is 1, 2, 3, or 4, Q 1 However, it is a leader array, Q 2 However, it is a part that can be cut, R 1 is -CO 2 H, -C(O)NHOH, -SO 2 NH 2 -, -SO 2 NHCOCH 3 -, -SO 3 H, -NHCONHSO 2 CH 3 -, -P(O)(OH) 2 and 【Chemistry 13】 Selected from the structures selected from, R 2 However, it is a residue in the side chain of an amino acid, provided that the amino acid is not isoleucine or threonine. R 3a and R 3b If each of these is present, it is independently selected from the C2-C5 alkynyl, C1-C5 azide, and amino acid side chain residues. R 4 However, selected from hydrogen and methyl, R 5 and R 5’ Each occurrence, if present, is independently a residue of the amino acid side chain. R 6 and R 6’ Each occurrence, if present, is independently selected from hydrogen and methyl, or R 6 Or R 6’ However, each of them is R 5 Or R 5’ It is covalently bonded to the intermediate atom, and together they form an unsubstituted five-membered heterocycle. R 7a and R 7b If each of these is present, it is independently selected from hydrogen and C1-C4 alkyl groups. A method provided that the compound is not PapA.

20. The method according to claim 19, wherein the method produces a thioether compound having a structure represented by the following formula. 【Chemistry 31】

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