WO2023201372A2 - Papb as a bimoiety-dependent thioether installation tool - Google Patents

Papb as a bimoiety-dependent thioether installation tool Download PDF

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WO2023201372A2
WO2023201372A2 PCT/US2023/065825 US2023065825W WO2023201372A2 WO 2023201372 A2 WO2023201372 A2 WO 2023201372A2 US 2023065825 W US2023065825 W US 2023065825W WO 2023201372 A2 WO2023201372 A2 WO 2023201372A2
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Prior art keywords
compound
thioether
formula
structure represented
amino acid
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PCT/US2023/065825
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French (fr)
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WO2023201372A3 (en
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Vahe BANDARIAN
Karsten A. S. EASTMAN
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University Of Utah Research Foundation
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Publication of WO2023201372A2 publication Critical patent/WO2023201372A2/en
Publication of WO2023201372A3 publication Critical patent/WO2023201372A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)

Definitions

  • Peptide-based therapeutics are growing due to their unique structure and ability to be produced via solid phase peptide synthesis (SPPS) or by recombinant DNA.
  • SPPS solid phase peptide synthesis
  • Many peptide therapeutics contain a disulfide bond in their active form. Disulfide bonds are susceptible to breakage via biological reductants such as glutathione. Additionally, many peptide therapeutics contain bulky or basic amino acid side chains which render them vulnerable to degradation by proteases. These factors contribute to their short serum half-lives.
  • RiPPs ribosomally synthesized and post-translationally modified peptides
  • rSAM radical S- adenosylmethionine
  • RiPP maturases have potential to offer biotechnological applications in peptide alterations such as thioether installation or peptide stapling.
  • rSAM enzymes use a radical intermediate to complete chemical transformations involved in natural product biosynthesis as well as 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.
  • the [4Fe-4S] RS cluster one iron coordinates the a-amino and a-carboxylate moieties of SAM.
  • the RS cluster When the RS cluster is catalytically active, it transfers an electron to bound SAM. Either chemical or biological reducing systems are useful for product turnover because the RS cluster is catalytically inactive in the +2 state. Homolytic cleavage of SAM forms the reactive 5’-deoxyadenosyl radical (5'- dAdo, FIG. 1).
  • 5'-dAdo' acts as a radical initiator by abstracting a hydrogen atom from a specific site on the substrate, thereby forming 5 ’-deoxyadenosine (5’-dAdoH, FIG. 1) and a theoretical RiPP radical intermediate.
  • the formed substrate radical is useful for substrate maturation. While only one [4Fe-4S] cluster is needed for reductive SAM cleavage, many rSAM enzymes also employ one or more auxiliary iron-sulf ur clusters (ACs) for substrate turnover (Fig. 4c). These ACs are coordinated to the enzyme by cysteine-rich C- terminal extensions from the RS canonical motif (FIG. 2).
  • rSAM maturases with multiple [Fe-S] clusters that form intrapeptide bonds between Ca, CB, or Cy on a specific residue and a cysteine thiol in the peptide substrate. Many of these thioether assembling maturases only form a single thioether in the mature peptide and are relatively slow in substrate turnover.
  • the RS cluster in addition to at least one AC cluster is necessary for thioether formation.
  • rSAM RiPP maturases also use a critical RiPP Recognition Element (RRE), that is responsible for binding to the leader sequence of the immature peptide (FIG. 2, left).
  • RRE critical RiPP Recognition Element
  • PapB is a RiPP maturase that catalyzes the insertion of six thioether crosslinks in the PapA polypeptide.
  • PapB catalyzes the insertion of links between the Cys thiol and the b- carbon of the Asp, where the residues being linked are in a CX 3 D motif.
  • the enzyme can also accept Glu at the modification site, and that PapB introduces the crosslink to the chemically analogous ⁇ -carbon.
  • PapB has also been shown to accept a shorter minimal substrate (msPapA), which only has a single pair of crosslinking amino acids in the CX 3 D motif.
  • PapB can catalyze both C13 and Cy thioether linkages, and forms six thioether linkages in the wild type PapA.
  • PapB contains a RS cluster and two ACs (FIG. 2). Replacing Asp residue(s) to Glu residue(s) in WT-PapA still results in successful crosslinking. Both CB and Cy thioether linkages were confirmed by 2D NMR.
  • the invention in one aspect, relates to methods of chemically modifying a peptide sequence to install one or more thioether linkages. Additionally disclosed are compounds formed using methods of chemically modifying a peptide sequence. Also disclosed are methods of chemically modifying a modified PapA sequence, and compounds formed using methods of chemically modifying a modified PapA sequence.
  • Also disclosed are methods of chemically modifying a compound to install a thioether linkage the method comprising reacting the compound with PapB, wherein the compound has a structure represented by a formula: wherein o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; wherein p is 1 or 2; wherein t is an integer from 0 to 500; wherein v is 1, 2, 3, 4, or 5; wherein A is S or Se; wherein Q 1 is a leader sequence; wherein Q 2 is a cleavable moiety; wherein R 1 is selected from -CO 2 H, -C(O)NHOH, - SO 2 NH 2 , -SO 2 NHC(O)CH 3 , -SO 3 H, -NHC(O)NHSO 2 CH 3 , -P(O)(OH) 2 , and a structure selected from: wherein R 4 is selected from hydrogen and methyl; wherein each occurrence of R 5 and R 5 , when present, is independently a residue of
  • Also disclosed are methods of chemically modifying a peptide sequence to install a thioether linkage, the method comprising reacting the peptide sequence with PapB, wherein the peptide sequence comprises X-Y n -Z; wherein X is a penicillamine or an amino acid residue comprising a -SH group or an amino acid residue comprising a -SeH group; wherein Y is a series of amino acid residues where n 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; wherein 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 wherein the peptide sequence is not PapA.
  • Also disclosed are methods of chemically modifying a modified PapA sequence to install a thioether linkage, the method comprising reacting the modified PapA sequence with PapB; wherein the modified PapA sequence comprises Cys-Y n -Asp, wherein Y is a series of amino acid residues and n 0, 1, 2, 4, 5, 6, or 7.
  • thioether compounds produced by a disclosed method.
  • compositions comprising an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • FIG. 1 is a schematic showing the proposed mechanism for beta-thioether crosslink.
  • FIG. 2 is a scheme showing the predicted structure of PapB.
  • FIG. 3 is a representative image showing SDS-PAGE analysis of reconstituted and purified PapB on a 12% crosslinked gel.
  • FIG. 4A and FIG. 4B show representative crosslinking data of minimal substrate PapA (msPapA) with PapB.
  • FIG. 4B shows the sequence of crosslinked PapB showing all of the observed b- and y- ions from tandem mass spectrometry.
  • FIG. 5 is a representative plot showing the comparison of activity of PapB processing Y17W msPapA with dithionite or FldA/FPR/NADPH.
  • FIG. 6 shows representative mass spectra demonstrating the effect of 2x and 4x enzyme concentration.
  • FIG. 7 shows representative mass spectra demonstrating the effect of 2x and 4x peptide concentration.
  • FIG. 8A-D show representative data for the Leader-C(X 0 -X 6 )D(Xm) crosslink formation.
  • FIG. 8A is a scheme showing the unmodified and modified peptide sequence illustrate the thioether crosslink based on the msPapA modification reported by Precord et al.
  • FIG. 8B shows representative mass spectra for CX 0 D-CX 2 D PapB modification.
  • FIG. 8C shows representative mass spectra for CX 4 D-CX 6 D PapB modification.
  • FIG. 8D are schematics showing that the expected 2 Da loss is seen in each b and y fragment in the tandem mass spectrometry.
  • FIG. 9A-B show representative data for the iodoacetic acid treatment for CX 0 D. Specifically, FIG. 9 A shows representative mass spectra data for CX 0 D without PapB. FIG. 9B shows shows representative mass spectra data for CX 0 D with PapB.
  • FIG. 10A-B show representative data for the iodoacetic acid treatment for CX 1 D. Specifically, FIG. 10A shows representative mass spectra data for CX 0 D without PapB. FIG. 10B shows shows representative mass spectra data for CX 1 D with PapB.
  • FIG. 11A-B show representative data for the iodoacetic acid treatment for CX2D. Specifically, FIG. 11A shows representative mass spectra data for CX 0 D without PapB. FIG. 11B shows shows representative mass spectra data for CX 2 D with PapB. [0035] FIG. 12A-B show representative data for the iodoacetic acid treatment for CX 4 D. Specifically, FIG. 12A shows representative mass spectra data for CX 0 D without PapB. FIG. 12B shows shows representative mass spectra data for CX 4 D with PapB.
  • FIG. 13A-B show representative data for the iodoacetic acid treatment for CX5D. Specifically, FIG. 13A shows representative mass spectra data for CX 0 D without PapB. FIG. 13B shows shows representative mass spectra data for CX 5 D with PapB.
  • FIG. 14A-B show representative data for the iodoacetic acid treatment for CX 6 D. Specifically, FIG. 14A shows representative mass spectra data for CX 0 D without PapB. FIG. 14B shows shows representative mass spectra data for CX 6 D with PapB.
  • FIG. 15A-C show representative data for leader extensions with single, nested, and in-line crosslinks.
  • FIG. 15A are peptide schemes showing the apparent crosslink locations that remain consistent after distancing the thioether motifs from the leader peptide.
  • FIG. 15B are representative mass spectra showing the isotopic distributions of the peptides; a shift of 2 Da in the case of single thioether motifs or 4 Da with double thioether motifs upon addition of PapB.
  • FIG. 13C are schematics showing a representation of the tandem mass spectrometry results.
  • FIG. 16A-B show representative data for the iodoacetic acid treatment for Leader- AAACSANDA.
  • FIG. 16A shows representative mass spectra data for Leader- AAACSANDA without PapB.
  • FIG. 16B shows shows representative mass spectra data for Leader-AAACSANDA with PapB.
  • FIG. 17A-B show representative data for the iodoacetic acid treatment for Leader- AAACSANDACSANDA.
  • FIG. 17A shows representative mass spectra data for Leader- AAACSANDACSANDA without PapB.
  • FIG. 17B shows shows representative mass spectra data for Leader-AAACSANDACSANDA with PapB.
  • FIG. 18A-B show representative data for the iodoacetic acid treatment for Leader- AAACSACDAADA.
  • FIG. 18A shows representative mass spectra data for Leader- AAACSACDAADA without PapB.
  • FIG. 18B shows shows representative mass spectra data for Leader- AAACSACDAAD A with PapB.
  • FIG. 19A-B show representative data for the iodoacetic acid treatment for Leader- AAAASACDAADA.
  • FIG. 19A shows representative mass spectra data for Leader- AAAASACDAADA without PapB.
  • FIG. 19B shows shows representative mass spectra data for Leader- AAAASACDAAD A with PapB.
  • FIG. 20A-B show representative data for the iodoacetic acid treatment for Leader- AAACSAADAADA.
  • FIG. 20A shows representative mass spectra data for Leader- AAACSAADAADA without PapB.
  • FIG. 20B shows shows representative mass spectra data for Leader- AAACSAADAADA with PapB.
  • FIG. 21A-C show representative data showing that PapB produces two thioether crosslinks in the AMK-1057 precursor peptide in vitro.
  • FIG. 21 A is a scheme showing that the AMK-1057 precursor peptide contains the leader peptide sequence, a TEV protease recognition sequence, and two CX 3 E motifs.
  • FIG. 21B shows representative mass spectra demonstrating that upon reaction with PapB in an in vitro assay, two crosslinks form. Additional processing with TEV protease produces the expected dicyclized peptide.
  • FIG. 21C is a scheme demonstrating the topology of the bonds as confirmed by tandem mass spectrometry.
  • FIG. 22A-C show representative data for PapB crosslinking D C and D D msPapA Peptides.
  • FIG. 22A is a scheme showing the thioether crosslink.
  • FIG. 22B are representative mass spectra showing formation of the thioether crosslinks.
  • FIG. 22C is a scheme demonstrating the topology of the bonds as confirmed by mass spectrometry.
  • FIG. 23A-B shows representative data for the iodoacetic acid treatment for Leader- D CSANDA.
  • FIG. 23A shows representative mass spectra data for Leader- D CSANDA without PapB.
  • FIG. 23B shows shows representative mass spectra data for Leader- D CSANDA with PapB.
  • FIG. 24A-B show representative data for the iodoacetic acid treatment for Leader- CSAN D DA.
  • FIG. 24A shows representative mass spectra data for Leader-CSAN D DA without PapB.
  • FIG. 24B shows shows representative mass spectra data for Leader- CSAN D DA with PapB.
  • FIG. 25A-B show representative data for the iodoacetic acid treatment for Leader- D CSAN D DA.
  • FIG. 25A shows representative mass spectra data for Leader- D CSAN D DA without PapB.
  • FIG. 25B shows shows representative mass spectra data for Leader- D CSAN D DA with PapB.
  • FIG. 26A-B show representative data for msPapA “DSANCA” peptides.
  • FIG. 26A shows representative mass spectra data for Leader-DSANCA and Leader- D DSANCA with and without PapB.
  • FIG. 26B shows representative mass spectra data for Leader-DSAN D CA and Leader- D DSAN D CA with and without PapB.
  • FIG. 27A-E show representative data for synthesis of an octreotide analog.
  • FIG. 27A is a structure of the FDA-approved therapeutic octreotide.
  • FIG. 27B is a schematic description of the designed peptides and the expected sites of modification upon modification with PapB. A TEV cleavage site is included in the second peptide to allow for liberation of the modified peptide sequence by PapB.
  • FIG. 27C is representative mass spectra data showing the isotopic envelope of these peptides indicating that a mixed population of processed and unprocessed peptides are present after modification by PapB.
  • FIG. 27D is representative mass spectra data showing that the TEV-cleaved peptide isotopic envelope reveals the anticipated 2 Da mass shift.
  • FIG. 27E is a scheme showing the anticipated loss of 2 Da in each y fragment after the C and in each b fragment after the C-terminal E as confirmed by tandem mass spectrometry.
  • FIG. 28 is a structure of the synthesized thioether-linked octreotide analog.
  • FIG. 29 is a scheme providing a brief summary of successful PapB-mediated thioether crosslinks in tested peptide sequences.
  • FIG. 30 shows representative data demonstrating that the leader peptide sequence is not required for modification via PapB.
  • FIG. 31 shows representative mass spectrometry data for a one-to-one interpeptide crosslink as well as polymerization-like addition of X-mer subunits.
  • FIG. 32 shows representative mass spectrometry results for a general assay peptide before and after PapB, demonstrating the presence of interpeptide products.
  • FIG. 33 shows representative mass spectra data showing evidence of simple and complex mass envelopes.
  • FIG. 34 is a schematic showing the experimental approaches to creating modified insulin analogs using PapB.
  • FIG. 35 shows representative mass spectra data for the synthesized insulin analogs.
  • FIG. 36 shows representative mass spectra data for crosslinking in peptides containing EneA.
  • FIG. 37 shows representative tandem mass spectrometry data for dAdo + D24EneA msPapA adduct.
  • FIG. 38 shows representative data, including mass spectrometry and EXAFS, for crosslinking in selenopeptides.
  • FIG. 39 shows representative tandem mass spectrometry data for C19U msPapA.
  • FIG. 40 shows representative mass spectrometry data demonstrating that aspartic acid may be replaced with glutamic acid, and cysteine may be replaced with homocysteine. Crosslinking is observed.
  • FIG. 41 shows representative mass spectrometry data demonstrating that ⁇ -amino acids may be incorporated in the peptide. Crosslinking is observed.
  • FIG. 42 shows representative mass spectrometry data demonstrating that no crosslinking was observed when altering the position of the C and D residues.
  • FIG. 43 shows representative data demonstrating the effect of components in the reduction system employed.
  • FIG. 44 is a schematic summarizing the findings of experiments conducted using prereduced PapB.
  • FIG. 45 is a scatterplot showing representative data of %product as a function of time for prereduced PapB experiments.
  • FIG. 46 shows representative data, including photodiode array chromatography, UV- Vis, and extracted ion chromatography, for PapB with and without reductant, as well as prereduced PapB.
  • FIG. 47 is a concept schematic for a bioreactor setup for peptide modification via PapB.
  • FIG. 48A-B show representative data for C-terminal glycine sequence.
  • FIG. 48A is a scheme showing the thioether crosslink.
  • FIG. 48B are representative mass spectra showing formation of the thioether crosslinks.
  • FIG. 49A-B show representative data for deuterium labeled C-terminal glycine analogs.
  • FIG. 49A is a scheme showing the thioether crosslink.
  • FIG. 49B are representative mass spectra showing formation of the thioether crosslinks.
  • FIG. 50A-B show representative data for C-terminal glycine carboxamide sequence.
  • FIG. 50A is the structure of the sequence.
  • FIG. SOB are representative mass spectra showing lack of formation of the thioether crosslinks.
  • FIG. 51A-C show representative data for crosslinking with C-terminal ⁇ -amino acids.
  • FIG. 50A is a scheme showing the generic thioether crosslink reaction for C-terminal ⁇ - amino acids.
  • FIG. SOB is a scheme showing the thioether crosslink reaction with C-terminal ⁇ -alanine.
  • FIG. 51C is the corresponding mass spectra data showing formation of the thioether crosslink.
  • FIG. 52A-D show representative data for the crosslinking with various C-terminal ⁇ - amino acids.
  • FIG. 52A is a scheme showing the absence of crosslink reaction with C- terminal 2,2-dimethyl-beta-alanine.
  • FIG. 52B is a scheme showing the absence of crosslink reaction with C-terminal (R)-3-amino-2-methylpropanoic acid.
  • FIG. 52C is a scheme showing the crosslink reaction with C-terminal (S)-3-amino-2-methylpropanoic acid.
  • FIG. 52D is the corresponding mass spectra data showing formation of the thioether crosslink.
  • FIG. 53 shows representative data for the crosslinking with common C-terminal ⁇ - amino acids.
  • FIG. 54A shows a schematic thioether crosslinking with a D-tryptophan ⁇ -amino acid.
  • FIG. 54B is the corresponding mass spectra data showing formation of the thioether crosslink
  • FIG. 55A-D show representative structures of thioether crosslinking of N-methyl amino acids.
  • FIG. 55A shows unsubstituted N-methylated thioether crosslinked product.
  • FIG. 55B shows substituted N-methylated thioether crosslinked product.
  • FIG. 55C shows a schematic thioether crosslinking with a substituted N-methylated substrate.
  • FIG. 55D is the corresponding mass spectra data showing formation of the thioether crosslink.
  • FIG. 56A-D show representative data for thioether crosslinking with C-terminal L- alanine or D-alanine.
  • FIG. 56A shows a schematic of a C-terminal L-alanine without thioether crosslink product.
  • FIG. 58B is the corresponding mass spectra data showing lack of formation of the thioether crosslink.
  • FIG. 56C shows a schematic of a C-terminal D- alanine with thioether crosslink product.
  • FIG. 56D is the corresponding mass spectra data showing formation of the thioether crosslink.
  • FIG. 57A-B show representative data for thioether crosslinking with deuterium labeled C-terminal D-alanine.
  • FIG. 57A shows a schematic of a deuterium labeled C- terminal D-alanine with thioether crosslink product.
  • FIG. 57B is the corresponding mass spectra data showing formation of the thioether crosslink and loss of the deuterium labeled confirmed by mass shift and loss 3Da.
  • FIG. 58A-B show representative data for thioether crosslinking with deuterium labeled C-terminal D-methionine.
  • FIG. 58A shows a schematic of a deuterium labeled C- terminal D-methionine with thioether crosslink product.
  • FIG. 58B is the corresponding mass spectra data showing formation of the thioether crosslink and loss of the deuterium labeled confirmed by mass shift and loss 3Da.
  • FIG. 59A-B show representative data for thioether crosslinking with d 2 -labeled D- valine.
  • FIG. 59A shows a structure of a deuterium labeled C-terminal D-valine.
  • FIG. 59B is the corresponding mass spectra data showing formation of the thioether crosslink however mass shift is indicative of no loss of deuterium.
  • FIG. 60A-B show representative data for thioether crosslinking with d 3 -labeled D- valine.
  • FIG. 60A shows a schematic of a deuterium labeled side chain C-terminal D-valine with thioether crosslink product.
  • FIG. 60B is the corresponding mass spectra data showing formation of the thioether crosslink and loss of the deuterium labeled confirmed by mass shift and loss 3Da.
  • FIG. 61A-D show representative data for thioether crosslinking with deuterium labeled C-terminal D-phenyl alanine.
  • FIG. 61A shows a structure of a deuterium labeled C a C-terminal D-phenyl alanine.
  • FIG. 61B is the corresponding mass spectra data showing formation of the thioether crosslink however mass shift is indicative of no loss of deuterium.
  • FIG. 61C shows a structure of a deuterium labeled aryl C-terminal D-phenyl alanine.
  • FIG. 61D is the corresponding mass spectra data showing formation of the thioether crosslink however mass shift is indicative of no loss of deuterium
  • FIG. 62A-B show representative data for thioether crosslinking with deuterium labeled d8-C-terminal D-phenylalanine.
  • FIG. 62A shows a schematic of a deuterium labeled d8-C-terminal D-methionine with thioether crosslink product.
  • FIG. 62B is the corresponding mass spectra data showing formation of the thioether crosslink and loss of the deuterium labeled confirmed by mass shift.
  • FIG. 63 shows structures of sactipeptide thioether crosslink of corresponding D- aminoacids
  • FIG. 64 shows structures of ranthipeptide thioether crosslink of corresponding D- aminoacids.
  • FIG. 65A-B show representative data for 6-membered non-peptidic thioether crosslinking.
  • FIG. 65 A shows scheme of Leader-Cys-Gly reaction.
  • FIG. 65B is the corresponding mass spectra data showing lack of formation of the thioether crosslink of 6- membered ring.
  • FIG. 66A-B show representative data for 7-membered non-peptidic thioether crosslinking.
  • FIG. 66A shows scheme of Leader-hCys-Gly reaction.
  • FIG. 66B is the corresponding mass spectra data showing formation of the thioether crosslink of 7-membered ring.
  • FIG. 67A-B show representative data for 7-membered non-peptidic thioether crosslinking.
  • FIG. 67 A shows scheme of Leader-Cys- ⁇ Ala reaction.
  • FIG. 67B is the corresponding mass spectra data showing formation of the thioether crosslink of 7-membered ring.
  • FIG. 68A-B show representative data for 8-membered non-peptidic thioether crosslinking.
  • FIG. 68A shows scheme of Leader-hCys- ⁇ Ala reaction.
  • FIG. 68B is the corresponding mass spectra data showing formation of the thioether crosslink of 8-membered ring.
  • FIG. 69A-B show representative data for 8-membered non-peptidic thioether crosslinking.
  • FIG. 69 A shows scheme of Leader-Cys-GABA reaction.
  • FIG. 69B is the corresponding mass spectra data showing formation of the thioether crosslink of 8-membered ring.
  • FIG. 70A-B show representative data for 9-membered non-peptidic thioether crosslinking.
  • FIG. 70 A shows scheme of Leader-hCys-GABA reaction.
  • FIG. 70B is the corresponding mass spectra data showing formation of the thioether crosslink of 9-membered ring.
  • FIG. 71A-B show representative data for 16-membered non-peptidic thioether crosslinking.
  • FIG. 71 A shows scheme of Leader-hCys-NH-PEG 3 -CO 2 H reaction.
  • FIG. 71 A is the corresponding mass spectra data showing formation of the thioether crosslink of 16- membered ring.
  • FIG. 72A-B show representative data for 20-membered non-peptidic thioether crosslinking.
  • FIG. 72A shows scheme of Leader-hCys-NH-PEG 4 -CO 2 H reaction.
  • FIG. 72B is the corresponding mass spectra data showing formation of the thioether crosslink of 20- membered ring.
  • FIG. 73A-B show representative data for unusual non-peptidic thioether crosslinking.
  • FIG. 73A shows scheme of Leader-Cys-Ser-Ala-Asn-2-(2-aminophenyl)acetic acid reaction.
  • FIG. 73B is the corresponding mass spectra data showing formation of the thioether crosslink of 17-membered ring.
  • FIG. 74A-B show representative data for unusual non-peptidic thioether crosslinking.
  • FIG. 74A shows scheme of Leader-Cys-Ser-Ala-Asn-2-(2-(aminomethyl)phenyl)acetic acid reaction.
  • FIG. 74B is the corresponding mass spectra data showing formation of the thioether crosslink of 18-membered ring.
  • FIG. 75A-B show representative data for coumarin thioether crosslinking.
  • FIG. 75A shows scheme of Leader-Cys-coumarin reaction.
  • FIG. 75B is the corresponding mass spectra data showing formation of the thioether crosslink of 12-membered ring.
  • FIG. 76A-C show representative data for the synthesis thioether peptidomimetic.
  • FIG. 76A is a structure of Setmalanotide, an FDA approved drug.
  • FIG. 76B shows a schematic thioether crosslinking with a modified peptide structure (e.g., an analog of Setmalanotide).
  • FIG. 76C is the corresponding mass spectra data showing formation of the thioether crosslink.
  • FIG. 77A-D show representative data for the synthesis thioether peptidomimetic.
  • FIG. 77A is a structure of a Novartis orally available peptide.
  • FIG. 77B is a structure of the designed peptides (an analog of the therapeutic peptide from FIG. 77A) and the expected product upon modification with PapB.
  • FIG. 77C shows a schematic thioether crosslinking with a modified peptide structure.
  • FIG. 77D is the corresponding mass spectra data showing formation of the thioether crosslink.
  • FIG. 78A-D show representative therapeutic cyclic peptides that can be mimicked by a thioether crosslink peptide.
  • FIG. 78A show the structure of a representative cyclic peptide, bremelanotide.
  • FIG. 78B shows a representative structure of the thioether crosslinked product, an analog of bremelanotide, which contains the amino acid sequence norleucine, cysteine, D-phenylalanine, arginine, tryptophan, and epsilon-amino hexanoic acid (ACP).
  • FIG. 78C shows a representative scheme of the Leader-XCDFRWZ XXX reaction.
  • FIG. 78D is the corresponding mass spectra data showing formation of the thioether crosslink of therapeutic analog.
  • FIG. 79A-E show representative data illustrating that PapB forms crosslinks in thiol- and carboxylate-containing extended sidechains.
  • FIG. 79B shows a 2 Da shift in the MS for the carboxylate-containing residue as Asp.
  • FIG. 79C shows a 2 Da shift in the MS for the carboxylate-containing residue as Glu.
  • FIG. 79D shows a 2 Da shift in the MS for the carboxylate-containing residue as homoGlu.
  • FIG. 79E shows the MS for the liberated macrocyclized peptide core from the leader sequence following cleavage of the TEV protease recognition sequence with TEV protease.
  • FIG. 80 shows a representative proton NMR spectrum of the linear G(hC)SAN(hE)A peptide.
  • FIG. 81 shows a representative proton NMR spectrum of the cyclized G(hC)SAN(hE)A peptide.
  • FIG. 82 shows a representative ROESY spectrum of the linear G(hC)SAN(hE)A peptide.
  • FIG. 83 shows a representative ROESY spectrum of the cyclized G(hC)SAN(hE)A peptide.
  • FIG. 84A-C show representative data pertaining to a carboxylate isostere (tetrazole moiety) crosslinked by PapB.
  • FIG. 84A shows a schematic of the linear and cyclized peptide illustrating the putative crosslink location.
  • FIG. 84B shows MS results illustrating a clear 2 Da loss between an assay without PapB (darker gray) and with the addition of PapB (lighter gray).
  • FIG. 84C shows the expected tandem mass spectrometry with no fragmentation between Cys and T4Az.
  • FIG. 85 shows representative fragmentation of reacted D23T4Az msPapA variant.
  • FIG. 86 shows representative fragments of a tetrazole loss in the D23T4Az msPapA variant
  • Ranges can be expressed herein 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 of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ⁇ 10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art.
  • an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a weight percent (wt. %) of a component is based on the total weight of the formulation or composition in which the component is included.
  • IC 50 is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc.
  • a substance e.g., a compound or a drug
  • an IC 50 can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein.
  • IC 50 refers to the half-maximal (50%) inhibitory concentration (IC) of a substance.
  • EC 50 is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% agonism of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc.
  • an EC 50 can refer to the concentration of a substance that is required for 50% agonism in vivo, as further defined elsewhere herein.
  • EC 50 refers to the concentration of agonist that provokes a response hallway between the baseline and maximum response.
  • the term “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the subject is a mammal.
  • a patient refers to a subject afflicted with a disease, disorder, or condition.
  • patient includes human and veterinary subjects.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.
  • the subject is a mammal such as a primate, and, in a further aspect, the subject is a human.
  • subject also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • domesticated animals e.g., cats, dogs, etc.
  • livestock e.g., cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.
  • prevent refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
  • diagnosis means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.
  • administering refers 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, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition.
  • a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration.
  • compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.
  • dosage form means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject.
  • a dosage forms can comprise inventive a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharmaceutically acceptable excipient, such as a preservative, buffer, saline, or phosphate buffered saline.
  • Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques.
  • Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonyl phenol, sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-
  • kit means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. [00132] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit.
  • kits may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble- shooting, references, technical support, and any other related documents.
  • Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.
  • therapeutic agent include any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action.
  • the term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like.
  • therapeutic agents include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
  • the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; anti-cancer and anti-neoplastic agents such as kinase inhibitors, poly ADP ribose polymerase (PARP) inhibitors and other DNA damage response modifiers, epigenetic agents such as bromodomain and extra-terminal (BET) inhibitors, histone deacetylase (HD Ac) inhibitors, iron chelotors and other ribonucleotides reductase inhibitors, proteasome inhibitors and Nedd8-activating enzyme (NAE) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, traditional cytotoxic agents such as paclitaxel, dox, irinotecan, and platinum compounds, immune checkpoint blockade agents such as cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibody (mAB), programme
  • the agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas.
  • therapeutic agent also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • pharmaceutically acceptable describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
  • sactipeptide refers to a sulfur-to-alpha carbon thioether cross-linked peptide belonging to the ribosomally synthesized post-translationally modified peptide (RiPP) superfamily. As illustrated by the structure below, a sactipeptide contains an intramolecular thioether bond that crosslinks the sulfur atom of a cysteine residue to the ⁇ -carbon of an acceptor amino acid.
  • ranthipeptide refers to a radical non-a thioether- containing peptide, which, similar to sactipeptides above, is also a member of the RiPP superfamily.
  • a ranthipeptide can contain an intramolecular thioether bond that crosslinks the sulfur atom of a cysteine residue to any carbon other than the a-carbon of an acceptor amino acid.
  • ranthipeptide residues containing an ⁇ - or ⁇ -carbon are shown below.
  • the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.
  • exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.
  • the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of 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 paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form 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). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. 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 bacterial-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 media just 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.
  • the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described below.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms, such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • substitution or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).
  • a 1 ,” “A 2 ,” “A 3 ,” and “A 4 ” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.
  • aliphatic or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • alkyl as used herein is 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.
  • 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 from one to six (e.g., from one to four) carbon atoms.
  • alkyl group can also be a Cl 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, Cl -CIO alkyl, and the like up to and including a C1-C24 alkyl.
  • alkyl is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group.
  • halogenated alkyl or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.
  • halogenated alkyl specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.
  • monohaloalkyl specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine.
  • polyhaloalkyl specifically refers to an alkyl group that is independently substituted with two or more halides, i.e.
  • alkoxyalkyl specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below.
  • aminoalkyl specifically refers to an alkyl group that is substituted with one or more amino groups.
  • hydroxyalkyl specifically refers to an alkyl group that is substituted with one or more hydroxy groups.
  • cycloalkyl refers to both unsubstituted and substituted cycloalkyl moieties
  • the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.”
  • a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy”
  • a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like.
  • the practice 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 also include the specific term.
  • cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms.
  • cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, and the like.
  • heterocycloalkyl is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where 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.
  • the cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted.
  • the cycloalkyl group and heterocycloalkyl 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.
  • polyalkylene group as used herein is a group having two or more CH 2 groups linked to one another.
  • the polyalkylene group can be represented by the formula -(CH 2 ) a - , where “a” is an integer of from 2 to 500.
  • Alkoxy also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as — OA 1 — OA 2 or — OA 1 — (OA 2 ) a — OA 3 , where “a” is an integer of from 1 to 200 and A 1 , A 2 , and A 3 are alkyl and/or cycloalkyl groups.
  • alkenyl as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.
  • the alkenyl group 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.
  • 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 here
  • Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbomenyl, and the like.
  • heterocycloalkenyl is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where 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.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted.
  • the cycloalkenyl group and heterocycloalkenyl group 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.
  • alkynyl as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond.
  • the alkynyl group 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.
  • 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
  • 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 bound.
  • cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like.
  • heterocycloalkynyl is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where 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.
  • the cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted.
  • the cycloalkynyl group and heterocycloalkynyl group 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.
  • aromatic group refers to a ring structure having cyclic clouds of delocalized ⁇ electrons above and below the plane of the molecule, where the ⁇ clouds contain (4n+2) ⁇ electrons.
  • aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference.
  • aromatic group is inclusive of both aryl and heteroaryl groups.
  • aryl as used herein is a group that contains 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, — NH 2 , carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.
  • biasryl is a specific type of aryl group and is included in the definition of “aryl.”
  • the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond.
  • biaryl can be two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
  • amine or “amino” as used herein are represented by the formula — NA 1 A 2 , where A 1 and A 2 can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is — NH 2 .
  • alkylamino as used herein is represented by the formula — NH(- alkyl) where alkyl is a described herein.
  • Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like.
  • dialkylamino as used herein is represented by the formula — N(- alkyl) 2 where alkyl is a described herein.
  • Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert- pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N- propylamino group, N-ethyl-N-propylamino group and the like.
  • esters as used herein is represented by the formula — (OC(O)A 1 or — C(O)OA 1 , where A 1 can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • polyester as used herein is represented by the formula — (A 1 O(O)C-A 2 -C(O)O) a — or (A 1 O(O)C-A 2 -OC(O)) a , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.
  • ether as used herein is represented by the formula A 1 OA 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein.
  • polyether as used herein is represented by the formula — (A 1 O-A 2 O) a — , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500.
  • Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.
  • halo halogen
  • halide halogen
  • pseudohalide pseudohalogen
  • pseudohalo pseudohalogen
  • pseudohalo pseudohalo
  • functional groups include, by way of example, cyano, thiocyanato, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.
  • heteroalkyl refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized. Heteroalkyls can be substituted as defined above for alkyl groups.
  • heteroaryl refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group.
  • heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfin- oxides, and dioxides are permissible heteroatom substitutions.
  • the heteroaryl group can be substituted or unsubstituted.
  • the heteroaryl 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.
  • 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.
  • 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[l ,2-a]pyrazinyl, benzo[c] [ 1 ,2,5]thiadiazolyl, benzo[c][l,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.
  • heterocycle or “heterocyclyl,” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon.
  • Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3- oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1 , 3, 4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including
  • heterocyclyl group can also be a 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, and the like up to and including a C2-C18 heterocyclyl.
  • a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like.
  • a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.
  • 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 encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring.
  • Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6- membered ring containing 1 , 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms.
  • Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[l,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-l,4-benzodioxinyl, 3,4-dihydro-2H- chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; lH-pyrrolo[3,2-b]pyridin-3-yl; and 1H- pyrazolo[3,2-b]pyridin-3-yl.
  • heterocycloalkyl refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems.
  • the heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted.
  • heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.
  • hydroxyl or “hydroxyl” as used herein is represented by the formula — OH.
  • ketone as used herein is represented by the formula A 1 C(O)A 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • nitro as used herein is represented by the formula NO 2 .
  • nitrile or “cyano” as used herein is represented by the formula CN.
  • sil as used herein is represented by the formula — SiA 1 A 2 A 3 , where A 1 , A 2 , and A 3 can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfo-oxo is represented by the formulas — S(O)A 1 , — S(O) 2 A 1 , — OS(O) 2 A 1 , or — OS(O) 2 OA 1 , where A 1 can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfonyl is used herein to refer to the sulfo-oxo group represented by the formula — S(O) 2 A 1 , where A 1 can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfone as used herein is represented by the formula A 1 S(O) 2 A 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfoxide as used herein is represented by the formula A 1 S(O)A 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • thiol as used herein is represented by the formula — SH.
  • R 1 ,” “R 2 ,” “R 3 ,” “R n ,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above.
  • R 1 is a straight chain alkyl group
  • one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like.
  • a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group.
  • an alkyl group comprising an amino group the amino group can be incorporated within the backbone of the alkyl group.
  • the amino group can be attached to the backbone of the alkyl group.
  • the nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.
  • compounds of the invention may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogen of the designated moiety are replaced with a suitable substituent.
  • 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 substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on R° are independently halogen, -(CH 2 ) 0-2 R ⁇ , -(haloR ⁇ ), -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR ⁇ , -(CH 2 ) 0-
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2) 2-3 O-, wherein each independent occurrence of R* is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, - R ⁇ , -(haloR ⁇ ), -OH, -OR ⁇ , -O(haloR ⁇ ), -CN, -C(O)OH, -C(O)OR ⁇ , -NH 2 , -NHR ⁇ , -NR ⁇ 2 , or -NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 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 substituents on a substitutable nitrogen of an “optionally substituted” group include is independently hydrogen, C 1-6 aliphatic which may be substituted as defined below, 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, notwithstanding the definition above, two independent occurrences of taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of are independently halogen, - R ⁇ , -(haloR ⁇ ), -OH, -OR ⁇ , -O(haloR ⁇ ), -CN, -C(O)OH, -C(O)OR ⁇ , -NH 2 , -NHR ⁇ , -NR ⁇ 2 , or -NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci 4 aliphatic, -CH 2 Ph, -O(CH 2 ) 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.
  • leaving group refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons.
  • suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate.
  • hydrolysable group and “hydrolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions.
  • hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999).
  • organic residue defines a carbon-containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove.
  • Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc.
  • Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
  • an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.
  • a very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared.
  • a 2,4-thiazolidinedione radical in a particular compound has the structure: regardless of whether thiazolidinedione is used to prepare the compound.
  • the radical for example an alkyl
  • the number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary elsewhere herein.
  • Organic radicals contain 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.
  • 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 bound to at least some of the carbon atoms of the organic radical.
  • an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2- naphthyl radical.
  • an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphoms, and the like.
  • organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, di- substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, wherein the terms are defined elsewhere herein.
  • organic radicals that include heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals and the like.
  • a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture.
  • Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers.
  • the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included.
  • the products of such procedures can be a mixture of stereoisomers.
  • a specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called 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 designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula.
  • one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane).
  • the Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.
  • the enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent.
  • a further step can liberate the desired enantiomeric form.
  • specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.
  • Designation of a specific absolute configuration at a chiral carbon in a disclosed compound is understood to mean that the designated enantiomeric form of the compounds can be provided in enantiomeric excess (e.e.).
  • Enantiomeric excess 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%.
  • the designated enantiomer is substantially free from the other enantiomer.
  • the “R : forms of the compounds can be substantially free from the “S” forms of the compounds and are, thus, in enantiomeric excess of the “S” forms.
  • “S” forms of the compounds can be substantially free of “R” forms of the compounds and are, thus, in enantiomeric excess of the “R” forms.
  • a disclosed compound When a disclosed compound has two or more chiral carbons, it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to four optical isomers and two pairs of enantiomers ((S,S)/(R,R) and (R,S)/(S,R)).
  • the pairs of enantiomers e.g., (S,S)/(R,R)
  • the 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, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. Unless otherwise specifically excluded, a disclosed compound includes each diastereoisomer of such compounds and mixtures thereof.
  • the compounds according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug forming moieties.
  • the hydroxymethyl position may form mono-, di-, or triphosphates and again these phosphates can form prodrugs.
  • Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO 2000/041531, p. 30).
  • the nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.
  • “Derivatives” of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof.
  • the “combinations” mentioned in this context refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, and solvates.
  • Examples of radio- actively labeled forms include compounds labeled with tritium, phosphorous-32, iodine- 129, carbon-11, fluorine- 18, and the like.
  • Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance.
  • the disclosed compounds can be isotopically- labeled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F and 36 Cl, respectively.
  • Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
  • Certain isotopically-labeled compounds of the present invention for example those into which radioactive isotopes such as 3 H and 14 C are 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.
  • isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.
  • the compounds described in the invention can be present as a solvate.
  • the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate.
  • the compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution.
  • one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates.
  • the invention includes all such possible solvates.
  • co-crystal means a physical association of two or more molecules which owe their stability through non-covalent interaction.
  • One or more components of this molecular complex provide a stable framework in the crystalline lattice.
  • the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. 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.
  • certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.
  • amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form.
  • pyrazoles can exist in two tautomeric forms, N 1 -unsubstituted, 3-A 3 and N 1 -unsubstituted, 5-A 3 as shown below.
  • the invention includes all such possible tautomers.
  • polymorphic forms or modifications It is known that chemical substances form solids, which are present in different states of order which are termed polymorphic forms or modifications.
  • the different modifications of a polymorphic substance can differ greatly in their physical properties.
  • the compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.
  • a structure of a compound can be represented by a formula: which is understood to be equivalent to a formula: wherein n is typically an integer. That is, R" is understood to represent five independent substituents, R n(a) , R n(b) , R n(c) , R n(d) , R n(e) .
  • independent substituents it is meant that each R substituent can be independently defined. For example, if in one instance R n(a) is halogen, then R n(b) is not necessarily halogen in that instance.
  • Certain 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.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
  • compositions of the invention Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary.
  • o is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; wherein p is 1 or 2; wherein t is an integer from 0 to 500; wherein v is 1, 2, 3, 4, or 5; wherein A is S or Se; wherein R 1 is selected from -CO 2 H, - C(O)NHOH, -SO 2 NH 2 , -SO 2 NHC(O)CH 3 , -SO 3 H, -NHC(O)NHSO 2 CH 3 , -P(O)(OH) 2 , and a structure selected from:
  • R 4 is selected from hydrogen and methyl; wherein each occurrence of R 5 and R 5 , when present, is independently a residue of a side chain of amino acid; wherein each occurrence of R 6 and R 6 ’, when present, is independently selected from hydrogen and methyl, or wherein R 6 or R 6 ’ is covalently bonded to R 5 or R 5 ’, respectively, and, together with the intermediate atoms, comprise an unsubstituted 5-membered heterocycle; wherein each of R 7a and R 7b , when present, is independently selected from hydrogen and C1-C4 alkyl; and wherein R 8 is selected from hydrogen and methyl, provided that the compound is not PapA.
  • R 4 is selected from hydrogen and methyl; wherein each occurrence of R 5 and R 5 , when present, is independently a residue of a side chain of amino acid; wherein each occurrence of R 6 and R 6 ’, when present, is independently selected from hydrogen and methyl, or wherein R 6 or R 6 ’ is covalently bonded to R 5 or R 5 ’, respectively, and, together with the intermediate atoms, comprise an unsubstituted 5-membered heterocycle; wherein each of R 7a and R 7b , when present, is independently selected from hydrogen and C1-C4 alkyl; and wherein R 8 is selected from hydrogen and methyl, provided that the compound is not PapA.
  • R 2 is a residue of a side chain of amino acid, provided that the amino acid is not isoleucine or threonine; wherein each of R 3a and R 3b , when present, is independently selected from C2-C5 alkynyl, C1-C5 azido, and a residue of a side chain of an amino acid; wherein R 4 is selected from hydrogen and methyl; wherein each occurrence of R 5 and R 5 , when present, is independently a residue of a side chain of amino acid; wherein each occurrence of R 6 and R 6 ’, when present, is independently selected from hydrogen and methyl, or wherein R 6 or R 6 is covalently bonded to R 5 or R 5 , respectively, and, together with the intermediate atoms, comprise an unsubstituted 5-membered heterocycle; wherein each of R 7a and R 7b , when present, is independently selected from hydrogen and C1-C4 alkyl, provided that the compound is not PapA.
  • o is independently 0, 1, 2, 3, 4, 5, 6, or 7.
  • t 0.
  • v is 1 or 2.
  • R 1 is -CO 2 H or a structure:
  • R 1 is -CO 2 H.
  • the cleavable moiety is -CO 2 -(C4-C8 alkylene)-OC(O)-.
  • the cleavable moiety is a protease recognition sequence.
  • the protease recognition sequence is TEV recognition sequence.
  • the compound comprises one or more D-amino acid residues. In a further aspect, the compound comprises one or more ⁇ -amino acid residues. In a still further aspect, the compound comprises one or more N-methylated amino acid residues.
  • PapB installs a single thioether linkage in the compound.
  • PapB installs two or more thioether linkages in the compound.
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • n is 0. In a further aspect, m is 1.
  • n is 0. In a further aspect, n is 1.
  • o is 0, 1, 2, 3, 4, 5, 6, or 7. In a further aspect, o is 1, 2, 3,
  • o 1, 2, 3, or 4.
  • p is 1. In a further aspect, p is 2.
  • A is S. In a further aspect, A is Se.
  • L is C2-C4 alkyl. In a further aspect, L is -(C1-C4 alkyl)(OCH 2 CH 2 ) q . In a still further aspect, L is a structure selected from:
  • the cleavable moiety is a protease recognition sequence.
  • the protease recognition sequence is a TEV protease recognition sequence.
  • the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), in which X is any amino acid and Z is any amino acid that contains a hydrophobic residue.
  • the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).
  • the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3).
  • the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2).
  • R 1 is selected from -CO 2 H and a structure:
  • R 1 is CO 2 H.
  • R 2 is a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • R 2 is a residue of a side chain of an amino acid selected from alanine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, and glycine.
  • one of R 3a and R 3b when present, is hydrogen, and one of R 3a and R 3b , when present, is a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • R 4 is hydrogen. In a further aspect, R 4 is methyl.
  • each occurrence of R 5 when present, is independently a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • each occurrence of R 6 when present, is hydrogen. In a further aspect, each occurrence of R 6 , when present, is methyl. [00240] In various aspects, each of R 7a and R 7b , when present, is hydrogen. In a further aspect, each of R 7a and R 7b , when present, is methyl.
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.
  • one of R 3a and R 3b when present, is hydrogen, and one of R 3a and R 3b , when present, is a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula: [00252] In various aspects, the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.
  • the compound has a structure represented by a formula: wherein r is 2, 3, or 4.
  • the compound has a structure represented by a formula: wherein s is 1 or 2.
  • the compound has a structure represented by a formula: c. THIOETHER COMPOUNDS
  • thioether compounds produced by a disclosed method.
  • the method produces a thioether compound having a structure represented by a formula: wherein v’ is 0, 1, 2, or 3.
  • the method further comprises addition of a reducing agent.
  • the method further comprises addition of a protease.
  • the method produces a thioether compound having a structure represented by a formula: wherein v’ is 0, 1, 2, or 3.
  • the thioether compound is selected from:
  • the method produces a thioether compound having a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula: [00266] In various aspects, the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound is a sactipeptide.
  • the sactipeptide has a structure represented by a formula selected from:
  • the thioether compound is a ranthipeptide.
  • the ranthipeptide has a structure represented by a formula selected from:
  • the method produces a thioether compound having a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound is a sactipeptide.
  • the sactipeptide has a structure represented by a formula selected from:
  • the thioether compound is a ranthipeptide.
  • the ranthipeptide has a structure represented by a formula selected from:
  • the thioether compound is selected from:
  • the thioether compound is selected from:
  • thioether compounds prepared by a disclosed method, wherein the thioether compound is an analog of a peptide therapeutic.
  • exemplary peptide therapeutics include, but are not limited to, octreotide, setmalanotide, romidepsin, bremelanotide, pramlintide, oxytocin, setmelanotide, or cyclosporin.
  • R 2 is a residue of a side chain of amino acid, provided that the amino acid is not isoleucine or threonine; wherein each of R 3a and R 3b , when present, is independently selected from C2-C5 alkynyl, C1-C5 azido, and a residue of a side chain of an amino acid; wherein R 4 is selected from hydrogen and methyl; wherein each occurrence of R 5 and R 5 , when present, is independently a residue of a side chain of amino acid; wherein each occurrence of R 6 and R 6 , when present, is independently selected from hydrogen and methyl, or wherein R 6 or R 6 is covalently bonded to R 5 or R 5 , respectively, and, together with the intermediate atoms, comprise an unsubstituted 5-membered heterocycle; wherein each of R 7a and R 7b , when present, is independently selected from hydrogen and C1-C4 alkyl, provided that the compound is not PapA.
  • o is independently 0, 1, 2, 3, 4, 5, 6, or 7.
  • t is 0.
  • v is 1 or 2.
  • R 1 is -CO 2 H or a structure:
  • R 1 is -CO 2 H.
  • the cleavable moiety is a chemically cleavable moiety.
  • Exemplary chemical cleavable moieties include, but are not limited to, -CO 2 -(C4-C8 alkylene)-OC(O)-.
  • the cleavable moiety is an enzymatically cleavable moiety such as, for example, a protease recognition sequence.
  • the protease recognition sequence is TEV recognition sequence.
  • the compound comprises one or more D-amino acid residues. In a further aspect, the compound comprises one or more ⁇ -amino acid residues. In a still further aspect, the compound comprises one or more N-methylated amino acid residues.
  • PapB installs a single thioether linkage in the compound.
  • PapB installs two or more thioether linkages in the compound.
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the method produces a thioether compound having a structure represented by a formula: wherein v’ is 0, 1, 2, or 3.
  • the method further comprises addition of a reducing agent.
  • the method further comprises addition of a protease.
  • the method produces a thioether compound having a structure represented by a formula: wherein v’ is 0, 1, 2, or 3.
  • the thioether compound is selected from:
  • n is 0. In a further aspect, m is 1.
  • n is 0. In a further aspect, n is 1.
  • o is 0, 1, 2, 3, 4, 5, 6, or 7. In a further aspect, o is 1, 2, 3,
  • o is 1, 2, 3, or 4.
  • p is 1. In a further aspect, p is 2.
  • A is S. In a further aspect, A is Se.
  • L is C2-C4 alkyl. In a further aspect, L is -(C1-C4 alkyl)(OCH 2 CH 2 ) q . In a still further aspect, L is a structure selected from:
  • the cleavable moiety is a protease recognition sequence.
  • the protease recognition sequence is a TEV protease recognition sequence.
  • the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), in which X is any amino acid and Z is any amino acid that contains a hydrophobic residue.
  • the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).
  • the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3).
  • the leader sequence is LKQINVIAGVKEPIRAYG (SEQ ID NO: 2).
  • R 1 is selected from -CO 2 H and a structure:
  • R 1 is -CO 2 H.
  • R 2 is a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • R 2 is a residue of a side chain of an amino acid selected from alanine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, and glycine.
  • one of R 3a and R 3b when present, is hydrogen, and one of R 3a and R 3b , when present, is a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • R 4 is hydrogen. In a further aspect, R 4 is methyl.
  • each occurrence of R 5 when present, is independently a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • each occurrence of R 6 when present, is hydrogen. In a further aspect, each occurrence of R 6 , when present, is methyl.
  • each of R 7a and R 7b when present, is hydrogen. In a further aspect, each of R 7a and R 7b , when present, is methyl.
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.
  • one of R 3a and R 3b when present, is hydrogen, and one of R 3a and R 3b , when present, is a residue of a side chain of an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • an amino acid selected from alanine, valine, leucine, serine, cysteine, methionine, arginine, lysine, asparagine, glycine, phenylalanine, tyrosine, and tryptophan.
  • the compound has a structure represented by a formula: [00343] In various aspects, the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • the compound has a structure represented by a formula:
  • o is 1, 2, 3, 4, 5, 6, 7, 8, or 9.
  • the compound has a structure represented by a formula: wherein r is 2, 3, or 4.
  • the compound has a structure represented by a formula: wherein s is 1 or 2. [00348] In various aspects, the compound has a structure represented by a formula:
  • PapB installs a single thioether linkage in the compound. In a further aspect, PapB installs two or more thioether linkages in the compound.
  • the method produces a thioether compound having a structure represented by a formula:
  • the thioether compound has a structure represented by a formula: [00352] In various aspects, the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound is a sactipeptide.
  • the sactipeptide has a structure represented by a formula selected from:
  • the thioether compound is a ranthipeptide.
  • the ranthipeptide has a structure represented by a formula selected from: [00366]
  • the method further comprises addition of a reducing agent.
  • the reducing agent comprises dithionite, flavodoxin, flavodoxin reductase, titanium citrate, reduced nicotinamide adenine dinucleotide phosphate, or any combination thereof.
  • the method further comprises addition of a protease.
  • the protease is TEV protease.
  • the method produces a thioether compound having a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound has a structure represented by a formula selected from:
  • the thioether compound is a sactipeptide.
  • the sactipeptide has a structure represented by a formula selected from:
  • the thioether compound is a ranthipeptide.
  • the ranthipeptide has a structure represented by a formula selected from:
  • the thioether compound is selected from:
  • the thioether compound is selected from:
  • the peptide sequence comprises octreotide or vapreotide. In a yet further aspect, the peptide sequence comprises octreotide. In a yet further aspect, the peptide sequence comprises vapreotide.
  • the peptide sequence comprises D FCF D WKTET (SEQ ID NO: 3), wherein the first and fourth positions are D-amino acids. [00391] In a further aspect, the peptide sequence comprises FCFAKTETA.
  • the peptide sequence further comprises a leader sequence of LKQINVIAGVKEPIRAYG (SEQ ID NO: 2) or LKQINVIAGVKPIRAYG (SEQ ID NO: 3). In a further aspect, the peptide sequence further comprises a leader sequence of LKQINVIAGVKEPIRAYG (SEQ ID NO: 3).
  • the peptide sequence further comprises a TEV protease recognition sequence.
  • the TEV protease recognition sequence is EXLYZQ (SEQ ID NO: 1), in which X is any amino acid and Z is any amino acid that contains a hydrophobic residue.
  • the TEV protease recognition sequence is ENLYFQ (SEQ ID NO: 1).
  • the peptide sequence comprises one or more D-amino acid residues.
  • the peptide sequence comprises one or more ⁇ -amino acid residues.
  • the peptide sequence comprises one or more N-methylated amino acids.
  • the modified PapA sequence comprises minimal substrate PapA.
  • 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).
  • the modified PapA sequence is LKQINVIAGVKEPIRAYGCDSNNAANA (SEQ ID NO: 6). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSDNNAAA (SEQ ID NO: 7). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSNDAA A (SEQ ID NO: 8). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSAANDA (SEQ ID NO: 9). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGCSAAANDA (SEQ ID NO: 10).
  • the modified PapA sequence is LKQINVIAGVKEPIRAYGCSAAAANDA (SEQ ID NO: 11).
  • 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).
  • the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSANDA (SEQ ID NO: 12). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSANDACSANDA (SEQ ID NO: 13). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSACDAADA (SEQ ID NO: 14). In a still further aspect, the modified PapA sequence is LKQINVIAGVKEPIRAYGAAAASACDAADA (SEQ ID NO: 15).
  • the modified PapA sequence is LKQINVIAGVKEPIRAYGAAACSAADAAADA (SEQ ID NO: 16). [00401] In a further aspect, the modified PapA sequence comprises one or more D- amino acid residues.
  • the modified PapA sequence comprises one or more ⁇ - amino acid residues.
  • the modified PapA sequence comprises one or more N- methylated amino acid residues.
  • X is a penicillamine or an amino acid residue comprising a -SH group or an amino acid residue comprising a -SeH group.
  • X is a penicillamine.
  • X is an amino acid residue comprising a -SH group.
  • X is cysteine, homocysteine, D-cysteine, or D-homocysteine.
  • X is homocysteine.
  • X is D-cysteine.
  • X is D-homocysteine.
  • X is cysteine.
  • X is an amino acid residue comprising a -SeH group.
  • X is selenocysteine or homoselenocysteine.
  • X is selenocysteine.
  • X is homoselenocysteine.
  • amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, ⁇ -amino acid residues, and N-methylated amino acid residues.
  • Natural amino acid residues may include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
  • Unnatural amino acid residues may include, but are not limited to, p- ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1 ,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine, O-(2- nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m- cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino- 1 -hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-
  • Y n comprises one or more D-amino acids.
  • Y n comprises one or more ⁇ -amino acids.
  • Y n comprises one or more N-methylated amino acids.
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. In a further aspect, n is 0, 1,
  • n is 0, 1, 2, 3, 4, 5, 6, 7, or 8.
  • n is 0, 1, 2, 3, 4, 5, 6, or 7.
  • n is 0, 1, 2, 3, 4, 5, or 6.
  • n is 0, 1, 2, 3, 4, or 5.
  • n is 0, 1, 2, 3, or 4.
  • n is 0, 1, 2, or 3.
  • n is 0, 1, or 2.
  • n is 0 or 1.
  • n is 0.
  • n is 1.
  • n is 2.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • n is 7.
  • n is 8.
  • n is 9. c. Z GROUPS
  • 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.
  • Z is aspartic acid or glutamic acid. In a yet further aspect, Z is aspartic acid. In a yet further aspect, Z is glutamic acid.
  • Z is a hydroxy-glutamic acid residue.
  • Z is 2-amino-3-(2H-tetrazol-5-yl)propanoic acid.
  • Z is a a carboxyl-functionalized amino acid residue.
  • 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.
  • (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
  • amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, ⁇ -amino acid residues, and N-methylated amino acid residues.
  • Natural amino acid residues may include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
  • Unnatural amino acid residues may include, but are not limited to, p- ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1 ,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine, O-(2- nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m- cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino-
  • Y a comprises one or more D-amino acids.
  • Y a comprises one or more ⁇ -amino acids.
  • Y a comprises one or more N-methylated amino acids.
  • a is 0, 1, 2, 3, 4, 5, 6, or 7. In a further aspect, a is 0, 1, 2, 3,
  • a is 0, 1, 2, 3, 4, or 5. In a still further aspect, a is 0, 1, 2, 3, or 4. In a still further aspect, a is 0, 1, 2, or 3. In a still further aspect, a is 0, 1, or 2. In a yet further aspect, a is 0 or 1. In a yet further aspect, a is 0. In a yet further aspect, a is 1. In a yet further aspect, a is 2. In a yet further aspect, a is 3. In a yet further aspect, a is 4. In a yet further aspect, a is 5. In a yet further aspect, a is 6. In a yet further aspect, a is 7. e. Y B GROUPS
  • amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, ⁇ -amino acid residues, and N-methylated amino acid residues.
  • Natural amino acid residues may include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
  • Unnatural amino acid residues may include, but are not limited to, p- ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1 ,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine, O-(2- nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m- cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino-
  • Yb comprises one or more D-amino acids.
  • Yb comprises one or more ⁇ -amino acids.
  • Yb comprises one or more N-methylated amino acids.
  • b is 0, 1, 2, 3, 4, 5, 6, or 7. In a further aspect, b is 0, 1, 2,
  • b is 0, 1, 2, 3, 4, or 5. In a still further aspect, b is 0, 1, 2, 3, or 4. In a still further aspect, b is 0, 1, 2, or 3. In a still further aspect, b is 0, 1, or 2. In a yet further aspect, b is 0 or 1. In a yet further aspect, b is 0. In a yet further aspect, b is 1. In a yet further aspect, b is 2. In a yet further aspect, b is 3. In a yet further aspect, b is 4. In a yet further aspect, b is 5. In a yet further aspect, b is 6. In a yet further aspect, b is 7. f. Y x GROUPS
  • amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, ⁇ -amino acid residues, and N-methylated amino acid residues.
  • Natural amino acid residues may include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
  • Unnatural amino acid residues may include, but are not limited to, p- ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1 ,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine, O-(2- nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m- cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino-
  • Y x comprises one or more D-amino acids.
  • Y x comprises one or more ⁇ -amino acids.
  • Y x comprises one or more N-methylated amino acids.
  • x is 0, 1, 2, 3, 4, 5, or 6. In a still further aspect, x is 0, 1, 2,
  • x is 0, 1, 2, 3, or 4. In a still further aspect, x is 0, 1, 2, or 3. In a still further aspect, x is 0, 1, or 2. In a yet further aspect, x is 0 or 1. In a yet further aspect, x is 0. In a yet further aspect, x is 1. In a yet further aspect, x is 2. In a yet further aspect, x is 3. In a yet further aspect, x is 4. In a yet further aspect, x is 5. In a yet further aspect, x is 6. g. Y Y GROUPS
  • amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, ⁇ -amino acid residues, and N-methylated amino acid residues.
  • Natural amino acid residues may include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
  • Unnatural amino acid residues may include, but are not limited to, p- ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1 ,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine, O-(2- nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m- cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino-
  • Y y comprises one or more D-amino acids.
  • Y y comprises one or more ⁇ -amino acids.
  • Y y comprises one or more N-methylated amino acids.
  • y is 0, 1, 2, 3, 4, 5, 6, 7, or 8. In a still further aspect, y is 0,
  • y is 0, 1, 2, 3, 4, 5, 6, or 7.
  • y is 0, 1, 2, 3, 4, 5, or 6.
  • y is 0, 1, 2, 3, 4, or 5.
  • y is 0, 1, 2, 3, or 4.
  • y is 0, 1, 2, or 3.
  • y is 0, 1, or 2.
  • y is 0 or 1.
  • y is 0.
  • y is 1.
  • y is 2.
  • y is 3.
  • y is 4.
  • y is 5.
  • y is 6.
  • y is 7.
  • y is 8. h. Y z GROUPS
  • amino acid residues include, but are not limited to, natural amino acid residues, unnatural amino acid residues, D-amino acid residues, ⁇ -amino acid residues, and N-methylated amino acid residues.
  • Natural amino acid residues may include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
  • Unnatural amino acid residues may include, but are not limited to, p- ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, 1 ,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine, O-(2- nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m- cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino- 1 -hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-
  • Y z comprises one or more D-amino acids.
  • Y z comprises one or more ⁇ -amino acids.
  • Y z comprises one or more N-methylated amino acids.
  • z is 0, 1, 2, 3, 4, 5, 6, or 7. In a further aspect, z is 0, 1, 2, 3,
  • z is 0, 1, 2, 3, 4, or 5. In a still further aspect, z is 0, 1, 2, 3, or 4. In a still further aspect, z is 0, 1, 2, or 3. In a still further aspect, z is 0, 1, or 2. In a yet further aspect, z is 0 or 1. In a yet further aspect, z is 0. In a yet further aspect, z is 1. In a yet further aspect, z is 2. In a yet further aspect, z is 3. In a yet further aspect, z is 4. In a yet further aspect, z is 5. In a yet further aspect, z is 6. In a yet further aspect, z is 7.
  • each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed compound can be provided by the disclosed methods.
  • the invention relates to product compounds having a structure selected from:
  • the compound is:
  • the compound is:
  • the compounds of this invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein.
  • Preferred methods include, but are not limited to, those described below.
  • the invention relates to methods of chemically modifying a peptide sequence to install a thioether linkage, the method comprising reacting the peptide substrate with PapB.
  • 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 full peptide sequence by PapB. However, the leader sequence is no