WO2016090305A1 - Synthèse en phase solide de peptides contenant des déhydroamino acides volumineux - Google Patents

Synthèse en phase solide de peptides contenant des déhydroamino acides volumineux Download PDF

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WO2016090305A1
WO2016090305A1 PCT/US2015/064091 US2015064091W WO2016090305A1 WO 2016090305 A1 WO2016090305 A1 WO 2016090305A1 US 2015064091 W US2015064091 W US 2015064091W WO 2016090305 A1 WO2016090305 A1 WO 2016090305A1
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peptide
synthetic peptide
amino acid
cio
formula
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PCT/US2015/064091
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Steven L. CASTLE
Jintao JIANG
Ankur JALAN
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Castle Steven L
Jiang Jintao
Jalan Ankur
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Publication of WO2016090305A1 publication Critical patent/WO2016090305A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention described herein relates to methods of incorporating bulky ⁇ , ⁇ -dehydroamino acids into peptides and those peptides.
  • the method involves using novel azlactone intermediates for coupling with amino acids. These methods are compatible with standard peptide synthesis methods, such as solid- phase peptide synthesis. Further, the resulting peptides and peptide compositions are reported herein. 2. Background Information
  • a method has been devised for incorporating bulky tetrasubstituted dehydroamino acids such as dehydrovaline and dehydroethylnorvaline into peptides via solid-phase peptide synthesis.
  • the method involves preparation of azlactone dipeptides containing the dehydroamino acid of choice, then subjecting the azlactones to ring opening reactions using resin-bound peptides as the nucleophiles.
  • One embodiment described herein is a synthetic peptide, including at least one dehydroamino acid, wherein the at least one dehydroamino acid is of Formula I:
  • B 1 and B 2 are each independently selected from hydrogen, C C 10 alkyl, Ci-Cio alkylene, Ci-Ci 0 alkanol, Ci-Ci 0 carboxylic, Ci-Ci 0 fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen.
  • the peptide has a molecular weight of from about 0.1 kDa to about 250 kDa.
  • B 1 and B 2 are each independently selected from C C 10 alkyl and Ci-Cio perfluoroalkyl.
  • B 1 and B 2 are different.
  • B 1 and B 2 are the same.
  • aromatic is phenyl and heterocyclic is selected from indole, pyrrole, azole and pyrrolidine.
  • Another embodiment described herein is a synthetic peptide, including one or more peptide subunits according to Formula II:
  • B 1 and B 2 are defined as for Formula I above and R 1 is hydrogen or naturally occurring proteinogenic amino acid side chain; n is integer greater than or equal to 1 ; and m is an integer greater than or equal to 1.
  • Another embodiment described herein is a synthetic peptide including a structure according to Formula III:
  • B 1 , B 2 , R 1 , n, and m are defined as for Formulas I and II above and wherein Y 2 includes at least one or more natural or non-natural amino acids; and X is an amino acid protecting group. In one aspect, Y 2 further includes one or more dehydroamino acids according to Formula I.
  • the amino acid protecting group of X is selected from the group consisting of Alloc, Fmoc, Cbz, Boc, Ddz, Bpoc, Nps, Nsc, Bsmoc, Trt, ivDde, Tcp, Pms, Esc, Sps, o bs, d bs, Bts, Troc, Dts, pNZ, Poc, oNZ, NVOC, NPPOC, Mnppoc, BrPhF, Azoc, Hfa, Phdec, Pydec, 2-Cl-Trt, Dmb, 2-Ph-Pr, Phenyl-Edotn, Fm, Dmab, Cam, Allyl, Bn, Pac, pNB, Tmse, Ptmse, Tmsi, Tee, cHx, Men, Mpe, Tegbz, Phth,
  • the amino acid protecting group is Alloc.
  • B 1 , B 2 , R 1 , n, and m are defined as for Formulas I, II, and III above, and wherein Y 1 and Y 2 are each a peptide chain including at least one or more natural or non-natural amino acids.
  • Y 1 and Y 2 or Y 1 or Y 2 optionally include one or more additional dehydroamino acids according to the structure of Formula I above.
  • B 1 and B 2 are both ethyl or B 1 and B 2 are both methyl.
  • R 1 is hydrogen or the side chain of phenylalanine.
  • Another embodiment described herein is a pharmaceutical composition including a synthetic peptide as described herein and a
  • X is selected from the group consisting of Alloc, Fmoc, Cbz, Boc, Ddz, Bpoc, Nps, Nsc, Bsmoc, Trt, ivDde, Tcp, Pms, Esc, Sps, oNbs, dNbs, Bts, Troc, Dts, pNZ, Poc, oNZ, NVOC, NPPOC, Mnppoc, BrPhF, Azoc, Hfa, Phdec, Pydec, 2-Cl-Trt, Dmb, 2-Ph-Pr, Phenyl-Edotn, Fm, Dmab, Cam, Allyl, Bn, Pac, pNB, Tmse, Ptmse, Tmsi, Tee, cHx, Men, Mpe, Tegbz, Phth, DBS, and triazone;
  • R 1 is hydrogen or any naturally occurring proteinogenic amino acid side
  • B 1 and B 2 are each independently selected from hydrogen, C C 10 alkyl, C C 10 alkylene, C C 10 alkanol, C C 10 carboxylic, C C 10 fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen; and
  • L is a leaving group selected from hydroxy, iodo, bromo, chloro and sulfonate
  • n is an integer greater than or equal to 1.
  • Another embodiment described herein is a synthetic peptide precursor including one or more azlactones according to Formula VI:
  • R 1 is hydrogen or any naturally occurring proteinogenic amino acid side chain
  • B 1 and B 2 are each independently selected from hydrogen, C C 10 alkyl, Ci-Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen; and
  • X is an amino acid protecting group selected from the group consisting of Alloc, Fmoc, Cbz, Boc, Ddz, Bpoc, Nps, Nsc, Bsmoc, Trt, ivDde, Tcp, Pms, Esc, Sps, oNbs, dNbs, Bts, Troc, Dts, pNZ, Poc, oNZ, NVOC, NPPOC, Mnppoc, BrPhF, Azoc, Hfa, Phdec, Pydec, 2-Cl-Trt, Dmb, 2-Ph-Pr, Phenyl- Edotn, Fm, Dmab, Cam, Allyl, Bn, Pac, pNB, Tmse, Ptmse, Tmsi, Tee, cHx, Men, Mpe, Tegbz, Phth, DBS, and triazone.
  • Another embodiment described herein is a method of making a synthetic peptide, including: (a) providing a substrate bound peptide having a free amine; (b) contacting the substrate bound peptide with an azlactone of Formula VI to form a reaction mixture; and (c) heating the reaction mixture.
  • the further includes isolating a dehydroamino acid linked substrate bound peptide.
  • the reaction mixture of (c) is heated to a temperature of about 40 °C to about 80 °C for about 10 minutes to about 24 hours.
  • the method further includes cleaving the amino acid protecting group X.
  • the method further includes subjecting the isolated dehydroamino acid linked substrate bound peptide to an in vitro peptide synthesis method including a solid phase peptide synthesis, a solution phase peptide synthesis, or a solid phase solution phase hybride peptide synthesis method to form an elongated synthetic peptide linked to the substrate.
  • the method further includes cleaving the elongated synthetic peptide from the substrate.
  • Another embodiment described herein is a method of increasing the stability of a synthetic peptide that includes substituting at least one amino acid of the synthetic peptide for at least one dehydro amino acid according to the Formula I.
  • the increased stability includes an increased half-life of the synthetic peptide.
  • the synthetic peptide includes at least one ⁇ -turn motif and the increased stability includes an increased stability of the ⁇ -turn motif of the synthetic peptide that includes the at least one ⁇ -turn motif.
  • FIG. 1 Illustration of the differences in dehydroamino acids having one or two ⁇ -carbon substituted groups.
  • FIG. 2 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 1a; chromatogram for crude 1 1a (Discovery C18 Column (25 cm x 4.6 mm, 5 ⁇ ), 10-60% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 50 min, 1 mL/min).
  • FIG. 3 HPLC chromatogram of elongated synthetic peptide
  • FIG. 4 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 lb; chromatogram for crude 1 lb (Discovery CI 8 Column (25 cm x 4.6 mm, 5 ⁇ ), 10-60% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 50 min, 1 mL/min).
  • FIG. 5 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 lb; chromatogram for purified 1 lb (Discovery CI 8
  • FIG. 6 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 lc; crude 1 lc (Discovery C18 Column (25 cm x 4.6 mm, 5 ⁇ ), 10-60% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 50 min, 1 mL/min).
  • FIG. 7 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 lc; purified 1 lc (Discovery CI 8 Column (25 cm x 4.6 mm, 5 ⁇ ), 10-60% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 50 min, 1 mL/min).
  • FIG. 8 HPLC chromatogram of elongated synthetic peptide corresponding to structure l id; crude 1 Id (Discovery C18 Column (25 cm x 4.6 mm, 5 ⁇ ), 10-60% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 50 min, 1 mL/min).
  • FIG. 9 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 Id; purified l id (Discovery C18 Column (25 cm x 4.6 mm, 5 ⁇ ), 10-60% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 50 min, 1 mL/min).
  • FIG. 10 Stabilization and reduction in proteolytic cleavage of ⁇ -turn containing synthetic peptides containing at least one dehydroamino acid.
  • dehydroamino acid or " ⁇ ” as used herein refers to any amino acid that has lost two vicinal hydrogen atoms to form a double bond.
  • these terms include ⁇ , ⁇ -dehydroamino acids, which refer to a-amino acids having a double bond between the a-carbon and the ⁇ -carbon of the amino acid.
  • the ⁇ -carbon of these amino acids are singly substituted with a substituent group or doubly substituted with a suitable substituent group.
  • the ⁇ -carbon of the amino acid having two substituted groups may be symmetrically substituted (i.e., the same) or asymmetrically substituted (i.e., different).
  • amino acid refers to compounds containing an amine (-NH 2 ) and a carboxylic acid (-COOH) group.
  • Amino acids described herein also typically contain a side-chain or "R-group" which in some instances is a hydrogen atom such as is present in the amino acid glycine.
  • R-group which in some instances is a hydrogen atom such as is present in the amino acid glycine.
  • amino acid is intended to include both the L-stereoisomer and the D- stereoisomer.
  • proteinogenic amino acid or "natural amino acid” as used herein refers to a-amino acids, wherein the amino acid R-group is attached to the a- carbon of the amino acid having a general formula of H 2 NCH(R)C(0)OH.
  • proteinogenic amino acid is intended to include both the L- stereoisomer and the D -stereoisomer.
  • non-natural amino acid refers to any non- protein coding or proteinogenic amino acid known in the art. These amino acids may be any alpha (a-), beta ( ⁇ -), gamma ( ⁇ -) or delta ( ⁇ -) amino acid and include any stereoisomer thereof.
  • alkyl refers to a straight or branched chain hydrocarbon containing from 1 to 10, 20, or 30 or more carbon atoms.
  • C n -C n+m refers to the number of carbons as a straight or branched alkyl chain, wherein n and m are integers greater than 1.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec -butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.
  • alkyl is intended to include substituted halo alkyls, such as
  • alkenyl or "alkylene” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10, 20, or 30 or more carbon atoms, which include 1 to 4, 5 or 6 or more double bonds in the normal chain.
  • alkenyl include, but are not limited to ethylene, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like.
  • alkenyl or “alkylene” is intended to include both substituted and unsubstituted alkeynyl or alkylene unless otherwise indicated.
  • alkynyl refers to a straight or branched chain hydrocarbon containing from 1 to 10, 20, 30 or 40 or more carbon atoms (or in lower alkynyl 1 to 4 carbon atoms) which include 1, 2, or 3 or more triple bonds in the normal chain.
  • Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2- butynyl, 4- pentynyl, 3-pentynyl, and the like.
  • aryl or “aromatic” as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings.
  • Representative examples of aryl include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • aryl or “aromatic” is intended to include both substituted and unsubstituted aryl or aromatic unless otherwise indicated.
  • cyclic refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below).
  • Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo.
  • cycloalkyl is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.
  • heterocyclic refers to an aliphatic (e.g., fully or partially saturated heterocyclic) or aromatic (e.g., heteroaryl) monocyclic- or bicyclic-ring system.
  • Monocyclic ring systems are exemplified by any 3, 4, 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms (i.e., other than a carbon atom) independently selected from oxygen, nitrogen and sulfur.
  • the 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Therefore the term "heterocyclic” as used herein also encompasses heteroaromatic and heteroaryl groups.
  • monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine,
  • Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl or cyclic group as defined herein, or another monocyclic ring system as defined herein.
  • bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline,
  • heterocyclic is a heteroaryl or heteroaromatic group such as azole, indole, and pyrrole.
  • alkoxy refers to an alkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -0-.
  • alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
  • alkoxy groups when part of a more complex molecule, include an alkoxy substituent attached to an alkyl via an ether linkage.
  • leaving group refers to any molecular fragment, which leaves with a loan pair of electrons.
  • the leaving group is a hydroxide, which undergoes an elimination dehydration reaction to form alkene containing amino acids (dehydroamino acid) that are described further herein.
  • halo or halogen as used herein refers to any suitable halogen, including -F, -CI, -Br, and -I.
  • amino as used herein means the radical -NH 2 .
  • amide as used herein alone or as part of another group refers to a -C(0)NR a R b radical, where R a and R b are independently any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • carboxylic or carboxylate as used herein alone or as part of another group refers to a -C(0)0 " or -C(0)OH radical.
  • ureido as used herein alone or as part of another group refers to an R a NC(0)NRb radical, where Ra and Rb are any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • AAAs ⁇ , ⁇ -Dehydroamino acids
  • AAAs ⁇ , ⁇ -Dehydroamino acids
  • these dehydroamino acids enhance the proteolytic stability of peptides that contain them, by altering the shape of the backbone and by stabilizing folded states through their rigidifying effect.
  • the conformational preferences of AAAs that contain at least one hydrogen atom at the ⁇ carbon e.g., AAla, AAbu, APhe
  • AAla, AAbu, APhe have been studied, and rules of their inclusion in natural peptide secondary structures have been established.
  • AEnv dehydroethylnorvaline
  • some embodiments described herein include dehydroamino acid - substituted synthetic peptides.
  • the dehydro amino acid substituted synthetic peptides include at least one dehydroamino acid.
  • the synthetic peptides having at least one dehydro amino acid as described herein do not include naturally occurring peptide, peptide fragment, or protein amino acid sequences.
  • the synthetic peptides include at least one naturally occurring dehydro amino acid as described herein, but do not include a 100% homologous naturally occurring peptide, peptide fragment, or protein amino acid sequence flanking the at least one dehydro amino acid.
  • the synthetic peptides have less than 90%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence.
  • the synthetic peptides have less than 80%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence.
  • the synthetic peptides have less than 70%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence.
  • the synthetic peptides have less than 50%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence. In some aspects, the synthetic peptides have less than 30%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence.
  • synthetic peptides having at least one dehydro amino acid described herein include a naturally occurring and non-naturally occurring peptide amino acid sequence.
  • the synthetic peptides include a portion or a fragment of a naturally occurring peptide or protein amino acid sequence, wherein a dehydro amino acid is part of the naturally occurring sequence.
  • the synthetic peptides include a synthetic or non-natural occurring amino acid sequence, wherein a dehydro amino acid is part of the synthetic or non-naturally occurring amino acid sequence.
  • the synthetic peptides of these embodiments include naturally occurring amino acid sequences and naturally occurring dehydro amino acids but because they include synthetic non-naturally occurring amino acid sequences these peptides are not naturally occurring.
  • the dehydroamino acid includes a structure according to Formula I below:
  • B 1 is selected from hydrogen, Ci-Cio alkyl, Ci-Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen; and
  • B 2 is selected from hydrogen, Ci-Cio alkyl, Ci-Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen.
  • the dehydroamino acid of Formula I may be present at the C-terminus, N-terminus, or at a location that is anywhere in between the C-terminus or N- terminus of the synthetic peptides described herein.
  • the synthetic peptides described herein may have more than one dehydroamino acid according to Formula I.
  • the synthetic peptides described herein may have at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or even at least 50 dehydroamino acids of Formula I.
  • B 1 and B 2 described above is the same or B 1 and B 2 are different.
  • the aromatic group for B 1 and/or B 2 is phenyl and the heterocyclic group for B 1 and/or B 2 is selected from indole, pyrrole, azole and pyrrolidine.
  • B 1 and B 2 are selected from Ci-Cio alkyl and Ci-Cio perfluoroalkyl.
  • B 1 and B 2 are both methyl.
  • B 1 and B 2 are both ethyl.
  • the synthetic peptides include any naturally occurring dehydroamino acid.
  • exemplary and non-limiting naturally occurring dehydroamino acids include dehydroalanine, (Z)-dehydrobutyrine, (E)- dehydrobutyrine, dehydrovaline, (E)-dehydroisoleucine, (E)-dehydroaspartic acid, (Z)-dehydrotryptophan, (E)-dehydrotryptophan, (E)-chlorodehydroalanine, (Z)- ureidodehydroalanine, (Z)-aminodehydroalanine, (Z)-3,4,5-trihydroxy- dehydrophenylalanine, 2-amino-3-hydroxymethyl-4,5-epoxy-dehydropentanoic acid, N-methyldehydroalanine, (Z)-N-methyldehydro-butyrine, (Z)-N- methyldehydrophenyla
  • the synthetic peptides described herein may include at least one peptide subunit that includes at least one dehydroamino acid, wherein the peptide subunit includes a structure according to Formula II below:
  • R 1 is selected from Ci-Cio alkyl, Ci-Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, halogen and a proteinogenic amino acid side chain;
  • n is zero or an integer greater than or equal to 1 ;
  • R 1 is hydrogen or a proteinogenic amino acid side chain. [0055] In some embodiments, R 1 is hydrogen or a proteinogenic amino acid side chain.
  • Naturally occurring proteinogenic amino acid side chains are well known and are typically referred to as the amino acid "R-group.”
  • the naturally occurring proteinogenic amino acid side chains or R-groups may be selected from arginine (arg), histidine (his), lysine (lys), aspartic acid (asp), glutamic acid (glu), serine (ser), threonine (thr), asparagine (asn), glutamine (gin), cysteine (cys), proline (pro), alanine (ala), valine (val), isoleucine (ile), leucine (leu), methionine (met), phenylalanine (phe), tyrosine (tyr), tryptophan (trp), formylmethionine (fmet), selenocysteine (sec), and pyrrolysine (pyl).
  • the amino acid glycine does not have a substituted R-group and has two hydrogen atoms on the amino acid ⁇ -carbon.
  • the side chains described herein may be present as R 1 in either the L or D enantiomer.
  • the proteinogenic amino acid R-groups useful for the synthetic peptides described herein are provided below.
  • the synthetic peptides described herein may include at least one peptide subunit that includes at least one dehydroamino acid, wherein the peptide subunit includes a structure according to Formula III below:
  • R 1 , B 1 , B 2 , n and m are defined as in Formulas I and II above;
  • X is an amino acid protecting group and
  • Y 2 is a peptide or peptide chain that includes at least one or more natural or non-natural amino acids; and
  • X is an amino acid protecting group.
  • Y 2 may further include one or more additional
  • Y 2 may further include one or more additional peptide subunits according to Formula II.
  • Exemplary and non-limiting amino acid protecting groups include tert-butyloxycarbonyl (Boc); Trityl (Trt); 3,5-dimethoxyphenylisoproxycarbonyl (Dbz); 2-(4-biphenyl)isopropoxycarbonyl (Bpoc); 2-nitrophenylsulfenyl (Nps); 9- fluoroenylmethoxycarbonyl (Fmoc); 2-4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc); (l, l-dioxobenzo[b]thiophene-2-yl)methyloxycarbonyl (Bsmoc); (1, 1- dixonaphtho[l,2-b]thiophene-2-yl)methyloxycarbonyl (a-Nsmoc); l-(4,4-dimethyl- 2,6-dioxocyclohex-l-ylidene)-3-methyl
  • allyloxycarbonyl (Alloc); o-nitrobenzenesuflonyl (oNBS); 2,4- dinitrobenzenesuflonyl (dNBS); benzothiazole-2-sulfonyl (Bts); 2,2,2- trichloroethyloxycarbonyl (Troc); dithiasuccinoyl (Dts); -nitrobenzyloxycarbonyl (pNZ);proparglyoxycarbonyl (Poc), o-nitrobenzyloxycarbonyl (oNZ), 4- nitroveratryloxycarbonyl (Nvoc), 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC), 2- (3 ,4-methylenedioxy-6-nitrophenyl)propyloxycarbonyl (Mnppoc), 9-(4- bromophenyl)-9-fluorenyl (BrPhF), azidomethoxycarbonyl (Azoc),
  • phenyldisulphanylethyloxycarbonyl (Phdec), 2-pyridyldisulphanylethyloxycarbonyl (Pydec), 2-chlorotrityl (2-Cl-Trt), 2,4-dimethoxybenzyl (Dmb), 2-phenylisopropyl (2-Ph-Pr), 5-phenyl-3,4-ethylenedioxythenyl (phenyl-EDOTn), 9-fluorenylmethyl (Fm), 4-(N-[l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]- amino)benzyl (Dmab), methyl (Me), ethyl (Et), carbamoylmethyl (Cam), Allyl (Al), Benzyl (Bn), phenacyl (Pac), / nitrobenzyl (pNB), 2-trimethylsilylethyl (TM
  • the synthetic peptides described herein includes a structure according to Formula IV below:
  • R 1 , B 1 , B 2 , n and m are defined as in Formulas I, II, and III above and Y 1 and Y 2 are each a peptide or peptide chain includes at least one or more natural or non-natural amino acids.
  • Y 1 and Y 2 may further include one or more additional dehydroamino acids according to Formula I.
  • Y 1 and Y 2 may further include one or more additional peptide subunits according to Formula II.
  • the synthetic peptide includes about 2 to about 2,500 amino acids, including at least one dehydroamino acid. In another aspect, the synthetic peptide includes about 2 to about 1,000 amino acids. In another aspect, the synthetic peptide includes about 2 to about 500 amino acids In another aspect, the synthetic peptide includes about 2 to about 250 amino acids In another aspect, the synthetic peptide includes about 2 to about 100 amino acids. In another aspect, the synthetic peptide includes about 2 to about 50 amino acids. In another aspect, the synthetic peptide includes about 2 to about 25 amino acids. In another aspect, the synthetic peptide includes about 2 to about 10 amino acids. In another aspect, the synthetic peptide includes about 2 to about 5 amino acids.
  • the synthetic peptide has a molecular weight of about 0.2 kDa to about 250 kDa, including each integer within the specified range. In one aspect, the synthetic peptide has a molecular weight of about 0.2 kDa to about 150 kDa, including each integer within the specified range. In another aspect, the synthetic peptide has a molecular weight of about 0.2 kDa to about 125 kDa, including each integer within the specified range. In another aspect, the synthetic peptide has a molecular weight of about 0.2 kDa to about 100 kDa, including each integer within the specified range.
  • the synthetic peptide has a molecular weight of about 0.2 kDa to about 75 kDa, including each integer within the specified range. In another aspect, the synthetic peptide has a molecular weight of about 0.2 kDa to about 50 kDa, including each integer within the specified range. In another aspect, the synthetic peptide has a molecular weight of about 0.2 kDa to about 25 kDa, including each integer within the specified range. In another aspect, the synthetic peptide has a molecular weight of about 0.2 kDa to about 10 kDa, including each integer within the specified range.
  • the synthetic peptides incorporating at least one dehydroamino acid imparts several functional benefits over traditional peptides.
  • the synthetic peptides described herein have an increased overall stability and half-life due to the incorporation of at least one dehydroamino acid (e.g., of Formula I) for several reasons. 1) Dehydroamino acids promote stability from increased A 1;3 strain rendering the peptide more rigid. 2) Peptides having dehydroamino acids stabilize the folded state of a peptide, which are degraded by proteases at a much slower rate. 3) The presence of an alkene at the a and ⁇ carbons gives rise to a planar and non-tetrahedral structure.
  • one embodiment described herein is a method for increasing the half-life of a synthetic peptide including substituting at least one amino acid of a target peptide with at least one
  • Another embodiment described herein is a method for decreasing proteolysis by peptidases of a susceptible synthetic peptide the method including substituting at least one amino acid of a target peptide with at least one
  • Another embodiment described herein is a method for increasing the stability of ⁇ -turn motif containing synthetic peptides including substituting at least one amino acid of a target peptide with at least one dehydroamino acid of Formula I or peptide subunit of Formula II by the methods described herein.
  • R 1 , B 1 , B 2 , n and m, and X are defined as in Formulas I, II, III, and rv above; and L is any leaving group. Suitable leaving groups include but are not limited to hydroxy, iodo, bromo, chloro, sulfonate, and the like.
  • the synthetic peptides of Formula V are used to generate the azlactone compounds described herein (e.g., of Formula VI).
  • Another embodiment described herein includes a synthetic peptide incorporating one or more of the structures below:
  • Another embodiment described herein is a synthetic peptide precursor having one or more azlactones according to Formula VI below:
  • R 1 , B 1 , B 2 , and X are defined as in Formulas I, II, III, IV, and V above.
  • Another embodiment described herein is a synthetic peptide precursor incorporating any one or more of the structures below:
  • Another embodiment described herein is a synthetic peptide linked to a resin including one or more azlactones according to Formula VII below:
  • R 1 , B 1 , B 2 , and X are defined as in Formulas I, II, III, IV, V, and VI above.
  • the synthetic peptides of Formula VII are further elongated using standard peptide synthesis methods.
  • compositions that include one or more of the synthetic peptides described herein and a pharmaceutically acceptable carrier.
  • the synthetic peptides used in the pharmaceutical compositions described herein have one or more substituted dehydroamino acids according to Formula I.
  • the synthetic peptides have a structure according to Formula II, III, or IV.
  • the synthetic peptides of the pharmaceutical compositions including at least one dehydroamino acid described herein are generated by the methods described herein.
  • compositions described herein are known and are suitable for the formulation and delivery of the synthetic peptides described herein.
  • the therapeutic peptides described herein may be delivered through parenteral routes, such as intravenously, intramuscularly, subcutaneously, or intradermally or other methods such as, transdermally, intra nasally, mucosally, sublingually, orally or through pulmonary delivery methods known in the art.
  • Some embodiments described herein are methods for synthesizing the synthetic peptides described herein that include at least one dehydroamino acid.
  • the synthetic strategy for generating peptides includes at least one dehydroamino acid as described herein is summarized in Scheme 1.
  • the peptide synthesis methodology is exemplified as solid phase peptide synthesis; however, other methods, such as solution phase peptide synthesis and solid phase solution phase hybrid peptide synthesis methods may also be used.
  • This synthetic strategy relies on the fact that C-terminal dehydroamino acids are rapidly transformed into azlactones (i.e., oxazolones) upon activation of their carboxylate group. This process can be used to facilitate couplings to dehydroamino acids.
  • azlactones i.e., oxazolones
  • This process can be used to facilitate couplings to dehydroamino acids.
  • symmetrical AAAs such as AVal and ⁇ .
  • dipeptides according to Formula V undergo cyclization in a single pot to generate azlactones according to Formula VI (e.g., dehydration and cyclization when L is selected as hydroxy).
  • Heating the azlactone according to Formula VI in the presence of a peptide triggers a coupling reaction, furnishing a dehydroamino acid linked peptides, for example, the resin-bound peptides according to Formula VII.
  • a peptide e.g., a resin-bound peptide
  • Subjection of the dehydroamino acid linked peptides to the standard peptide synthesis protocols described herein generates a peptide having one or more dehydroamino acids at the desired locations.
  • resin linked peptides having a dehydro amino acid according to Formula VII are used to generate desired peptides via solid-phase peptide synthesis (SPPS).
  • SPPS solid-phase peptide synthesis
  • the desired peptide with one or more dehydroamino acids located at the desired positions is generated following cleavage of this peptide from the resin and purification.
  • Scheme 1 Strategy for synthesis of ⁇ -containing peptides via SPPS.
  • Some embodiments described herein are methods of making a synthetic peptide having at least one dehydroamino acid including (a) providing a peptide having a free amine; (b) contacting the free amine containing peptide with the azlactone of Formula VI to form a reaction mixture; and (c) heating the reaction mixture. Following step (c), the peptide now includes a linked dehydroamino acid.
  • the free amine containing peptide is bound to a suitable substrate or resin for subsequent peptide synthesis methods (e.g., SPPS).
  • this process relies on the contacting of the substrate bound peptide with an azlactone under conditions that promote a ring opening reaction of the azlactone. Following this reaction, coupling with substrate bound amino acids having a free amine group occurs.
  • the ring opening is promoted by contacting the azlactone with the peptide, wherein the free amine serves as a nucleophile. The ring opening is further promoted with heating. The ring opening may further be promoted by the addition of a suitable catalyst or base.
  • the temperature condition for promoting azlactone ring opening is about 40 °C to about 75°C. In one aspect, the temperature is about 50 °C to about 75°C. In another aspect, the temperature is about 50 °C to about 60°C. In another aspect, the temperature is about 60 °C to about 70°C. In another aspect, the temperature is about 60 °C. [0078] In some embodiments described herein, the time condition for promoting azlactone ring at the temperatures described herein is from about 10 minutes to about 48 hours. In one aspect, the time is about 10 minutes to about 24 hours. In another aspect, the time is about 1 hour to about 24 hours. In another aspect, the time is about 2 hours to about 24 hours.
  • the time is about 5 hours to about 24 hours. In another aspect, the time is about 10 hours to about 24 hours. In another aspect, the time is about 5 hours to about 24 hours. In another aspect, the time is about 15 hours to about 24 hours. In another aspect, the time is about 20 hours to about 24 hours. In another aspect, the time is about 24 hours.
  • the efficiency of the coupling reaction is increased by the inclusion of a catalyst.
  • the catalyst may be any nucleophilic catalyst (e.g., a base) known in the art.
  • exemplary and non-limiting catalysts may include 4-dialkylaminopyrimidine catalysts, such as 4- (dimethylamino)pyridine and 4-pyrrolidinopyridine, and a base, such as triethylamine (Et 3 N).
  • the catalyst includes 4- (dimethylamino)pyridine (DMAP) or any chiral derivative thereof.
  • the base is triethylamine (Et 3 N).
  • the coupling reaction described above may be carried out in any suitable solvent.
  • exemplary and non-limiting solvents may include N- methylpyrrolidone ( ⁇ ) or dimethylformamide (DMF).
  • DMF dimethylformamide
  • the solvent is N-methylpyrrolidone ( ⁇ ).
  • the amino acid protecting group during coupling of the dehydroamino acid to a substrate bound peptide includes Alloc.
  • the amino acid protecting group e.g., X of Formulas III, V, VI, and VII
  • Protecting group removal reactions are known in the art and are carried out with either a suitable acid or base or through the use of a catalyst and appropriate scavengers and nucleophiles.
  • exemplary and non-limiting acids such as trifluoroacetic acid (TFA), hydrochloric acid (HQ), or trichloroacetic acid (TCA) may be used.
  • Suitable bases may include piperidine, ammonia, N- methylpyrrolidine, hydrazine, sodium hydroxide, or sodium carbonate.
  • Suitable catalyst/nucleophiles include palladium catalysts, such as Pd(PPh 3 ) 4 and nucleophile/scavengers, such as ⁇ 3 ⁇ 3 , Me 2 H-BH 3 or PhSiH 3 .
  • additional amino acids are added to extend the substrate bound peptide including a dehydroamino acid to generate elongated peptides.
  • Peptide synthesis techniques that may be used include solid phase peptide synthesis, solution phase peptide synthesis or a solid phase solution phase hybrid peptide synthesis method.
  • Solution phase peptide synthesis methods are the least complex and are known in the art. As described herein, solution phase peptide synthesis involves the coupling of amino acids in a solvent one at a time typically followed by purification of intermediates. For directionality, suitable protecting groups for the growing N-terminus (e.g., N-Boc or N-Fmoc) and C-terminus (e.g., Trityl, ?-butyl, or Boc) may be used. Other protecting groups, such as those described as being useful for the amino acid protecting group X of Formula III above may also be used. Continuous solution phase peptide synthesis methods are also known and may be used to synthesize the elongated peptides described herein.
  • suitable protecting groups for the growing N-terminus e.g., N-Boc or N-Fmoc
  • C-terminus e.g., Trityl, ?-butyl, or Boc
  • Other protecting groups such as those described as being useful for the amino acid protecting group X
  • next generation solution phase peptide synthesis methods may be used, which are known in the art, See, for example, Takahashi et al., Organic Letters. 2012, 14 (17), pp. 4514-4517; Carpino et al, B. Org. Process Res. Dev. 2003, each of which is incorporated by reference herein for its teachings thereof.
  • Solid phase peptide synthesis methods include the following steps.
  • An amino acid corresponding to the C-terminus of the target peptide is covalently attached to an insoluble polymeric substrate or support (e.g., a resin).
  • the next amino acid, with a protected a-amino acid e.g., Fmoc or Boc
  • a protected a-amino acid e.g., Fmoc or Boc
  • Excess reactants and co-products are removed by filtration and washing.
  • Suitable solid phase peptide synthesis substrates are resins or small porous beads. The size of the bead allows for a rapid penetration of reagents in SPPS. Peptides to be elongated are covalently attached to the porous beads via a suitable covalent linker.
  • Exemplary and non-limiting resins may include a Pam resin, a Wang resin, a Rink amide resin, a PAL resin, a Sieber resin, a MBHA resin, a trityl resin, DHP resin, Weinreb aminomethyl resin, or Polyethylene
  • the solid phase peptide synthesis method may be carried out via manual peptide synthesis using suitable reaction vessels, such as a fritted-filter reaction vessel or by using commercially available peptide synthesizers.
  • Solid phase solution phase hybrid peptide synthesis methods are known in the art. These methods are a hybrid approach between solid phase and solution phase methodologies described herein. These strategies typically involve condensing in solution two peptide fragments prepared through solid phase synthesis using known coupling reagents or through chemical ligation techniques. These hybrid approaches may be used for assembling larger peptides.
  • a synthetic peptide comprising:
  • At least one dehydroamino acid wherein the at least one dehydroamino acid is of Formula I:
  • B 1 and B 2 are each independently selected from hydrogen, Ci-Cio alkyl, Ci-Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen.
  • R 1 is hydrogen or any naturally occurring proteinogenic amino acid side chain
  • n is integer greater than or equal to 1 ;
  • n is an integer greater than or equal to 1.
  • Y 2 comprises at least one or more natural or non-natural amino acids; and X is an amino acid protecting group.
  • amino acid protecting group of X is selected from the group consisting of Alloc, Fmoc, Cbz, Boc, Ddz, Bpoc, Nps, Nsc, Bsmoc, Trt, ivDde, Tcp, Pms, Esc, Sps, oNbs, dNbs, Bts, Troc, Dts, pNZ, Poc, oNZ, NVOC, NPPOC, Mnppoc, BrPhF, Azoc, Hfa, Phdec, Pydec, 2-Cl-Trt, Dmb, 2-Ph-Pr, Phenyl-Edotn, Fm, Dmab, Cam, Allyl, Bn, Pac, pNB, Tmse, Ptmse, Tmsi, Tee, cHx, Men, Mpe, Tegbz, Phth, DBS, and triazone
  • Y 1 and Y 2 are each a peptide chain comprising at least one or more natural or non-natural amino acids.
  • a pharmaceutical composition comprising the synthetic peptide according to any one of statements 1-15 and a pharmaceutically acceptable carrier.
  • X is selected from the group consisting of Alloc, Fmoc, Cbz, Boc, Ddz, Bpoc, Nps, Nsc, Bsmoc, Trt, ivDde, Tcp, Pms, Esc, Sps, oNbs, dNbs, Bts, Troc, Dts, pNZ, Poc, oNZ, NVOC, NPPOC, Mnppoc, BrPhF, Azoc, Hfa, Phdec, Pydec, 2-Cl-Trt, Dmb, 2-Ph-Pr, Phenyl-Edotn, Fm, Dmab, Cam, Allyl, Bn, Pac, pNB, Tmse, Ptmse, Tmsi, Tee, cHx, Men, Mpe, Tegbz, Phth, DBS, and triazone;
  • B 1 and B 2 are each independently selected from hydrogen, Ci-Cio alkyl, Ci- Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen; and
  • L is a leaving group selected from hydroxy, iodo, bromo, chloro and sulfonate; and n is an integer greater than or equal to 1.
  • a synthetic peptide precursor comprising one or more azlactones according to Formula VI:
  • R is hydrogen or any naturally occurring proteinogenic amino acid side chain
  • B 1 and B 2 are each independently selected from hydrogen, Ci-Cio alkyl, Ci- Cio alkylene, Ci-Cio alkanol, Ci-Cio carboxylic, Ci-Cio fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen; and
  • X is an amino acid protecting group selected from the group consisting of Alloc, Fmoc, Cbz, Boc, Ddz, Bpoc, Nps, Nsc, Bsmoc, Trt, ivDde, Tcp, Pms, Esc, Sps, oNbs, dNbs, Bts, Troc, Dts, pNZ, Poc, oNZ, NVOC, NPPOC, Mnppoc, BrPhF, Azoc, Hfa, Phdec, Pydec, 2-Cl-Trt, Dmb, 2-Ph-Pr, Phenyl-Edotn, Fm, Dmab, Cam, Allyl, Bn, Pac, pNB, Tmse, Ptmse, Tmsi, Tee, cHx, Men, Mpe, Tegbz, Phth, DBS, and triazone.
  • [00112] 25 A method of increasing the stability of a synthetic peptide comprising substituting at least one amino acid of the synthetic peptide for at least one dehydro amino acid according to the Formula I of statement 1.
  • Ci 3 H 18 N 2 0 4 H + requires 267.1345).
  • the crude peptide was lyophilized and purified by HPLC (Discovery BIO Wide Pore C18-10 Column (25 cm x 10 mm, 10 ⁇ ), 30- 50% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 16 min, 6 mL/min flow rate, then 100% CH 3 CN (0.1% TFA) for 15 min, 15 mL/min flow rate).
  • HPLC Discovery BIO Wide Pore C18-10 Column (25 cm x 10 mm, 10 ⁇ ), 30- 50% CH 3 CN (0.1% TFA) in H 2 0 (0.1% TFA) gradient over 16 min, 6 mL/min flow rate, then 100% CH 3 CN (0.1% TFA) for 15 min, 15 mL/min flow rate).
  • HRMS high resolution mass spectrometry
  • Rink amide MB HA resin (100-200 mesh, 100 ⁇ ) was added to a fritted polypropylene syringe. The resin was swelled in (3 ⁇ 4(3 ⁇ 4 (10 min), and then in DMF (3 min). The swelling solvents were drained from the resin using a vacuum manifold. After Fmoc deprotection (see below for procedure, repeated twice), the amino acid was coupled to the resin (see below for procedure, repeated twice).
  • the activated amino acid solution was then added to the resin, and the resulting mixture was heated to 70 °C with stirring in the microwave (2 min ramp to 70 °C, 2 min hold at 70 °C). All amino acids were double coupled to allow the coupling reaction to proceed to completion.
  • the resin was
  • Structure 18 includes the azlactone structure 13; structure 19 includes the azlactone structure 15; structure 20 includes the azlactone structure 17.
  • the bonds presented as dashed lines indicate areas of the peptide undergoing a ⁇ -turn.
  • Example 22 Increased stability of ⁇ -turn containing peptides with at least one dehydroamino acid
  • Stabilization of secondary structures is one of the important benefits of incorporating dehydroamino acids into the primary structure of a synthetic peptide. As described herein, peptides with stable secondary structures are less likely to be degraded by proteases. Therefore, the effects of dehydroamino acids in reducing proteolysis of synthetic peptides having a ⁇ -turn secondary structure were investigated.
  • Pronase E from Streptomyces griseus (EC 3.4.24.31) was purchased from EMD Millipore. It was brought up in 10 mM sodium phosphate, 140 mM NaCl buffer, pH 7.4, to ⁇ 0. lmg/mL. A total of 6 ⁇ ⁇ of this enzyme mixture was used in proteolysis experiments. [00168] A total of 1.5 ml of 0.5 mM of each peptide in the phosphate buffer (140 mM Na+/K+Cl- buffer pH 7.4) was reacted with 6 ⁇ ⁇ of pronase E mixture at 37 °C. A 50 ⁇ .
  • peptides according to structures 18-20 were synthesized according to the methods described herein. Each of these peptides demonstrated a common ⁇ -turn secondary structure as shown.
  • the stability of peptides according to structure 18 and 20 were compared to a model synthetic peptide known to have ⁇ -turn structure that does not include a dehydroamino acid.
  • the amino acid residue sequence Asn-Gly of this model peptide was substituted for either AVal-Gly (18) or Pro-AVal (20).
  • Each of the peptides was placed in a protease solution and the percentage of peptide remaining was measured. As shown in Figure 6, both peptides 18 and 20 were more resilient to protease clevage as evidenced by the increased amount of whole peptide remaining compared to the reference model peptide.

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Abstract

L'invention concerne des procédés qui permettent d'incorporer des α,β-déhydroamino acides volumineux, de type déhydrovaline et déhydroéthylnorvaline dans des peptides par synthèse peptidique en phase solide et les compositions peptidiques obtenues. Le procédé de synthèse consiste à utiliser des intermédiaires d'azlactone à coupler à des peptides liés à une résine.
PCT/US2015/064091 2014-12-04 2015-12-04 Synthèse en phase solide de peptides contenant des déhydroamino acides volumineux WO2016090305A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2018156500A1 (fr) * 2017-02-21 2018-08-30 The Board Of Regents For Oklahoma State University Structure et synthèse de dérivés d'acides aminés hautement fluorés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165132A1 (en) * 1991-08-09 2002-11-07 Murray Goodman Lanthionine bridged peptides

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165132A1 (en) * 1991-08-09 2002-11-07 Murray Goodman Lanthionine bridged peptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RICH ET AL.: "Synthesis of Dehydro Amino Acids and Peptides by Dehydrosulfenylation. Rate Enhancement Using Sulfenic Acid Trapping Agents", J. ORG. CHEM., vol. 42, no. 24, 1977, pages 3815 - 3820 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018156500A1 (fr) * 2017-02-21 2018-08-30 The Board Of Regents For Oklahoma State University Structure et synthèse de dérivés d'acides aminés hautement fluorés
US10676423B2 (en) 2017-02-21 2020-06-09 The Board Of Regents For Oklahoma State University Structure and synthesis of highly fluorinated amino acid derivatives

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