WO2016090305A1 - Solid-phase synthesis of peptides containing bulky dehydroamino acids - Google Patents

Abstract

Methods of incorporating bulky α,β-dehydroamino acids such as dehydrovaline and dehydroethylnorvaline into peptides via solid-phase peptide synthesis and the resulting peptide compositions is reported. The synthesis method involves using azlactone intermediates for coupling with resin bound peptides.

Description

SOLID-PHASE SYNTHESIS OF PEPTIDES CONTAINING

BULKY DEHYDROAMINO ACIDS

RELATED APPLICATIONS

[0001] The present application claims the benefit of the filing date under 35 U.S.C. § 119(e) of Provisional U.S. Patent Application No. 62/123,998, filed December 4, 2014, which is hereby incorporated by reference.

BACKGROUND

1. Technical Field

[0002] The invention described herein relates to methods of incorporating bulky α,β-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

[0003] Bulky dehydroamino acids impart rigidity and stability to peptides. Both of these characteristics are valuable, as the former characteristic improves the peptide's ability to bind to biological targets such as other proteins, and the latter characteristic improves the peptide's stability to in vivo degradation by proteases. Thus, the inclusion of dehydroamino acids into therapeutic peptides should render them more useful as drugs. Unfortunately, it is very difficult to synthesize peptides that contain bulky dehydroamino acids. Solid-phase peptide synthesis has never been used to construct this type of peptide. The present disclosure addresses these problems by providing a rapid and efficient synthetic route to generating peptides that contain bulky dehydroamino acids.

BRIEF SUMMARY

[0004] 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.

[0005] 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:

wherein B¹ and B² are each independently selected from hydrogen, C C₁₀ alkyl, Ci-Cio alkylene, Ci-Ci₀ alkanol, Ci-Ci₀ carboxylic, Ci-Ci₀ fluoroalkyl, alkoxy, cyclic, heterocyclic, ureido, amino, -OH, aromatic, and halogen. In one aspect, the peptide has a molecular weight of from about 0.1 kDa to about 250 kDa. In another aspect, B¹ and B² are each independently selected from C C₁₀ alkyl and Ci-Cio perfluoroalkyl. In another aspect B¹ and B² are different. In another aspect, B¹ and B² are the same. In another aspect, aromatic is phenyl and heterocyclic is selected from indole, pyrrole, azole and pyrrolidine.

[0006] Another embodiment described herein is a synthetic peptide, including one or more peptide subunits according to Formula II:

wherein B¹ and B² are defined as for Formula I above and R¹ 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.

[0007] Another embodiment described herein is a synthetic peptide including a structure according to Formula III:

wherein B¹, B², R¹ , n, and m are defined as for Formulas I and II above and wherein Y² includes at least one or more natural or non-natural amino acids; and X is an amino acid protecting group. In one aspect, Y² further includes one or more dehydroamino acids according to Formula I. In another aspect, 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,

DBS, and triazone. In another aspect, the amino acid protecting group is Alloc.

[0008] Another embodiment described herein is a synthetic peptide including a structure according to Formula IV:

wherein B¹, B², R¹, n, and m are defined as for Formulas I, II, and III above, and wherein Y¹ and Y² are each a peptide chain including at least one or more natural or non-natural amino acids. In one aspect, Y¹ and Y² or Y¹ or Y² optionally include one or more additional dehydroamino acids according to the structure of Formula I above. In another aspect, B¹ and B² are both ethyl or B¹ and B² are both methyl. In another aspect, R¹ is hydrogen or the side chain of phenylalanine.

[0009] Another embodiment described herein is a pharmaceutical composition including a synthetic peptide as described herein and a

pharmaceutically acceptable carrier. [0010] Another embodiment described herein is a synthetic peptide according to Formula V:

wherein 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;

R¹ is hydrogen or any naturally occurring proteinogenic amino acid side

B¹ and B² are each independently selected from hydrogen, C C₁₀ alkyl, C C₁₀ alkylene, C C₁₀ alkanol, C C₁₀ carboxylic, C C₁₀ 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.

[0011] Another embodiment described herein is a synthetic peptide precursor including one or more azlactones according to Formula VI:

wherein R¹ is hydrogen or any naturally occurring proteinogenic amino acid side chain; B¹ and B² are each independently selected from hydrogen, C C₁₀ 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.

[0012] 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. In one aspect, the further includes isolating a dehydroamino acid linked substrate bound peptide. In another aspect, 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. In another aspect, the method further includes cleaving the amino acid protecting group X. In another aspect, 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. In another aspect, the method further includes cleaving the elongated synthetic peptide from the substrate.

[0013] 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. In one aspect, the increased stability includes an increased half-life of the synthetic peptide. In another aspect, the synthetic peptide includes at least one β-turn motif and the increased stability includes an increased stability of the β-turn motif of the synthetic peptide that includes the at least one β-turn motif.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 Illustration of the differences in dehydroamino acids having one or two β-carbon substituted groups.

[0015] 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, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0016] FIG. 3 HPLC chromatogram of elongated synthetic peptide

corresponding to structure 1 1a; chromatogram for purified 1 1a (Discovery CI 8 Column (25 cm x 4.6 mm, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0017] 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, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0018] FIG. 5 HPLC chromatogram of elongated synthetic peptide corresponding to structure 1 lb; chromatogram for purified 1 lb (Discovery CI 8

Column (25 cm x 4.6 mm, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0019] 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, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0020] 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, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min). [0021] 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, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0022] 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, 5μιη), 10-60% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 50 min, 1 mL/min).

[0023] FIG. 10 Stabilization and reduction in proteolytic cleavage of β-turn containing synthetic peptides containing at least one dehydroamino acid.

DETAILED DESCRIPTION

[0024] The following paragraphs define in more detail the embodiments of the invention described herein. The following embodiments are not meant to limit the invention or narrow the scope thereof, as it will be readily apparent to one of ordinary skill in the art that suitable modifications and adaptations may be made without departing from the scope of the invention, embodiments, or specific aspects described herein. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof where incorporation is permitted.

[0025] For purposes of interpreting this specification, the following terms and definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0026] The term "dehydroamino acid" or "ΔΑΑ" as used herein refers to any amino acid that has lost two vicinal hydrogen atoms to form a double bond. As used herein, these terms include α,β-dehydroamino acids, which refer to a-amino acids having a double bond between the a-carbon and the β-carbon of the amino acid. In some aspects described herein, the β-carbon of these amino acids are singly substituted with a substituent group or doubly substituted with a suitable substituent group. Further, the β-carbon of the amino acid having two substituted groups may be symmetrically substituted (i.e., the same) or asymmetrically substituted (i.e., different).

[0027] The term "amino acid" as used herein refers to compounds containing an amine (-NH₂) 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. As used herein, the term amino acid is intended to include both the L-stereoisomer and the D- stereoisomer.

[0028] The term "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 H₂NCH(R)C(0)OH. As used herein, the term proteinogenic amino acid is intended to include both the L- stereoisomer and the D -stereoisomer.

[0029] The term "non-natural amino acid" as used herein refers to any non- protein coding or proteinogenic amino acid known in the art. These amino acids may be any alpha (a-), beta (β-), gamma (γ-) or delta (δ-) amino acid and include any stereoisomer thereof.

[0030] The term "alkyl" 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. As used herein, the denotation 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. Representative examples of 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. In some aspects, the term "alkyl" is intended to include substituted halo alkyls, such as

perfluoroalkyl and fluoroalkyl.

[0031] The term "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. Representative examples of 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. The term "alkenyl" or "alkylene" is intended to include both substituted and unsubstituted alkeynyl or alkylene unless otherwise indicated.

[0032] The term "alkynyl" as used herein alone or as part of another group, 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.

[0033] The term "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. The term "aryl" or "aromatic" is intended to include both substituted and unsubstituted aryl or aromatic unless otherwise indicated.

[0034] The term "cyclic" "cycloalkyl" as used herein alone or as part of another group, 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. The term "cycloalkyl" is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.

[0035] The term "heterocyclic" as used herein alone or as part of another group, 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. Representative examples of 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, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. 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.

Representative examples of 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,

thiopyranopyridine, and the like. These rings include quaternized derivatives thereof. In some aspects the heterocyclic is a heteroaryl or heteroaromatic group such as azole, indole, and pyrrole.

[0036] The term "alkoxy" as used herein alone or as part of another group, 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-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like. In some aspects, alkoxy groups, when part of a more complex molecule, include an alkoxy substituent attached to an alkyl via an ether linkage.

[0037] The term "leaving group" as used herein refers to any molecular fragment, which leaves with a loan pair of electrons. In one aspect described herein 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.

[0038] The term "halo" or "halogen" as used herein refers to any suitable halogen, including -F, -CI, -Br, and -I. [0039] The term "amino" as used herein means the radical -NH₂.

[0040] The term "amide" as used herein alone or as part of another group refers to a -C(0)NR_aR_b radical, where R_a and R_b are independently any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0041] The term "carboxylic" or carboxylate as used herein alone or as part of another group refers to a -C(0)0^" or -C(0)OH radical.

[0042] The term "ureido" as used herein alone or as part of another group refers to an R_aNC(0)NRb radical, where Ra and Rb are any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0043] Terms such as "include," "including," "contain," "containing," "has," or "having" and the like mean "comprising."

Dehydroamino Acid Substituted Synthetic Peptides

[0044] α,β-Dehydroamino acids (AAAs) are characterized by planar geometry and restricted rotational freedom relative to standard amino acids. As described herein, 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 β carbon (e.g., AAla, AAbu, APhe) have been studied, and rules of their inclusion in natural peptide secondary structures have been established. However, much less is known about bulky AAAs that are fully substituted at the β carbon (e.g., AVal), which exhibit a more pronounced rigidifying effect on peptides due to their high levels of A_1;3 strain (Figure 1). While the structures of simple AVal derivatives and AVal-containing di-, tri-, and tetrapeptides have been examined, we are unaware of any studies of larger peptides that include this residue. Further, there is a lack of methods for incorporating bulky AAAs into peptides via peptide synthesis techniques.

[0045] In synthesizing peptides containing tetrasubstituted AAAs, we examined the effects of bulky residues such as, for example, AVal and its homologue dehydroethylnorvaline (AEnv) on the structures of peptides. As described herein, we have developed protocols for the efficient synthesis of peptides that include these bulky AAAs. [0046] Thus, some embodiments described herein include dehydroamino acid - substituted synthetic peptides. In some aspects described herein, the dehydro amino acid substituted synthetic peptides include at least one dehydroamino acid.

[0047] In some embodiments, 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.

[0048] In some embodiments, 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. In some aspects, the synthetic peptides have less than 90%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence. In some aspects, the synthetic peptides have less than 80%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence. In some aspects, the synthetic peptides have less than 70%, homology with any naturally occurring peptide, peptide fragment, or protein amino acid sequence. In some aspects, 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.

[0049] In some embodiments, synthetic peptides having at least one dehydro amino acid described herein include a naturally occurring and non-naturally occurring peptide amino acid sequence. In one aspect, 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. In another aspect, the synthetic peptides include a synthetic or non- naturally occurring amino acid sequence, wherein a dehydro amino acid is part of the synthetic or non-naturally occurring amino acid sequence. Thus, 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. [0050] In some embodiments described herein, the dehydroamino acid includes a structure according to Formula I below:

Formula I

wherein:

B¹ 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

B² 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.

[0051] 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. In some aspects, the synthetic peptides described herein may have more than one dehydroamino acid according to Formula I. In some aspects, 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.

[0052] In some embodiments described herein, B¹ and B² described above is the same or B¹ and B² are different. In some aspects, the aromatic group for B¹ and/or B² is phenyl and the heterocyclic group for B¹ and/or B² is selected from indole, pyrrole, azole and pyrrolidine. In some aspects, B¹ and B² are selected from Ci-Cio alkyl and Ci-Cio perfluoroalkyl. In one aspect described herein, B¹ and B² are both methyl. In another aspect described herein, B¹ and B² are both ethyl.

[0053] In some embodiments described herein, 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- methyldehydrophenylalanine, (Z)-N'-methyldehydrobutyrine, dehydroalanine ester, (Z)-dehydrobutyrine ester, (E)-dehydrobutyrine ester, dehydroproline ester, (Z)-O- methyl-dehydroserine ester, O-aryl-N-methyldehydrotyrosine ester, N- methyldehydroalanine thioester, thiazole-dehydroalanine, thiazole-(Z)- dehydrobutyrine, thiazole-(E)-0-methyl-dehydrothreonine, thiazoline-(Z)- dehydrobutyrine, oxazole-dehydroalanine, oxazole-(Z)-dehydrobutyrine, oxazole- (Z)-dehydro-homobutyrine, oxazole-dehydroleucine, 5-methyl-oxazole- dehydroalanine, 5-methyl-oxazole-(Z)-dehydrobutyrine, 5-methyl-oxazole-(Z)- dehydrohomobutyrine, 5-methyl-oxazole-(Z)-dehydrohomoserine. See, Siodlak, D. Amino Acids. 2015, 47, pp. 1-17, which is incorporated by reference herein for its teachings thereof.

[0054] In some embodiments described herein, 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:

Formula II

wherein B¹ and B² are as defined in Formula I above;

R¹ 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 ; and

m is an integer greater than or equal to 1. In some aspects described herein, n and m are both equal to the integer 1. In some aspects, R¹ is hydrogen or a proteinogenic amino acid side chain. [0055] In some embodiments, R¹ is hydrogen or a proteinogenic amino acid side chain.

[0056] 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 (gly) does not have a substituted R-group and has two hydrogen atoms on the amino acid β-carbon. The side chains described herein may be present as R¹ in either the L or D enantiomer. The proteinogenic amino acid R-groups useful for the synthetic peptides described herein are provided below.

[0057] In some embodiments described herein, 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:

Formula III

wherein R¹, B¹, B², n and m are defined as in Formulas I and II above; X is an amino acid protecting group and Y² 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. In some aspects, Y² may further include one or more additional

dehydroamino acids according to Formula I. In some aspects, Y² may further include one or more additional peptide subunits according to Formula II.

[0058] Exemplary and non-limiting amino acid protecting groups (X) 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-methylbutyl (ivDde); 2,7-di-tert-butyl-Fmoc; 2- fluoro-Fmoc (Fmoc(2F)); 2-monoisooctyl-Fmoc (mio-Fmoc); 2,7-diiooctyl-Fmoc (dio-Fmoc); tetrachlorophthaloyl (TCP); 2-[phenyl(methyl)sulfonio])ethyloxy carbonyl tetrafluoroborate (Pms); ethanesulfonyl ethoxycarbonyl (Esc); 2-(4- sulfophenylsulfonyl)ethoxycarbonyl (Sps); benzyloxycarbonyl (CbZ);

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),

hexafluoroacetone (HFA), 2-chlorobenzyloxycarbonyl (Cl-Z),

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 (TMSE), (2-phenyl-2-trimethylsilyl)ethyl (Ptmse), 2-(trimethylsilyl)isopropyl (Tmsi), 2,2,2-trichloroethyl (Tee), cyclohexyl (cHx), β-menthyl (Men), β-3- methylpent-3-yl (Mpe), 4-(3,6,9-trioxadecyl)oxybenzyl (TEGBz; TEGBn), phthalimide (Phth), dibenzosuberyl (DBS), and triazone. See also, Isidro-Llobet et al, Chem. Rev. 2009 109, pp. 2455-2504, which is incorporated by reference herein for its teachings thereof.

[0059] In some embodiments described herein, the synthetic peptides described herein includes a structure according to Formula IV below:

Formula IV

wherein R¹, B¹, B², n and m are defined as in Formulas I, II, and III above and Y¹ and Y² are each a peptide or peptide chain includes at least one or more natural or non-natural amino acids. In some aspects, Y¹ and Y² may further include one or more additional dehydroamino acids according to Formula I. In some aspects, Y¹ and Y² may further include one or more additional peptide subunits according to Formula II.

[0060] In some embodiments described herein, 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.

[0061] In some embodiments described herein, 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. In another aspect, 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.

[0062] 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 β carbons gives rise to a planar and non-tetrahedral structure. This structure further prevents proteolysis because most peptidase active sites typically recognize tetrahedral structures and not planar structures. In addition, it was discovered that synthetic peptides having a β-turn are stabilized by the incorporation of at least one dehydroamino acid according to Formula I. [0063] Thus, some embodiments described herein are related to imparting these benefits to a target synthetic peptide. In particular, 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

dehydroamino acid of Formula I or peptide subunit of Formula II by the methods described herein.

[0064] 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

dehydroamino acid of Formula I or peptide subunit of Formula II by the methods described herein.

[0065] Another embodiment described herein is a method for increasing the stability of β-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.

[0066] Another embodiment described herein is a synthetic peptide according to Formula V below:

Formula V

wherein R¹, B¹, B², 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. In some aspects, described herein, the synthetic peptides of Formula V are used to generate the azlactone compounds described herein (e.g., of Formula VI).

[0067] Another embodiment described herein includes a synthetic peptide incorporating one or more of the structures below:

[0068] Another embodiment described herein is a synthetic peptide precursor having one or more azlactones according to Formula VI below:

Formula VI

R

wherein R¹, B¹, B², and X are defined as in Formulas I, II, III, IV, and V above.

[0069] Another embodiment described herein is a synthetic peptide precursor incorporating any one or more of the structures below:

17

[0070] Another embodiment described herein is a synthetic peptide linked to a resin including one or more azlactones according to Formula VII below:

Formula VII

wherein R¹, B¹, B², and X are defined as in Formulas I, II, III, IV, V, and VI above. In some aspects, as described further herein, the synthetic peptides of Formula VII are further elongated using standard peptide synthesis methods.

[0071] Some embodiments described herein are pharmaceutical compositions that include one or more of the synthetic peptides described herein and a pharmaceutically acceptable carrier. In some aspects, the synthetic peptides used in the pharmaceutical compositions described herein have one or more substituted dehydroamino acids according to Formula I. In one aspect, the synthetic peptides have a structure according to Formula II, III, or IV. In another aspect, the synthetic peptides of the pharmaceutical compositions including at least one dehydroamino acid described herein are generated by the methods described herein.

[0072] Pharmaceutically acceptable carriers for peptide therapeutics 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.

Methods for Synthesizing Dehydroamino Acid Substituted Synthetic Peptides

[0073] 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.

[0074] 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. For example, symmetrical AAAs, such as AVal and ΔΕην. Thus, 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 (e.g., a resin-bound peptide) triggers a coupling reaction, furnishing a dehydroamino acid linked peptides, for example, the resin-bound peptides according to Formula VII.

[0075] 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. In some embodiments, resin linked peptides having a dehydro amino acid according to Formula VII are used to generate desired peptides via solid-phase peptide synthesis (SPPS). 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 ΔΑΑ-containing peptides via SPPS.

I

Peptide synthesis, then cleavage Formula VII

from resin and purification

ΔΑΑ-containing peptides

[0076] 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. In some aspects, the free amine containing peptide is bound to a suitable substrate or resin for subsequent peptide synthesis methods (e.g., SPPS). In some aspects, 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. In some aspects, 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.

[0077] In some embodiments described herein, 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. In another aspect, 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.

[0079] In some embodiments described herein, 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 (Et₃N). In one aspect, the catalyst includes 4- (dimethylamino)pyridine (DMAP) or any chiral derivative thereof. In another aspect, the base is triethylamine (Et₃N).

[0080] The coupling reaction described above may be carried out in any suitable solvent. Exemplary and non-limiting solvents may include N- methylpyrrolidone ( ΜΡ) or dimethylformamide (DMF). In some aspects, described herein the solvent is N-methylpyrrolidone ( ΜΡ).

[0081] During the azlactone/peptide coupling process, it was found that standard solid phase peptide synthesis Fmoc protecting groups were relatively unstable and provided relatively low yields of coupled products. Without wishing to be bound by any theory it is believed that Fmoc is unstable under the weakly basic coupling conditions. Therefore, it was found that the Alloc protecting group allows for more efficient coupling most likely due to its increased stability under the coupling conditions. Thus, in some aspects described herein, the amino acid protecting group during coupling of the dehydroamino acid to a substrate bound peptide includes Alloc.

[0082] Prior to subsequent peptide synthesis elongation reactions, the amino acid protecting group (e.g., X of Formulas III, V, VI, and VII) is removed. 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(PPh₃)₄ and nucleophile/scavengers, such as Η₃Ν·ΒΗ₃, Me₂ H-BH₃ or PhSiH₃.

[0083] In some embodiments, 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.

[0084] 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. See, for example, Carpino et al, Org. Proc. Res. & Dev. 2003, 7, pp. 28-37, which is incorporated by reference herein for its teachings thereof. Other 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.

[0085] Solid phase peptide synthesis methods are known in the art and 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), is activated and reacted with the resin-bound amino acid to yield an amino protected dipeptide on the resin. Excess reactants and co-products are removed by filtration and washing. After the target protected peptide chain has been built up in this stepwise fashion, all side chain groups are removed and the anchoring bond between the peptide and the resin is cleaved by suitable chemical means thereby releasing the crude peptide product into solution. See, for example, U.S. Patent No. 5,026,773 and Stawikowski, M and Fields, G.B. Curr Protoc. Protein Set 2002, each of which are incorporated by reference herein for their teachings thereof.

[0086] 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

glycol/polystyrene grafted resins. 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.

[0087] 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.

Statements

[0088] 1. A synthetic peptide, comprising:

at least one dehydroamino acid, wherein the at least one dehydroamino acid is of Formula I:

wherein B¹ and B² 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.

[0089] 2. The synthetic peptide of statement 1, wherein the peptide has a molecular weight of from about 0.1 kDa to about 250 kDa.

[0090] 3. The synthetic peptide according to any one of statements 1-2, wherein B¹ and B² are each independently selected from Ci-Cio alkyl and Ci-Cio

perfluoroalkyl.

[0091] 4. The synthetic peptide of statement 1, wherein aromatic is phenyl and heterocyclic is selected from indole, pyrrole, azole and pyrrolidine.

[0092] 5. The synthetic peptide according to any one of statements 1-4, wherein B¹ and B² are different.

[0093] 6. The synthetic peptide according to any one of statements 1-5, wherein B¹ and B² are the same.

[0094] 7. The synthetic peptide according to any one of statements 1-6 comprising one or more peptide subunits according to Formula II

wherein R¹ is hydrogen or any 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.

[0095] 8. The synthetic peptide according to any one of statements 1-7 comprising a structure according to Formula III:

wherein Y² comprises at least one or more natural or non-natural amino acids; and X is an amino acid protecting group.

[0096] 9. The synthetic peptide of statement 8, wherein Y² further comprises one or more dehydroamino acids according to Formula I.

[0097] 10. The synthetic peptide of statement 8, wherein 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, 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.

[0098] 1 1. The synthetic peptide of statement 10, wherein the amino acid protecting group is Alloc.

[0099] 12. The synthetic peptide according to any one of statements 1-11 comprising a structure according to Formula IV:

wherein Y¹ and Y² are each a peptide chain comprising at least one or more natural or non-natural amino acids.

[00100] 13. The synthetic peptide of statement 12, wherein Y¹ and Y² or Y¹ or Y² optionally comprise one or more additional dehydroamino acids according to the structure of Formula I.

[00101] 14. The synthetic peptide according to any one of statements 1-13, wherein B¹ and B² are both ethyl or B¹ and B² are both methyl.

[00102] 15. The synthetic peptide according to any one of statements 7-14, wherein R¹ is hydrogen or the side chain of phenylalanine.

[00103] 16. A pharmaceutical composition comprising the synthetic peptide according to any one of statements 1-15 and a pharmaceutically acceptable carrier.

[00104] 17. A synthetic peptide according to formula V:

wherein 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;

hydrogen or any naturally occurring proteinogenic amino acid side chain;

B¹ and B² 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.

[00105] 18. A synthetic peptide precursor comprising one or more azlactones according to Formula VI:

wherein R is hydrogen or any naturally occurring proteinogenic amino acid side chain;

B¹ and B² 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.

[00106] 19. A method of making a synthetic peptide according to any one of statements 1-16 comprising:

(a) providing a substrate bound peptide having a free amine;

(b) contacting the substrate bound peptide with an azlactone according to Formula VI of statement 18 to form a reaction mixture;

(c) heating the reaction mixture.

[00107] 20. The method of statement 19 further comprising isolating a dehydroamino acid linked substrate bound peptide.

[00108] 21. The method according to any one of statements 19-20, wherein 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.

[00109] 22. The method according to any one of statements 19-21 further comprising cleaving the amino acid protecting group X.

[00110] 23. The method according to any one of statements 19-22, further comprising subjecting the isolated dehydroamino acid linked substrate bound peptide to an in vitro peptide synthesis method comprising 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.

[00111] 24. The method of statement 23, further comprising cleaving the elongated synthetic peptide from the substrate.

[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.

[00113] 26. The method of statement 25, wherein the substituting comprises the method according to statements 19-24.

[00114] 27. The method according to any one of statements 25-26, wherein the increased stability comprises an increased half-life of the synthetic peptide

[00115] 28. The method according to any one of statements 25-27, wherein the synthetic peptide comprises at least one β-turn motif and the increased stability comprises an increased stability of the β-turn motif of the synthetic peptide comprising the at least one β-turn motif.

EXAMPLES

Example 1. Synthesis of AVal-based azlactones.

[00116] Initial attempts to execute the synthetic strategy outlined in Scheme 1 employed Fmoc-protected dipeptides of type 1. Although formation of the azlactones was facile, heating solutions of these species in the presence of resin- bound peptides yielded only trace amounts of the coupled products according to MS analysis. Further investigation suggested that the Fmoc group was not stable under the reaction conditions.

[00117] Accordingly, we examined Alloc-protected substrates. This group was more stable than the Fmoc group under the weakly basic coupling conditions. Moreover, it is possible to cleave the Alloc moiety from solid-supported peptides, which has been accomplished under conditions that are compatible with the acid- labile side-chain protecting groups that are typically employed in Fmoc-based SPPS.

[00118] Therefore, we constructed AVal-containing dipeptides 1 and azlactones 2 as outlined in Scheme 2. Hydrogenolysis of racemic Cbz-protected β-OHVal derivative 4 and coupling of the resulting amine with Alloc-Gly or Alloc-Phe afforded dipeptides 5a and 5b, respectively, in good yields. Saponification furnished the free acids la and lb, which underwent facile dehydration and cyclization upon exposure to AC2O and NaOAc. The azlactones 2 were typically used in subsequent reactions without purification due to their sensitivity to S1O2.

Scheme 2. Synthesis of AVal-based azlactones.

1a (96%) 2a (90%;

1 b (88%) 2b (72%

Example 2 Synthesis of ΔΕην-based azlactones.

[00119] In preparation for synthesizing the corresponding ΔΕην-containing azlactones, we prepared racemic β-hydroxy ethylnorvaline derivative 7 in one-step via base- free aminohydroxylation of known enoate 6 (Scheme 3). This compound was transformed into azlactones 2c and 2d via the same sequence employed to convert 4 into 2a and 2b. COMU was found to be superior to EDOHC1 and HOBt for mediating peptide couplings of the hindered amine derived from 7. The slightly lower yields of the couplings and dehydration-cyclization reactions in the ΔΕην series are presumably a consequence of the bulkier nature of this amino acid relative to AVal.

Scheme 3. Synthesis of ΔΕην-based azlactones.

,CO_zEt CbzHN-OMs ^{1■ H}2 (⁶⁵⁰ P^si)

Os0₄ (10 mol %) 10% Pd/C, MeOH

CH₃CN-H₂0 8: 1 2. Alloc-Gly or Phe

45 °C

COMU, sym-collidine

5c (R = H, 77%) 1c (93%)

5d (R = Bn, 63%) 1d (84%)

Example 3 Incorporation of azlactones into resin-bound peptides

[00120] We explored the incorporation of azlactones 2a-d into resin-bound peptides. We used a therapeutically relevant peptide as a system for determining the viability of our synthetic strategy. Accordingly, we decided to synthesize analogues of the C-terminal region of enfuvirtide, an FDA-approved inhibitor of HIV membrane fusion. Thus, attachment of Fmoc-Phe-OH to Rink amide resin and elaboration using standard Fmoc-based SPPS techniques afforded resin-bound pentapeptide 9 (Scheme 4). Different solvents (DMF versus NMP), reaction temperatures (50 °C versus 60 °C), and additives (DMAP versus no additive) were then evaluated in the coupling of9 with azlactone 2a. Analysis of the reactions by LC/MS revealed that heating the mixture at 60 °C in NMP for 24 h in the presence of DMAP yielded the best results, although the coupling also proceeded in the absence of the additive. Therefore, these conditions were employed to mediate the ring openings of azlactones 2b-2d by 9. Each reaction afforded the desired product as evidenced by MS, but the yields decreased somewhat as the size of the azlactones increased (vide infra).

[00121] The Alloc groups of peptides lOa-d were then removed using conditions (Pd(PPh₃)₄, PhSiH₃) that were previously developed for the deprotection of resin-bound peptides. This process was repeated to ensure complete conversion. Coupling of the resulting resin-bound amines to Fmoc-Trp(Boc)-OH was then accomplished under standard conditions. Finally, TFA-mediated deprotection and cleavage of the octapeptides from the solid support furnished the targeted compounds lla-lld, which were purified via reverse-phase HPLC.

[00122] The nature of SPPS renders it impossible to precisely determine the yield of a single step in a synthetic sequence. Notwithstanding, we were interested in comparing the yields of the couplings of azlactones 2a-2d with 9. The HPLC traces of the crude final products lla-lld provided us with a qualitative means of making this comparison. The trace for crude 11a, which was derived from the smallest azlactone 2a, was very clean with only a handful of minor impurities present. The traces for crude lib and 11c, which are derived from medium-sized azlactones 2b and 2c, were similar to each other and showed a few more impurities than the trace for 11a. Still more impurities were visible in the trace for crude lid, which is obtained from the largest azlactone 2d. Importantly, all four targeted octapeptides were abundant and were easily purified to homogeneity.

Scheme 4. Solid-phase synthesis of ΔΑΑ-containing peptides.

[00123] These results demonstrate that we have devised a protocol for incorporating bulky tetrasubstituted α,β-dehydroamino acids into peptides via solid-phase peptide synthesis. Further, these methods may be used to generate therapeutically relevant peptides. Our method capitalizes on the facile generation of azlactones (i.e., oxazolones) from dipeptides that possess C-terminal β-hydroxy amino acids via a one-pot dehydration-cyclization process. These azlactone rings are readily opened by the amino groups of resin-bound peptides. Example 4 Experimental Details

[00124] Dimethylformamide and methanol were dried by passage through a solvent drying system containing cylinders of activated alumina. Other solvents and reagents were purchased from commercial vendors and used without purification. Flash chromatography was carried out using 60-230 mesh silica gel Microwave-promoted reactions were conducted in a CEM Corporation Mars Microwave Reaction System at constant temperature. ^XH NMR spectra were acquired on a 500 MHz spectrometer with chloroform (7.27 ppm) as internal reference. Signals are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), br s (broad singlet), m (multiplet). Coupling constants are reported in hertz (Hz). ¹³C NMR spectra were acquired on a spectrometer operating at 125 MHz with chloroform (77.23 ppm) as internal reference. Infrared spectra were obtained on an FT-IR spectrometer. Mass spectral data were obtained using ESI techniques.

Example 5 Generation of Ethyl 2-(((benzyloxy)carbonyl)amino)-3-ethyl-3- hydroxypentanoate

Structure 7

[00125] A solution of benzyl ((methylsulfonyl)oxy)carbamate (See, Qin, L.; Zhou, Z.; Wei, J.; Yan, T.; Wen, H. Synth. Commun.2010, 40, 642 for teachings thereof) (1.472 g, 6.00 mmol, 1.3 equiv) in CH₃CN (50 mL) at rt was treated with Os0₄ (4 wt % solution in ¾0, 2.9 mL, 0.46 mmol, 0.10 equiv), stirred for 10 min, and then treated with a solution of ethyl 3-ethylpent-2-enoate (See, Rawat, V.;

Chouthaiwale, P. V.; Chavan, V. B.; Suryavanshi, G.; Sudalai, A. Tetrahedron Lett. 2010, 51, 6565 for teachings thereof) (6, 710 mg, 4.54 mmol) in CH₃CN (10.0 mL) and ¾0 (4.7 mL). The resulting mixture was stirred at 45 °C for 4 d, treated with sat aq K2S2O5 (20 mL), and stirred for an additional 20 min. It was then diluted with H2O (40 mL) and extracted with EtOAc (4 x 80 mL). The combined organic layers were washed with sat aq aHC0₃ (2 x 40 mL) and brine (40 mL), dried ( a2S0₄), and concentrated in vacuo. Flash chromatography (300 mL of Si0₂, 0.25-5% MeOH in CH₂C1₂ gradient elution) afforded 7 (1.10 g, 3.40 mmol, 75%) as a yellow oil: ^XH NMR (CDC1₃, 500 MHz) δ 7.27-7.20 (m, 5H), 5.94 (d, J= 9.0 Hz, 1H), 5.20 (s, 2H), 4.29 (d, J= 9.5 Hz, 1H), 4.17-4.05 (m, 2H), 2.75 (br s, 1H), 1.54-1.38 (m, 4H), 1.19 (t, J= 7.0 Hz, 3H), 0.85 (t, J= 7.5 Hz, 3H), 0.78 (t, J= 7.5 Hz, 3H); ¹³C MR (CDC1₃, 125 MHz) δ 172.3, 156.3, 136.2, 128.5 (2C), 128.2 (2C), 128.1, 76.1, 67.1, 61.5, 58.5, 27.31, 27.29, 14.1, 7.59, 7.55; IR (film) v_max 3366, 3033, 2972, 1719, 1509, 1334, 1051 cm^"1; HRMS (ESI) m/z 324.1808 (MH⁺, Ci₇H₂5 0₅H⁺ requires 324.181 1).

Example 6 Generation of Ethyl 2-(2-(((allyloxy)carbonyl)amino)acetamido)-3- hydroxy-3 -methylbutanoate

Structure 5 a

[00126] A solution of 4 (See, Ma, Z.; Naylor, B. C; Loertscher, B. M.; Hafen, D.D.; Li, J. M.; Castle, S. L. J. Org. Chem.2012, 77, 1208 for teachings thereof) (353 mg, 1.20 mmol) in MeOH (10 mL) was treated with 10% Pd/C (1 14.6 mg, 0.32 wt equiv) and stirred at rt under H₂ (650 psi) for 1 d. The mixture was filtered through a pad of Celite (washed with 125 mL of MeOH), and the filtrate was concentrated in vacuo to afford the crude amine (198 mg, 193 mg theoretical yield, quant.), a portion of which was used without further purification in the next reaction.

[00127] The crude amine (81 mg, 0.50 mmol, 1.0 equiv) was dissolved in DMF (2 mL) and added to a solution of ((allyloxy)carbonyl)glycine (88 mg, 0.55 mmol, 1.1 equiv) in DMF (3 mL) at 0 °C under Ar. The mixture was stirred for 10 min, then treated with EDOHC1 (115.7 mg, 0.604 mmol, 1.2 equiv) and HOBt (81.5 mg, 0.532 mmol, 1.1 equiv). The resulting mixture was stirred at 0 °C to rt under Ar for 24 h, then diluted with H₂0 (15 mL) and extracted with EtOAc (4 x 30 mL). The combined organic layers were washed with brine (15 mL), dried (Na₂S0₄), and concentrated in vacuo. Flash chromatography (50 mL of Si0₂, 0.5-5 % MeOH in CH2CI2 gradient elution) afforded 5a (122 mg, 0.404 mmol, 80%) as a light yellow oil: 'H NMR (CDCI3, 500 MHz) δ 6.91 (d, J= 9.0 Hz, 1H), 5.96-5.88 (m, 1H), 5.49 (br s, 1H), 5.33 (d, J= 17.0 Hz, 1H), 5.24 (d, J= 13.5 Hz, 1H), 4.64-4.56 (m, 3H), 4.54 (d, J= 9.0 Hz, 1H), 4.30-4.20 (m, 2H), 3.95 (d, J= 6.0 Hz, 2H), 1.32 (t, J= 7.0 Hz, 3H), 1.30 (s, 3H), 1.26 (s, 3H); ¹³C NMR (CDC1₃, 125 MHz) δ 171.2, 169.3, 156.5, 132.5, 118.0, 72.0, 66.1, 61.8, 59.8, 44.4, 26.7, 26.6, 14.1 ; IR (film) v_max 3338, 2982, 1728, 1531, 1213 cm^"1; HRMS (ESI) m/z 303.1534 (MH⁺, Ci₃H₂₂N₂0₆H⁺ requires 303.1556).

Example 7 Generation of Ethyl 2-((5)-2-(((allyloxy)carbonyl)

phenylpropanamido)-3 -hydroxy-3 -methylbutanoate

Structure 5b.

[00128] A solution of the crude amine prepared via hydrogenolysis of 4 (73 mg, 0.45 mmol, 1.0 equiv) in DMF (2 mL) was added to a solution of

((allyloxy)carbonyl)-L-phenylalanine (127.7 mg, 0.512 mmol, 1.1 equiv) in DMF (3 mL) at 0 °C under Ar. The mixture was stirred for 10 min, then treated with EDOHC1 (96.1 mg, 0.501 mmol, 1.1 equiv) and HOBt (68.4 mg, 0.447 mmol, 0.99 equiv). The resulting mixture was stirred at 0 °C to rt under Ar for 24 h, then diluted with H₂0 (15 mL) and extracted with EtOAc (4 x 40 mL). The combined organic layers were washed with brine (10 mL), dried (Na₂S0₄), and concentrated in vacuo. Flash chromatography (50 mL of Si0₂, 0.5-5 % MeOH in CH₂C1₂ gradient elution) afforded 5b (132 mg, 0.336 mmol, 74%) as a light yellow oil that was a 1 : 1 mixture of diastereomers: ¾ NMR (CDC1₃, 500 MHz) δ 7.31-7.19 (m, 5H), 6.81-6.75 (m, 1H), 5.94-5.82 (m, 1H), 5.47 (d, J= 7.0 Hz, 1H), 5.29 (d, J = 10.0 Hz, 1H), 5.26 (d, J= 8.5 Hz, 1H), 4.71-4.64 and 4.59-4.40 (2m, 5H), 4.23- 4.17 (m, 2H), 3.25-3.19 (m, 1H), 3.16-3.11 (m, 1H), 1.29 (t, J= 7.5 Hz, 3H), 1.23 (s, 3H), 1.19 (s, 3H); ¹³C NMR (CDC1₃, 125 MHz) δ 171.4, 171.0 and 170.7, 155.7, 136.2 and 136.1, 132.7 and 132.4, 129.5 and 129.3 (2C), 128.8 and 128.5 (2C), 127.1 and 127.0, 1 17.9 and 1 17.7, 72.0 and 71.9, 66.0, 61.62 and 61.58, 56.2, 54.6, 38.6 and 38.3, 26.63 and 26.58, 26.5 and 26.4, 14.1; IR (film) v_max 3338, 2982, 1728, 1531, 1213, 1050 cm^"1; HRMS (ESI) m/z 393.1989 (MH⁺, C₂oH₂₈N₂0₆H⁺ requires 393.2026). Example 8 Generation of Ethyl 2-(2-(((allyloxy)carbonyl)amino)acetamido)-3-ethyl- 3 -hydroxypentanoate

Structure 5c

[00129] A solution of 7 (470 mg, 1.45 mmol) in MeOH (14 mL) was treated with 10% Pd/C (190 mg, 0.4 wt equiv) and stirred at rt under H₂ (650 psi) for 2 d. The mixture was filtered through a pad of Celite (washed with 200 mL of MeOH), and the filtrate was concentrated in vacuo to afford the crude amine (258 mg, 1.36 mmol, 94%), a portion of which was used without further purification in the next reaction.

[00130] The crude amine (41 mg, 0.22mmol, 1.0 equiv) was dissolved in DMF (1 mL) and added to a solution of ((allyloxy)carbonyl)glycine (103.4 mg, 0.650 mmol, 3.0 equiv) in DMF (1 mL) at 0 °C under Ar. The mixture was stirred for 10 min, then treated with COMU (279.7 mg, 0.653 mmol, 3.0 equiv) and 2,4,6- collidine (172 μί, 158 mg, 1.30 mmol, 6.0 equiv). The resulting mixture was warmed to rt and stirred for 12 h. It was then cooled to 0 °C, quenched with sat aq aHC0₃ (3 mL), and extracted with EtOAc (4 x 10 mL). The combined organic layers were washed with brine (5 mL), dried (Na₂S0₄), and concentrated in vacuo. Flash chromatography (50 mL of Si0₂, 0.5-5 % MeOH in CH₂C1₂ gradient elution) afforded 5c (59 mg, 0.18 mmol, 82%, 77% from 7) as a light yellow oil: ^XH NMR (CDC1₃, 500 MHz) δ 6.79 (d, J= 8.5 Hz, 1H), 5.97-5.88 (m, 1H), 5.40 (br s, 1H), 5.32 (d, J= 17.0 Hz, 1H), 5.23 (d, J= 1 1.0 Hz, 1H), 4.64^1.58 (m, 3H), 4.32-4.28 (m, 1H), 4.27-4.19 (m, 2H), 3.94 (s, 2H), 1.58-1.46 (m, 4H), 1.31 (t, J= 7.5 Hz, 3H), 0.94 (t, J= 7.5 Hz, 3H), 0.87 (t, J= 7.5 Hz, 3H); ¹³C NMR (CDC1₃, 125 MHz) δ 171.8, 169.0, 156.4, 132.5, 118.0, 76.3, 66.7, 61.6, 56.7, 44.4, 28.4, 26.6, 14.1, 7.6, 7.5; IR (film) v_max 3347, 2975, 1729, 1521, 1255 cm^"1; HRMS (ESI) m/z 331.1879 (MH⁺, Ci₅H₂₆N₂0₆H⁺ requires 331.1869).

Example 9 Generation of Ethyl 2-((5)-2-(((allyloxy)carbonyl)amino)-3- phenylpropanamido)-3 -ethyl-3 -hydroxypentanoate

Structure 5d

[00131] A solution of the crude amine prepared via hydrogenolysis of 7 (50 mg, 0.26 mmol, 1.0 equiv) in DMF (1 mL) was added to a solution of

((allyloxy)carbonyl)-L-phenylalanine (198.5 mg, 0.796 mmol, 3.0 equiv) in DMF (1 mL) at 0 °C under Ar. The mixture was stirred for 10 min, then treated with COMU (340.2 mg, 0.794 mmol, 3.0 equiv) and 2,4,6-collidine (210 μί, 193 mg, 1.59 mmol, 6.0 equiv). The resulting mixture was warmed to rt and stirred for 12 h. It was then cooled to 0 °C, quenched with sat aq aHC0₃ (3 mL), and extracted with EtOAc (4 x 10 mL). The combined organic layers were washed with brine (5 mL), dried ( a2S0₄), and concentrated in vacuo. Flash chromatography (50 mL of Si0₂, 0.5-5 % MeOH in CH₂C1₂ gradient elution) afforded 5d (75 mg, 0.18 mmol, 68%, 63% from 7) as a light yellow oil that was a 1 : 1 mixture of diastereomers: 1H

NMR (CDC1₃, 500 MHz) δ 7.30-7.17 (m, 5H), 6.95 and 6.91 (2d, J= 9.0 and 8.5 Hz, 1H), 5.89-5.79 (m, 1H), 5.57 and 5.52 (2d, J= 7.0 and 7.5 Hz, 1H), 5.26-5.16 (m, 2H), 4.61 (d, J= 9.0 Hz, 1H), 4.57^1.46 (m, 3H), 4.23-4.14 (m, 2H), 3.14-3.03 (m, 2H), 2.85 (s, 1H), 1.51-1.37 (m, 4H), 1.28 (t, J= 7.5 Hz, 3H), 0.93-0.73 (m, 6H); ¹³C NMR (CDCI3, 125 MHz) δ 171.8, 171.2 and 171.1, 155.8 and 155.7, 136.4 and 136.3, 132.5, 129.4 and 129.3 (2C), 128.6 and 128.5 (2C), 126.9, 1 17.8, 76.4, 65.9, 61.5 and 61.4, 57.0 and 56.8, 56.1 and 55.9, 38.7 and 38.3, 26.6, 26.5, 14.1 and 14.0, 7.63 and 7.60, 7.57 and 7.50; IR (film) v_max 3320, 2972, 2942, 1728, 1532, 1258, 1032 cm^"1; HRMS (ESI) m/z 421.2338 (MH⁺, C₂₂H₃₂N₂0₆H⁺ requires 421.2339). Example 10 Generation of Allyl ((5-oxo-4-(propan-2-ylidene)-4,5-dihydrooxazol-2- yl)methyl)carbamate

Structure 2a.

[00132] A solution of5a (112 mg, 0.370 mmol) in ?-BuOH-H₂0 (3 : 1, 4 mL) at 0 °C under Ar was treated with LiOH^»H₂0 (70 mg, 1.7 mmol, 4.5 equiv), then stirred at 0 °C for 5 h. The resulting mixture was acidified to pH 1-2 by the addition of 2 N HC1 (1.5 mL) and extracted with EtOAc (6 x 6 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid la (98 mg, 0.36 mmol, 96%) was used directly in the next reaction without further purification.

[00133] A solution of la (55 mg, 0.20 mmol) in acetic anhydride (1.2 mL) at rt under Ar was treated with sodium acetate (25 mg, 0.30 mmol, 1.5 equiv). The resulting mixture was stirred at 50 °C for 7 h, at which time the starting material had disappeared as evidenced by MS. The excess acetic anhydride was decomposed by the addition of H₂0 (0.8 mL). The mixture was then extracted with EtOAc (4 5 mL), washed with sat aq Na₂C0₃ (3 x 3 mL) and brine (1.5 mL), dried (Na₂S0₄), and concentrated in vacuo to afford 2a (43 mg, 0.18 mmol, 90%) as a light yellow solid: ^XH NMR (CDC1₃, 500 MHz) δ 5.96-5.91 (m, 1H), 5.39- 5.32 (m, 2H), 5.25 (d, J= 10.5 Hz, 1H), 4.63 (br s, 2H), 4.30 (d, J= 5.5 Hz, 2H), 2.36 (s, 3H), 2.26 (s, 3H); HRMS (ESI) m/z 239.1076 (MH⁺, CnH₁₄N₂0₄H⁺ requires 239.1032).

Example 1 1 Generation of Allyl (5)-(l-(5-oxo-4-(propan-2-ylidene)-4,5- dihydrooxazol-2-yl)-2-phenylethyl)carbamate

Structure 2b.

[00134] A solution of 5b (60 mg, 0.15 mmol) in ?-BuOH-H₂0 (3 : 1, 2 mL) at 0 °C under Ar was treated with LiOH^»H₂0 (30.7 mg, 0.732 mmol, 4.8 equiv), then stirred at 0 °C for 5 h. The resulting mixture was acidified to pH 1-2 by the addition of 2 N HCl (0.8 mL) and extracted with EtOAc (6 x 6 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid lb (49 mg, 0.13 mmol, 88%) was used directly in the next reaction without further purification.

[00135] A solution of lb (49 mg, 0.13 mmol) in acetic anhydride (1 mL) at rt under Ar was treated with sodium acetate (17.4mg, 0.212 mmol, 1.6 equiv). The resulting mixture was stirred at 50 °C for 7 h, at which time the starting material had disappeared as evidenced by MS. The excess acetic anhydride was decomposed by the addition of H₂0 (0.5 mL). The mixture was then extracted with EtOAc (4 x 5 mL), washed with sat aq a₂C0₃(3x 3 mL) and brine (1.5 mL), dried ( a₂S0₄), and concentrated in vacuo to afford 2b (32 mg, 0.097 mmol, 72%) as a light yellow solid: ¾ NMR (CDC1₃, 500 MHz) δ 7.30-7.20 (m, 5H), 5.94-5.85 (m, 1H), 5.40-5.24 (m, 2H), 5.21 (d, J= 10.5 Hz, 1H), 4.99^1.93 (m, 1H), 4.60-4.54 (m, 2H), 3.27-3.21 (m, 1H), 3.14-3.10 (m, 1H), 2.34 (s, 3H), 2.20 (s, 3H); HRMS (ESI) m/z 329.1517 (MH⁺, Ci₈H₂₀N₂O₄H⁺ requires 329.1501). Example 12 Generation of Allyl ((5-oxo-4-(pentan-3-ylidene)-4,5-dihydrooxazol-2- yl)methyl)carbamate

Structure 2c.

[00136] A solution of 5c (55 mg, 0.17 mmol) in ?-BuOH-H₂0 (3 : 1, 2 mL) at 0 °C under Ar was treated with LiOH^»H₂0 (32.4 mg, 0.772 mmol, 4.6 equiv), then stirred at 0 °C for 5 h. The resulting mixture was acidified to pH 1-2 by the addition of 2 N HCl (0.8 mL) and extracted with EtOAc (6 x 6 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid lc (47 mg, 0.16 mmol, 93%) was used directly in the next reaction without further purification. [00137] A solution of lc (47 mg, 0.16 mmol) in acetic anhydride (1 mL) at rt under Ar was treated with sodium acetate (19.7 mg, 0.240 mmol, 1.5 equiv). The resulting mixture was stirred at 50 °C for 7 h, at which time the starting material had disappeared as evidenced by MS. The excess acetic anhydride was decomposed by the addition of H₂0 (0.5 mL). The mixture was then extracted with EtOAc (4 5 mL), washed with sat aq Na₂C0₃ (3x 3 mL) and brine (1.5 mL), dried (Na₂S0₄), and concentrated in vacuo to afford 2c (35 mg, 0.13 mmol, 85%) as a light yellow solid: ^XH NMR (CDC1₃, 500 MHz) δ 5.99-5.90 (m, 1H), 5.40- 5.35 (m, 1H), 5.32 (d, J= 10.5 Hz, 1H), 5.25 (d, J= 10.5 Hz, 1H), 4.63 (br s, 2H), 4.30 (d, J= 5.0 Hz, 2H), 2.80 (q, J= 7.5 Hz, 2H), 2.66 (q, J= 7.5 Hz, 2H), 0.89 (t, J= 7.0 Hz, 3H), 0.87-0.82 (m, 3H); HRMS (ESI) m/z 267.1363 (MH⁺,

Ci₃H₁₈N₂0₄H⁺ requires 267.1345).

Example 13 Generation of Allyl (S)-(l-(5-oxo-4-(pentan-3-ylidene)-4,5- dihydrooxazol-2-yl)-2-phenylethyl)carbamate

Structure 2d.

[00138] A solution of 5d (52 mg, 0.12 mmol) in ?-BuOH-H₂0 (3: 1, 2 mL) at 0 °C under Ar was treated with LiOH^»H₂0 (26.6 mg, 0.634 mmol, 5.1 equiv), then stirred at 0 °C for 5 h. The resulting mixture was acidified to pH 1-2 by the addition of 2 N HCl (0.8 mL) and extracted with EtOAc (6 x 6 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid Id (41 mg, 0.10 mmol, 84%) was used directly in the next reaction without further purification.

[00139] A solution of Id (41 mg, 0.104 mmol) in acetic anhydride (1 mL) at rt under Ar was treated with sodium acetate (14.1 mg, 0.172 mmol, 1.7 equiv). The resulting mixture was stirred at 50 °C for 7 h, at which time the starting material had disappeared as evidenced by MS. The excess acetic anhydride was decomposed by the addition of H₂0 (0.5 mL). The mixture was then extracted with EtOAc (4 5 mL), washed with sat aq a₂C0₃ (3 x 3 mL) and brine (1.5 mL), dried ( a2S0₄), and concentrated in vacuo to afford 2d (24 mg, 0.067 mmol, 64%) as a light yellow solid: ^XH NMR (CDC1₃, 500 MHz) δ 7.33-7.22 (m, 5H), 5.96- 5.85 (m, 1H), 5.34-5.19 (m, 3H), 5.00^1.95 (m, 1H), 4.60-4.54 (m, 2H), 3.29-3.22 (m, 1H), 3.14-3.08 (m, 1H), 2.81 (q, J= 7.5 Hz, 2H), 2.59 (q, J= 8.0 Hz, 2H), 0.91-0.82 (m, 6H); HRMS (ESI) m/z 357.1832 (MH⁺, C₂oH₂₄N₂0₄H⁺ requires 357.1814).

Example 14 Generation of Ethyl 2-(2-(((allyloxy)carbonyl) methylbutanamido)-3 -hydroxy-3 -methylbutanoate

Structure 12

12

[00140] ((Allyloxy)carbonyl)valine (658.3 mg, 3.27 mmol, 1.0 equiv.) was dissolved in dry DMF (10 ml) and cooled to 0 °C. EDC.HC1 (672.5 mg, 3.27 mmol, 1.0 equiv.) and HOBt (552.9 mg, 3.28 mmol, 1.0 equiv.) were added to the cold solution and the reaction mixture was allowed to stir for an hour under Ar at 0 °C. A solution of ethyl 2-amino-3 -hydroxy-3 -methylbutanoate (351.8 mg, 2.18 mmol, 0.67 equiv) in DMF (5 mL) was added to the above reaction mixture and allowed to stir at 0 °C to rt under Ar for 14 h. Then, the reaction was quenched with sat aq. aHC0₃ (15 mL) and extracted with EtOAc (4 x 25 mL). The combined organic layers were washed with brine (3 x 15 mL), dried over Na₂S0₄, and concentrated in vacuo. Flash chromatography (75.0 mL of Si0₂, 0.5-2.5 % MeOH in CH₂C1₂ gradient elution) afforded (12) (531.2 mg, 1.54 mmol, 71%) as a light yellow oil: ¾ NMR (CDC1₃, 500 MHz) δ 6.84 (br s, 1H), 5.98-5.87 (m, 1H), 5.32 (d, J= 17.7 Hz, 1H), 5.23 (d, J = 10.9 Hz, 1H), 4.64^1.58 (d, J = 5.7 Hz, 2H), 4.50 (d, J = 8.6 Hz, 1H), 4.30^1.20 (m, 2H), 2.25-2.09 (m, 1H) , 1.36-1.23 (m, 9H), 1.04-0.91 (m, 6H).HRMS (ESI) m/z 345.2223 (MH⁺, Ci₆H₂₈N₂0₆H⁺ requires 345.2026).

Example 15 Generation of Allyl (2-methyl-l-(5-oxo-4-(propan-2-ylidene)-4,5- dihydrooxazol-2-yl)propyl)carbamate tructure 13

13

[00141] A solution of 12 (713.8 mg, 2.07 mmol, 1.0 equiv.) in ?-BuOH-H₂0 (3 : 1, 16 mL) at 0 °C under Ar was treated with LiOH^»H₂0 (347.9 mg, 8.29 mmol, 4.0 equiv), then stirred at 0 °C for 4 h. The resulting mixture was acidified to pH 1-2 by the addition of 1 N HC1 (7.5 mL) and extracted with EtOAc (4 x 25 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid obtained (633.9 mg 2.0 mmol) was used directly in the next reaction without further purification.

[00142] A solution of the carboxylic acid (633.9 mg, 2.0 mmol) in acetic anhydride (8 mL) at rt under Ar was treated with sodium acetate (246.5 mg, 3.0 mmol, 1.5 equiv). The resulting mixture was stirred at rt for 12 h under Ar. The excess acetic anhydride was decomposed by the addition of H₂0 (15 mL). The mixture was then extracted with EtOAc (4 x 25 mL), washed with sat aq aHC0₃ (3 10 mL) and brine (25 mL), dried (Na₂S0₄), and concentrated in vacuo. Flash chromatography (50.0 mL of Si0₂, 1.0-8.0 % EtOAc in hexanes + 1% NEt₃ gradient elution) afforded (13) (413.4 mg, 1.47 mmol, 71%) as a light yellow oil: ¾ NMR (CDC1₃, 500 MHz) δ 5.96-5.81 (m, 1H), 5.39-5.21 (m, 2H), 4.66^1.57 (br s, 2H), 2.36 (s, 3H), 2.26 (s, 3H), 2.23-2.19 (m, 1H), 1.02 (d, J= 6.9 Hz, 3H), 0.96 (d, J= 6.9 Hz, 3H); HRMS (ESI) m/z 281.1495 (MH⁺, Ci₄H₂oN₂0₄H⁺ requires 281.1501).

Example 16 Generation of Ethyl 2-(2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-3 -ethyl-3 -hydroxypentanoate

Structure 14

[00143] ((Allyloxy)carbonyl)valine (688.6 mg, 3.42 mmol, 1.0 equiv.) was dissolved in dry DMF (10 ml) and cooled to 0 °C. EDC.HC1 (656.1 mg, 3.42 mmol, 1.0 equiv.) and HOBt (578.1 mg, 3.42 mmol, 1.0 equiv.) were added to the cold solution and the reaction mixture was allowed to stir for an hour under Ar at 0 °C. A solution of ethyl 2-amino-3-ethyl-3-hydroxypentanoate (434.2 mg, 2.29 mmol, 0.67 equiv) in DMF (5 mL) was added to the above reaction mixture and allowed to stir at 0 °C to rt under Ar for 14 h. Then, the reaction was quenched with sat aq. aHC0₃ (15 mL) and extracted with EtOAc (4 x 25 mL). The combined organic layers were washed with brine (3 x 15 mL), dried over Na₂S0₄, and concentrated in vacuo. Flash chromatography (80.0 mL of Si0₂, 0.5-2.0 % MeOH in CH₂C1₂ gradient elution) afforded (14) (495.6 mg, 1.33 mmol, 58%) as a light yellow oil: ¾ NMR (CDC1₃, 500 MHz) δ 5.99-5.85 (m, 1H), 5.25 (d, J= 16.9 Hz, 1H), 5.16 (d, J= 10.2 Hz, 1H), 4.75^1.54 (m, 3H), 4.37-4.18 (m, 2H), 4.14-4.06 (m, 1H), 2.25-2.09 (m, 1H) , 1.61-1.43 (m, 4H), 1.21-1.11 (m, 3H), 1.02-0.83 (m, 12H); HRMS (ESI) m/z 373.2403 (MH⁺, Ci₈H₃₂N₂0₆H⁺ requires 373.2339).

Example 17 Generation of Allyl (2-methyl-l-(5-oxo-4-(pentan-3-ylidene)-4,5- dihydrooxazol-2-yl)propyl)carbamate

Structure 15

15

[00144] A solution of 14 (473.1 mg, 1.27 mmol, 1.0 equiv.) in ?-BuOH-H₂0 (3 : 1, 12mL) at 0 °C under Ar was treated with LiOH^»H₂0 (213.2 mg, 5.08 mmol, 4.0 equiv), then stirred at 0 °C for 4 h. The resulting mixture was acidified to pH 1-2 by the addition of 1 N HC1 (5.0 mL) and extracted with EtOAc (4 x 20 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid obtained (327.1 mg, 0.95 mmol) was used directly in the next reaction without further purification.

[00145] A solution of the carboxylic acid (327.1 mg, 0.95 mmol) in acetic anhydride (8 mL) at rt under Ar was treated with sodium acetate (1 16.9 mg, 1.43 mmol, 1.5 equiv). The resulting mixture was stirred at rt for 12 h under Ar. The excess acetic anhydride was decomposed by the addition of H₂0 (15 mL). The mixture was then extracted with EtOAc (4 x 15 mL), washed with sat aq aHC0₃ (3 x 10 mL) and brine (25 mL), dried (Na₂S0₄), and concentrated in vacuo. Flash chromatography (50.0 mL of Si0₂, 1.0-5.0 % EtOAc in hexanes + 1 % NEt₃ gradient elution) afforded (15) (211.9 mg, 0.69 mmol, 54%) as a light yellow oil: ^ MR CDCL,, 500 MHz) δ 5.94-5.77 (m, 1H), 5.25 (d, J= 15.7 Hz, 1H), 5.14 (d, J= 10.8 Hz, 1H)„ 4.55-4.41 (m, 2H), 4.17^1.06 (m, 1H), 2.29-2.24 (m, 2H) 2.23-2.20 (m, 1H), 2.18-2.06 (m, 2H) 1.00-0.72 (m, 12H); HRMS (ESI) m/z 309.1843 (MH⁺, Ci₆H₂₄N₂0₄H⁺ requires 309.1814).

Example 18 Generation of Allyl 2-((l-ethoxy-3-hydroxy-3-methyl-l-oxobutan-2- yl)carbamoyl)pyrrolidine- 1 -carboxylate

Structure 16

[00146] ((Allyloxy)carbonyl)proline (1.973 g, 9.90 mmol, 1.0 equiv.) was dissolved in dry DMF (45 ml) and cooled to 0 °C. COMU (4.241 g, 9.90 mmol, 1.0 equiv.) and 2,4,6-collidine (1.3 mL, 9.84 mmol, 0.99 equiv.) were added to the cold solution and the reaction mixture was allowed to stir for half an hour under Ar at 0 °C. A solution of ethyl 2-amino-3-hydroxy-3-methylbutanoate (886.8 mg, 5.50 mmol, 0.56 equiv) in DMF (10 mL) was added to the above reaction mixture and allowed to stir at 0 °C to rt under Ar for 12 h. Then, the reaction was quenched with sat aq. NaHCC (25 mL) and extracted with EtOAc (4 x 25 mL). The combined organic layers were washed with brine (3 x 25 mL), dried over Na₂S0₄, and concentrated in vacuo. Flash chromatography (100.0 mL of Si0₂, 1.0-7.0 %

MeOH in CH₂C1₂ gradient elution) afforded (5) (996.4 mg, 2.91 mmol, 53%) as a light yellow oil: 'H NMR (CDC1₃, 500 MHz) δ 6.00-5.82 (m, 1H), 5.32-5.18 (m, 2H), 4.72^1.50 (m, 2H), 4.43^.33 (m, 1H), 4.30-4.17 (m, 2H), 3.65-3.31 (m, 2H), 2.37-2.08 (m, 2H), 1.98-1.84 (m, 2H), 1.36-1.21 (m, 9H); HRMS (ESI) m/z 343.1907 (MH⁺, Ci₆H₂₆N₂0₆H⁺ requires 343.1869). Example 19 Generation of Allyl 2-(5-oxo-4-(propan-2-ylidene)-4,5-dihydrooxazol- 2-yl)pyrrolidine- 1 -carboxylate

Structure 17

17

[00147] A solution of 16 (981.7 mg, 2.87 mmol, 1.0 equiv.) in ?-BuOH-H₂0 (3 : 1, 12mL) at 0 °C under Ar was treated with LiOH^»H₂0 (481.7 mg, 11.48 mmol, 4.0 equiv), then stirred at 0 °C for 4 h. The resulting mixture was acidified to pH 1-2 by the addition of 1 N HC1 (7.5.0 mL) and extracted with EtOAc (4 x 25 mL). The combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. The crude carboxylic acid obtained (646.9 mg, 2.06 mmol) was used directly in the next reaction without further purification.

[00148] A solution of the carboxylic acid (646.9, 2.06 mmol) in acetic anhydride (10 mL) at rt under Ar was treated with sodium acetate (253.2 mg, 3.09 mmol, 1.5 equiv). The resulting mixture was stirred at rt for 12 h under Ar. The excess acetic anhydride was decomposed by the addition of H₂0 (15 mL). The mixture was then extracted with EtOAc (4 x 20 mL), washed with sat aq NaHC0₃ (3 10 mL) and brine (25 mL), dried (Na₂S0₄), and concentrated in vacuo. Flash chromatography

(50.0 mL of Si0₂, 1.0-15.0 % EtOAc in hexanes + 1% NEt₃ gradient elution) afforded (17) (368.1 mg, 1.32 mmol, 46%) as a light yellow oil: ^XH NMR (CDC1₃, 500 MHz) δ 6.01-5.78 (m, 1H), 5.32-5.12 (m, 2H), 4.78-4.46 (m, 3H), 3.72-3.47 (m, 2H), 2.40-2.27 (m, 2H), 2.24 (s, 3H), 2.10 (s, 3H), 2.02-1.91 (m, 2H); HRMS (ESI) m/z 309.1843 (MH⁺, Ci₆H₂₄N₂0₄H⁺ requires 309.1814).

Example 20 Procedures for Solid-Phase Peptide Synthesis of Peptides

Attachment of C-terminal amino acid to resin

[00149] Rink amide MB HA resin (100-200 mesh, 10 μτηοΐ) was added to a fritted polypropylene syringe. The resin was swelled in CH₂C1₂ (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). Fmoc Deprotection

[00150] The resin (10 μιηοΐ) was treated with piperidine (20% solution in DMF, 250 μΚ) and allowed to stand for 1 min. The solution was drained from the resin using a vacuum manifold, and additional piperidine (20% solution in DMF, 250 μΚ) was added. The resulting mixture was stirred at 80 °C in a microwave oven for 4 min. The solution was drained from the resin using a vacuum manifold, and the resin was rinsed with DMF (5 x 3 mL).

Peptide coupling

[00151] The Fmoc -protected amino acid (50 μιηοΐ, 5 equiv) and HBTU (9.5 mg, 50 μιηοΐ, 5 equiv) were dissolved in a 0.1 M HOBt solution in NMP (250 μΐ,, 0.25 μιηοΐ, 5 equiv). z^'Pr₂NEt (8.5 μΐ,, 50 μιηοΐ, 10 equiv) was added to this solution, and it was allowed to stand for ca. 1 min. The solution was added to the resin (10 μιηοΐ), and the resulting mixture was stirred at 70 °C in a microwave oven for 10 min. The solution was drained from the resin using a vacuum manifold, and the resin was rinsed with DMF (5 x 3 mL).

Coupling of azlactone 2 with the resin-bound peptide

[00152] A solution of 2 (50 μιηοΐ, 5 equiv) and DMAP (5 μιηοΐ, 0.1 equiv) in NMP (300 μΚ) was added to the resin-bound peptide 9 (10 μιηοΐ). The resulting mixture was stirred at 60 °C for 24 h. The solution was drained from the resin using a vacuum manifold, and the resin was rinsed with DMF (5 x 2-3 mL).

Alloc deprotection

[00153] The resin-bound peptide 10 (10 μιηοΐ) was washed with CH₂CI₂ (10 x 4 mL) and placed under an Ar atmosphere. A solution of PhSiH₃ (29.7 μί, 26.1 mg, 241 μηιοΐ, 24 equiv) in CH₂CI₂ (1 mL) was added with stirring, followed by a solution of Pd(PPh₃)₄ (2.4 mg, 2 μηιοΐ, 0.2 equiv) in CH₂CI₂ (2 mL). The resulting mixture was stirred at rt under Ar for 10 min. The resin was washed with CH₂CI₂ (8 x 4 mL), and the deprotection reaction was repeated once.

10a (R = Me, R² = H)

10b (R¹ = Me, R² = Bn)

10c (R¹ = Et, R² = H)

10d (R¹ = Et, R² = Bn)

Cleavage of elongated peptides 10a- lOd from resin and purification

[00154] The resin was rinsed with CH₂CI₂ (10 x 2 mL), then treated carefully with a solution of phenol (50 mg, 530 μιηοΐ), H₂0 (50 μΚ), thioanisole (50 μΐ,, 53 mg, 430 μιηοΐ), ethanedithiol (25 μί, 28 mg, 300 μιηοΐ), and triisopropylsilane (10 μί, 7.7 mg, 50 μιηοΐ) in trifluoroacetic acid (800 μΚ) to avoid the buildup of excess

CO₂ pressure in the reaction vessel. The resulting mixture was stirred at rt for 4 h, and the peptide was precipitated by filtering the mixture and pouring the filtrate into cold Et20 (40 mL). The resin was rinsed with TFA (1 mL), and the precipitate was collected by centrifugation. The crude peptide was lyophilized and purified by HPLC (Discovery BIO Wide Pore C18-10 Column (25 cm x 10 mm, 10 μιη), 30- 50% CH₃CN (0.1% TFA) in H₂0 (0.1% TFA) gradient over 16 min, 6 mL/min flow rate, then 100% CH₃CN (0.1% TFA) for 15 min, 15 mL/min flow rate).The high resolution mass spectrometry (HRMS) characterization corresponding to each of the crude and purified peptides for each structure 11 a- 11 d is presented below.

[00155] Structure 11a: HRMS (ESI) m/z 1326.6124 (MH⁺, C₇4H7₉ i₃0iiH⁺ requires 1326.6100)

[00156] Structure lib: HRMS (ESI) m/z 1416.6556 (MH⁺, C₈iH₈₅Ni₃0iiH⁺ requires 1416.6570)

[00157] Structure 11c: HRMS (ESI) m/z 1354.6430 (MH⁺, C₇6H₈₃ i30iiH⁺ requires 1354.6413)

[00158] Structure lid: HRMS (ESI) m/z 1444.6921 (MH⁺, C₈₃H₈₉ i₃0iiH⁺ requires 1444.6883)

Example 21 Additional Procedures for Solid-Phase Peptide Synthesis of Peptides Attachment of C-terminal amino acid to resin

[00159] Rink amide MB HA resin (100-200 mesh, 100 μιηοΐ) was added to a fritted polypropylene syringe. The resin was swelled in (¾(¾ (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).

Fmoc Deprotection

[00160] To remove the Fmoc protecting group on the resin-linked amino acid, 5 mL of 20% piperidine in DMF was added to 100 μιηοΐ resin, and the resulting mixture was allowed to stand at room temperature for 1 min. The piperidine solution was drained from the resin with a vacuum manifold. Then, an additional 5 mL of 20% piperidine in DMF was added to the resin, and the resulting mixture was heated to 80 °C with stirring in a CEM MARS V multimode microwave (2 min ramp to 80 °C, 2 min hold at 80 °C). The piperidine solution was then drained from the resin using a vacuum manifold, and the resin was rinsed five times with DMF.

Peptide coupling

[00161] For coupling of an activated amino acid to a newly deprotected amine on resin, the desired Fmoc -protected amino acid (500 μιηοΐ, 5 eq) and HBTU (190mg, 500 μιηοΐ, 5 eq) were dissolved by vortexing in 15 mL 0.1 M HOBt (500 μιηοΐ, 5 eq) in NMP. To the dissolved amino acid solution was added 174 μΐ_^ DIEA (1000 μιηοΐ, 10 eq). The resulting mixture was vortexed briefly and allowed to react for at least 1 min. 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

subsequently rinsed five times with DMF. The cycles of deprotection and coupling were alternately repeated to give the desired full-length protein.

Coupling of azlactone with the resin-bound peptide

[00162] A solution of azlactone according to structures 13, 15, or 17 (500-750 μιηοΐ, 5 equiv) and Et₃ (250^150 L, 1.79-3.23 mmol, 17.9-32.3 equiv) in MP (10 mL) was added to the resin-bound peptide (100 μιηοΐ). The resulting mixture was stirred slowly at 80 °C for 36-48 h. The solution was drained from the resin using a vacuum manifold, and the resin was rinsed with DCM (5 x 10 mL).

Alloc deprotection

[00163] The resin-bound peptide (100 μιηοΐ) was washed with dry CH2CI2 (5 x 10 mL), placed under an Ar atmosphere and suspended the resin in dry DCM (5 mL). A solution of PhSiH₃ (0.75 mL, 657.75 mg, 6.08 mmol, 60.8 equiv) in CH₂C1₂ (4 mL) was added with stirring, followed by a solution of Pd(PPh₃)₄ (60.1 mg, 52 μιηοΐ, 0.52 equiv) in CH2CI2 (4 mL). The resulting mixture was stirred at rt under Ar for 20 min. The resin was washed with CH2CI2 (5 x 10 mL), and the deprotection reaction was repeated once.

Synthesis of an elongated synthetic peptide

[00164] Following Alloc deprotection, solid phase peptide synthesis as described herein was carried out to produce peptides according to structures 18, 19, and 20 below. 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 β-turn.

Cleavage of elongated peptides 18, 19, and 20 from resin and purification

[00165] The resin was rinsed with CH2CI2 (5 x 10 mL), then treated carefully with a solution of phenol (500 mg, 5.3 mmol), H₂0 (500 μΚ), thioanisole (500 μί, 530 mg, 4.3 mmol), ethanedithiol (250 μί, 230 mg, 3 mmol), and triisopropylsilane (100 μί, 77 mg, 500 μιηοΐ ) in trifluoroacetic acid (8 mL) to avoid the buildup of excess CO2 pressure in the reaction vessel. The resulting mixture was stirred at rt for 4-8 h, and the peptide was precipitated by filtering the mixture and pouring the filtrate into two centrifuge tubes containing cold Et20 (35 mL). The resin was rinsed with TFA (2 x 3 mL), and the precipitate was collected by centrifugation. The crude peptide was lyophilized and purified by HPLC (Discovery BIO Wide Pore CI 8- 10 Column (25 cm x 10 mm, 10 μιη), gradient elution with CH₃CN (0.1% TFA) in H₂0 (0.1% TFA)-15 mL/min flow rate. Each of the cleaved peptides were characterized by HPLC and HRMS; results indicated below.

Structure 18; Retention time: 18.428 min (15-35 % CH₃CN), 15 mL/min flow rate. HRMS (ESI) m/z = 1479.87, [(m/z)/2] = 740.45 [C₆₉Hii₆ 2oOi₆ ²⁺ m/z = 1481.8099]

Structure 19; Retention time: 16.557 min (21^15 % CH₃CN), 15 mL/min flow rate. HRMS (ESI) [(m/z)/2] = 754.45, [C₇iH₁₂oN₂oOi₆ ²⁺ m/z = 1509.8639]

Structure 20; Retention time: 10.929 min (21-31 % CH₃CN), 15 mL/min flow rate. HRMS (ESI) [(m/z)/2] = 760.94 [C₇2H₁₂oN₂₀Oi₆ ²+ = 1521.8749]

©

Example 22 Increased stability of β-turn containing peptides with at least one dehydroamino acid

[00166] 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 β-turn secondary structure were investigated.

Experimental Methods

[00167] 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 μϊ_^ 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 μϊ_^ of pronase E mixture at 37 °C. A 50 μΐ. aliquot was removed after 0, 5, 10, 15, 30, 45, 60, 120, 180, 300 and 420 min time points. The aliquots were quenched with 10 μϊ_^ glacial acetic acid and the volumes were made up to 75 μΐ, with the phosphate buffer. 40 μΐ, was injected on the analytical RP-HPLC [Column-Phenomenex JuPiter 5μ CI 8 300 A (250 x 4.60 mm, 5 micron)] using a gradient of 10%-40% acetonitrile over 30 minutes, 95% acetonitrile from 30-35 minutes; flow rate: 1 mL/min. The acetic acid peak was used as an internal control to account for the differences in the volumes of injections from the auto-injector.

Results

[00169] Three peptides according to structures 18-20 were synthesized according to the methods described herein. Each of these peptides demonstrated a common β-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 β-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.

Claims

CLAIMS What is claimed is:

1. A synthetic peptide, comprising:

at least one dehydroamino acid, wherein the at least one dehydroamino acid is of Formula I:

wherein B¹ and B² 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.

2. The synthetic peptide of claim 1, wherein the peptide has a molecular weight of from about 0.1 kDa to about 250 kDa.

3. The synthetic peptide of claim 1, wherein B¹ and B² are each independently selected from Ci-Cio alkyl and Ci-Cio perfluoroalkyl.

4. The synthetic peptide of claim 1, wherein aromatic is phenyl and heterocyclic is selected from indole, pyrrole, azole and pyrrolidine.

5. The synthetic peptide of claim 1, wherein B¹ and B² are different.

6. The synthetic peptide of claim 1, wherein B¹ and B² are the same.

7. The synthetic peptide of claim 1, comprising one or more peptide subunits according to Formula II

wherein R¹ is hydrogen or any 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.

8. The synthetic p ing to Formula III:

wherein Y² comprises at least one or more natural or non-natural amino acids; and X is an amino acid protecting group.

9. The synthetic peptide of claim 8, wherein Y² further comprises one or more dehydroamino acids according to Formula I.

10. The synthetic peptide of claim 8, wherein 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, 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.

11. The synthetic peptide of claim 10, wherein the amino acid protecting group is Alloc.

12. The synthetic peptide of claim 7 comprising a structure according to Formula TV:

wherein Y¹ and Y² are each a peptide chain comprising at least one or more natural or non-natural amino acids.

13. The synthetic peptide of claim 12, wherein Y¹ and Y² or Y¹ or Y² optionally comprise one or more additional dehydroamino acids according to the structure of Formula I.

14. The synthetic peptide of claim 12, wherein B¹ and B² are both ethyl or B¹ and B² are both methyl.

15. The synthetic peptide of claim 12, wherein R¹ is hydrogen or the side chain of phenylalanine.

16. A pharmaceutical composition comprising the synthetic peptide of claim 12 and a pharmaceutically acceptable carrier.

17. A synthetic peptide accordin to formula V:

wherein 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;

R¹ is hydrogen or any naturally occurring proteinogenic amino acid side chain; B¹ and B² 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;

L is a leaving group selected from hydroxy, iodo, bromo, chloro and sulfonate; and

n is an integer greater than or equal to 1.

18. A synthetic peptide precursor comprising one or more azlactones according to Formula VI:

R

wherein R¹ is hydrogen or any naturally occurring proteinogenic amino acid side chain;

B¹ and B² 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.

19. A method of making a synthetic peptide, comprising:

(a) providing a substrate bound peptide having a free amine;

(b) contacting the substrate bound peptide with an azlactone of Formula VI according to claim 18 to form a reaction mixture;

(c) heating the reaction mixture.

20. The method of claim 19 further comprising isolating a dehydroamino acid linked substrate bound peptide.

21. The method of claim 19, wherein 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.

22. The method of claim 19, further comprising cleaving the amino acid protecting group X.

23. The method of claim 20, further comprising subjecting the isolated

dehydroamino acid linked substrate bound peptide to an in vitro peptide synthesis method comprising 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.

24. The method of claim 23, further comprising cleaving the elongated synthetic peptide from the substrate.

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 claim 1.

26. The method of claim 25, wherein the increased stability comprises an increased half-life of the synthetic peptide.

27. The method of claim 25, wherein the synthetic peptide comprises at least one β- turn motif and the increased stability comprises an increased stability of the β-turn motif of the synthetic peptide comprising the at least one β-turn motif.