Post peptide cyclization to form disulfide bond mimetics

Abstract

The invention discloses a construction method of disulfide bond mimetics for later cyclization of peptides. The disclosed methods provide for late cyclization by thioacetalation of a peptide comprising two cysteine residues to form a thioacetal group. These bonds are all stable under acidic, alkaline and reducing conditions. The reaction may use a variety of cyclic ketones, even acetone, as cross-linking agents. Trifluoroacetic acid (TFA) alone can act as a catalyst and sole solvent, such that most of the peptide sequences are cyclized by the disclosed methods. The disclosed methods have numerous benefits and advantages, such as the fact that trifluoroacetic acid is used as a robust catalyst for the reaction and the sole solvent required, acetone and various cyclic ketones used as reactants are readily available, good chemoselectivity and good resistance of natural peptides to the reaction, and the fact that the resulting cross-links are structural mimetics of disulfide bonds.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 467,583, filed May 18, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0003] Field of invention

[0004] The disclosed invention generally falls within the field of protein and peptide crosslinking, and specifically within the field of peptide crosslinking using late disulfide bond mimics.

[0005] Background of the invention

[0006] In late-stage peptide modification, cyclization is a crucial strategy for enhancing conformational rigidity compared to linear peptides. Reduced flexibility locks the peptide in its conformation and improves stability and resistance to protease degradation. Peptide disulfide cyclization is a powerful technique in drug development, involving the formation of cyclic peptides by forming disulfide bonds between two cysteine ​​residues in the peptide chain. This process yields more stable and conformationally restricted molecules that exhibit improved pharmacological properties, such as enhanced potency, selectivity, and stability. In recent years, peptide disulfide cyclization has gained significant attention due to its potential in developing novel therapies for various diseases, including cancer, infectious diseases, and metabolic disorders. This method has been successfully used to develop several FDA-approved drugs, such as insulin, oxytocin, and vasopressin. However, the inherent instability of disulfide bonds in reducing environments limits its widespread application and broad drug distribution.

[0007] Any discussion of documents, actions, materials, devices, articles, etc. included in this specification shall not be construed as an admission that any or all of these matters constituted part of the prior art or common general knowledge in the field relating to this disclosure because of their existence prior to the priority date of each claim of this application.

[0008] Throughout this specification, the word “comprise” and its variations such as “comprises” or “comprising” should be understood to mean including the stated element, integer or step, or group of elements, integers or steps, but not excluding any other element, integer or step, or group of elements, integers or steps. Invention Overview

[0010] This application discloses a method for constructing disulfide bond mimics for late-stage peptide cyclization. Natural disulfide bonds are unstable in reducing environments, limiting their widespread application. The disclosed late-stage cyclization involves the thioacetalization of a peptide containing two cysteine ​​residues to form a thioacetal group, which is stable under acid, base, and reducing conditions. Compared to existing methods, the disclosed thioacetalization of peptides can generate novel disulfide bond mimics. This reaction can use a variety of cyclic ketones, even acetone, as a crosslinking agent. Almost all peptide sequences can be used in the disclosed method, as trifluoroacetic acid (TFA) can be used alone as both a catalyst and the sole solvent.

[0011] The disclosed method offers numerous benefits and advantages, such as the simultaneous use of trifluoroacetic acid as a robust catalyst and the only solvent required for the reaction, the readily available availability of acetone and various cyclic ketones as reactants, good chemoselectivity and tolerance of natural peptides to the reaction, and the fact that the resulting crosslinks are structural mimics of disulfide bonds.

[0012] This application discloses a method for late cyclization of peptides and the reagents used therein. Generally, the method involves maintaining a reaction mixture at a sufficient temperature for a sufficient time to form a product. Generally, the reaction mixture comprises a peptide containing two or more cysteine ​​residues, a cyclic ketone reagent or acetone, and a solvent. Generally, the product comprises a thioacetalized peptide, wherein thioacetalization couples two cysteine ​​residues of the peptide.

[0013] In some forms, peptides are linear or cyclic (including monocyclic, bicyclic, etc.). In some forms, peptides are random peptides or peptide drugs. In some forms, peptides are formed from natural amino acids.

[0014] In some forms, cyclic ketone reagents can be cyclic ketone reagents having the following structures:

[0015]

[0016] R and R' are independently alkyl (e.g., C1-C6 alkyl) and ----- is absent, or R and R' together form a cyclic moiety A, wherein A is cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, polyaryl, heteroaryl, heteropolyaryl, or heterocyclic, wherein R'' represents hydrogen or a substituent on the cyclic moiety A, and each occurrence of R" is independently benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl (e.g., phenyl), and wherein n is an integer from 0 to 10, 0 to 8, 0 to 6, 0 to 4, or 0 to 2. When R'' is alkyl, the alkyl group may be substituted or unsubstituted, and the substituent(s) present may be any of the substituent(s) disclosed herein, such as halides, azides, or alkynyl groups (e.g., -CCH or -CH2CCH).

[0017] In some forms, R and R' are independently alkyl, such as methyl. In some forms, R and R' are independently alkyl, such as methyl, and ----- is absent. In some forms, A is cyclobutane, azacyclobutane, cyclopentane, fluorenone, cyclohexane, or piperidine.

[0018] In some forms, the solvent is trifluoroacetic acid. In other forms, trifluoroacetic acid acts as a catalyst and is the sole solvent in the reaction mixture.

[0019] In some forms, the reaction mixture is kept at a temperature ranging from 20°C to 35°C, such as about 30°C, for up to 1 hour, up to 2 hours, up to 3 hours, or for a time ranging from 10 minutes to 1 hour, 20 minutes to 2 hours, or 30 minutes to 3 hours.

[0020] In some forms, the molar ratio of peptide to cyclic ketone reagent (peptide:cyclic ketone reagent) is 0.1 to 1, for example, about 0.2.

[0021] In some forms, cyclic ketone reagents have any of the following structures:

[0022]

[0023] Wherein R1 and R3 are H, benzyl, allyl, alkyl (e.g., C1-C6 alkyl), and each of R2 is independently H, benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl. When any of R1-R3 is alkyl, the alkyl group may be substituted or unsubstituted, and the substituent(s) may, when present, be any of the substituents disclosed herein, such as halides, azides, or alkynyl groups (e.g., -CCH or -CH2CCH).

[0024] In some forms, thioacetalized peptides have any of the following structures:

[0025]

[0026] Other advantages of the disclosed methods and compositions will be set forth in part in the following description and in part will be learned from that description or by practicing the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and obtained through the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only, and are not intended to limit the claimed invention. Brief description of the attached diagram

[0028] The accompanying drawings illustrate several embodiments of the disclosed methods and compositions, and together with the description, serve to explain the principles of the disclosed methods and compositions.

[0029] Figure 1 This is a diagram of scheme 2 (late-stage peptide cyclization using cyclic ketones).

[0030] Figure 2 This is a diagram illustrating examples of late-stage peptide cyclization using different cyclic ketones.

[0031] Figure 3 This is a diagram illustrating an example of late-stage peptide cyclization of a natural peptide using cyclic ketones.

[0032] Figure 4 This is a diagram of scheme 3 (late-stage peptide cyclization using acetone).

[0033] Figure 5 This is a diagram illustrating an example of late-stage peptide cyclization of a natural peptide using acetone.

[0034] Figure 6 The image shows the UV (190-400 nm) and MS (250-3000 m / z) spectra obtained by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of 2a (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). C 60 H 79 N 13 O 14 Calculated value of S2 by electrospray ionization mass spectrometry (ESI-MS): [M+H] + Mass-to-charge ratio (m / z) = 1271.4, measured value 1270.5; calculated value: [M+2H] 2+ The mass-to-charge ratio is 636.2, while the measured value is 636.1.

[0035] Figure 7The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analysis of 2b (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). C 30 H 36 Calculated values ​​for electrospray ionization mass spectrometry of N6O6S2: [M+H] + The mass-to-charge ratio is 641.2, while the measured value is 641.3.

[0036] Figure 8 The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) of 2c obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) are shown (containing a 10-50% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). 59 H 73 N 15 O 12 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1249.4, measured value 1248.9; calculated value: [M+2H] 2+ The mass-to-charge ratio is 625.2, and the measured value is 625.0.

[0037] Figure 9 The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis over 2 days are shown (containing a 10-50% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). C 66 H 83 N 15 O 15 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1391.5, measured value 1391.9; calculated value: [M+2H] 2+ The mass-to-charge ratio is 696.3, and the measured value is 696.7.

[0038] Figure 10 The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analysis of 2e (containing a 10-50% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). C 62 H 76 N 18 Calculated value of O9S2 by electrospray ionization mass spectrometry: [M+2H]2+ Mass-to-charge ratio = 641.7, measured value = 641.9; calculated value: [M+3H] 3+ The mass-to-charge ratio is 428.1, and the measured value is 428.2.

[0039] Figure 11 The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of 2f (containing a 15-60% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). C 67 H 77 N 11 O 10 Calculated values ​​of S2 by electrospray ionization mass spectrometry: [M+H ]+ Mass-to-charge ratio = 1172.3, measured value 1171.7; calculated value: [M+2H] 2+ The mass-to-charge ratio is 586.7, and the measured value is 586.6.

[0040] Figure 12 The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) of 2 g obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analysis (containing a 20-70% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, flow rate 0.4 mL / min). C 67 H 77 N 11 O 10 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1261.5, measured value 1260.8; calculated value: [M+2H] 2+ The mass-to-charge ratio is 631.2, and the measured value is 631.3.

[0041] Figure 13 The UV spectrum (190-400 nm) and mass spectrum (300-2000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry analysis over 2 hours are shown (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). 51 H 77 N 13 O 14 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1161.3, measured value 1160.6; calculated value: [M+2H] 2+ The mass-to-charge ratio is 581.1, and the measured value is 581.1.

[0042] Figure 14 The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of 2i are shown (containing a 10-50% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). C 52 H 80 N 14 O 14 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1190.4, measured value 1189.8; calculated value: [M+2H] 2+ The mass-to-charge ratio is 595.7, and the measured value is 595.6.

[0043] Figure 15 The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) are shown (containing a 10-50% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). C 55 H 82 N 14 O 14 Calculated value of S2 by electrospray ionization mass spectrometry: [M+Na] + Mass-to-charge ratio = 1249.9, measured value is 1249.9; calculated value: [M+H] + The mass-to-charge ratio is 1228.4, the measured value is 1227.8; calculated value: [M+2H] 2+ The mass-to-charge ratio is 614.7, and the measured value is 614.6.

[0044] Figure 16 The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) at 2k are shown (containing a 10-60% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 8 minutes, at a flow rate of 0.4 mL / min). 57 H 89 N 17 O 14 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1301.5, measured value 1300.8; calculated value: [M+2H] 2+ The mass-to-charge ratio is 651.2, and the measured value is 651.1.

[0045] Figure 17The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) of ring-(AcHN-GCYIQNCPLG-CONH2) obtained by ultra-high performance liquid chromatography-mass spectrometry analysis are shown (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). C 50 H 77 N 13 O 14 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1149.3, measured value 1148.6; calculated value: [M+2H] 2+ The mass-to-charge ratio is 575.1, and the measured value is 575.1.

[0046] Figure 18 The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of 3b (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). C 56 H 81 N 15 O 15 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1269.4, measured value 1270.2; calculated value: [M+2H] 2+ The mass-to-charge ratio is 635.2, and the measured value is 635.6.

[0047] Figure 19 The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) of 3c obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) are shown (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). 52 H 74 N 18 Calculated values ​​for the electrospray ionization mass spectrometry of O9S2: [M+H+TFA] + Mass-to-charge ratio = 1274.4, measured value 1273.7; calculated value: [M+H] + Mass-to-charge ratio = 1160.3, measured value 1159.5; calculated value: [M+2H] 2+ The mass-to-charge ratio is 580.7, the measured value is 580.6; calculated value: [M+3H] 3+ The mass-to-charge ratio is 387.4, and the measured value is 387.6.

[0048] Figure 20The UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis over 3 days are shown (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). 57 H 75 N 11 O 10 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1139.4, measured value 1138.6; calculated value: [M+2H] 2+ The mass-to-charge ratio is 570.2, and the measured value is 570.0.

[0049] Figure 21 The image shows the UV spectrum (190-400 nm) and mass spectrum (300-2000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of 3e (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, flow rate 0.4 mL / min). C 52 H 72 N 10 O 10 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1062.32, measured value 1061.7; calculated value: [M+2H] 2+ The mass-to-charge ratio is 531.6, and the measured value is 531.7.

[0050] Figure 22 The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of 3f (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, at a flow rate of 0.4 mL / min). C 66 H 124 N 24 O 13 Calculated value of S2 by electrospray ionization mass spectrometry: [M+2H] 2+ Mass-to-charge ratio = 763.9, measured value = 764.0; calculated value: [M+3H] 3+ The mass-to-charge ratio is 509.6, the measured value is 509.8; calculated value: [M+3H] 3+ The mass-to-charge ratio is 382.5, and the measured value is 382.8.

[0051] Figure 23The image shows the UV spectrum (190-400 nm) and mass spectrum (250-3000 m / z) of 3g obtained by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analysis (containing a 5-95% CH3CN / H2O gradient of 0.1% trifluoroacetic acid, lasting 5 minutes, flow rate 0.4 mL / min). C 79 H 110 N 18 O 19 Calculated values ​​for the electrospray ionization mass spectrometry of S2: [M+H] + Mass-to-charge ratio = 1680.9, measured value 1680.3; calculated value: [M+2H] 2+ The mass-to-charge ratio is 840.9, the measured value is 840.9; the calculated value is [M+3H]. 3+ The mass-to-charge ratio is 560.9, and the measured value is 561.0.

[0052] Detailed description of the invention

[0053] The disclosed methods and compositions can be more readily understood by referring to the following detailed description of specific embodiments and the examples included therein, as well as the accompanying drawings and the description preceding and following them.

[0054] This application discloses a method for constructing disulfide bond mimics for late-stage cyclization of peptides. Natural disulfide bonds are unstable in reducing environments, limiting their widespread application. The disclosed late-stage cyclization reaction forms a thioacetal group through the thioacetalization of a peptide containing two cysteine ​​residues. This thioacetal group is stable under acidic, basic, and reducing conditions. Compared to existing methods, the disclosed thioacetalization of peptides can generate novel disulfide bond mimics. This reaction can use a variety of cyclic ketones, even acetone, as cross-linking agents. Almost all peptide sequences can be used in the disclosed method because trifluoroacetic acid can be used alone as both a catalyst and the sole solvent.

[0055] The disclosed method has many benefits and advantages, such as the use of trifluoroacetic acid as a robust catalyst and the only solvent required for the reaction, the easy availability of acetone and various cyclic ketones used as reactants, good chemoselectivity and the tolerance of natural peptides to the reaction, and the fact that the resulting crosslinks are structural mimics of disulfide bonds.

[0056] To overcome the shortcomings of natural disulfide bonds, various stapling methods based on cysteine ​​peptides have been developed. Cysteine ​​peptides exhibit high reactivity and selectivity, making them suitable for forming disulfide bond mimics. Stapling is achieved using symmetrical linkers, such as dichloroacetone (DCA), dichloroacetophenone, dibromobenzyl linkers, or aryl linkers substituted with S-alkylated or S-arylated leaving groups. Recently, Cramer and colleagues reacted cysteine ​​peptides with a CH2I2 reagent to form a thiocarbenium intermediate, which is converted into a methylene thioacetal, eliminating the reducing tendency of the disulfide group and enhancing structural stability. In contrast, in the disclosed methods, cyclic ketones and acetone can be used as crosslinking agents for late-stage peptide cyclization based on cysteine ​​residues under trifluoroacetic conditions, generating a series of thioacetal disulfide bond mimics with good chemoselectivity and tolerance to natural peptides.

[0057] Scheme 1 illustrates exemplary general steps for the later cyclization of disulfide bond mimics using cyclic ketones or acetones, wherein R1-R3 are defined as follows:

[0058] R1 and R3 are each independently H, Bn, allyl, or C1-C6 alkyl;

[0059] R2 is a mono- or poly-substituted fluorenone, such as H, halogen, CN, CF3, NO2, alkyl, alkoxy, or aryl.

[0060] Scheme 1 utilizes the late-stage cyclization of disulfide bonds formed by cyclic ketones and acetone to mimic the process of cyclization.

[0061]

[0062] Scheme 2 shows an exemplary general method for late-stage peptide cyclization ( Figure 1 Natural peptide sequences, such as linear vasopressin, terlipressin, sematolide, oxytocin, and lanreotide, obtained by solid-phase peptide synthesis (SPPS) and purification, were dissolved in trifluoroacetic acid at a final concentration of 10 mM. A cyclic ketone (20.0 equivalents) at a concentration of 200 mM was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction was complete, the solvent was evaporated under a stream of N2, and then the ether was decanted. The residue was dissolved in a mixed solvent of acetonitrile (ACN) and water (H2O), and then purified by preparative high-performance liquid chromatography (HPLC). The corresponding products were obtained by freeze-drying. Detailed synthetic steps for later peptide modification with cyclic ketones are described below as examples.

[0063] Scheme 3 illustrates another exemplary general method for late-stage peptide cyclization using acetone. Figure 4Natural peptide sequences obtained through solid-phase peptide synthesis and purification, such as terlipressin, semaphorin, lanreotide, octreotide, bovine antimicrobial peptide (Bactenecin), and somatostatin, were dissolved in a mixed solvent of trifluoroacetic acid and acetone (1:1, v / v) to a final concentration of 10 mM. The reaction mixture was stirred at room temperature for approximately 8 h. After the reaction was complete, the solvent was evacuated under a stream of N2, and then the crude peptide product was precipitated by diethyl ether. The residue was dissolved in a mixed solvent of acetonitrile (ACN) and water (H2O), and then purified by preparative high-performance liquid chromatography. The target product was obtained by freeze-drying. Here, the detailed synthetic steps for late-stage peptide modification with cyclic ketones are described as examples below.

[0064] This application discloses a method for late cyclization of peptides and the reagents used therein. Generally, the method involves maintaining a reaction mixture at a sufficient temperature for a sufficient time to form a product. Generally, the reaction mixture comprises a peptide containing two or more cysteine ​​residues, a cyclic ketone reagent or acetone, and a solvent. Generally, the product comprises a thioacetalized peptide, wherein thioacetalization couples two cysteine ​​residues of the peptide.

[0065] In some forms, peptides are linear or cyclic (including monocyclic, bicyclic, etc.). In some forms, peptides are random peptides or peptide drugs. In some forms, peptides are formed from natural amino acids.

[0066] In some forms, cyclic ketone reagents or acetone are cyclic ketone reagents having the following structures:

[0067]

[0068] R and R' are independently alkyl (e.g., C1-C6 alkyl) and ----- is absent, or R and R' together form a cyclic moiety A, wherein A is cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, polyaryl, heteroaryl, heteropolyaryl, or heterocyclic, wherein R” represents hydrogen or a substituent on the cyclic moiety A, and each occurrence of R'' is independently benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl (e.g., phenyl), and wherein n is an integer from 0 to 10, 0 to 8, 0 to 6, 0 to 4, or 0 to 2. When R'' is alkyl, the alkyl group may be substituted or unsubstituted, and the substituent(s) present may be any of the substituents disclosed herein, such as halides, azides, or alkynyl (e.g., -CCH or -CH2CCH).

[0069] In some forms, R and R' are independently alkyl, such as methyl. In some forms, R and R' are independently alkyl, such as methyl, and ----- is not present. In some forms, A is cyclobutane, azacyclobutane, cyclopentane, fluorenone, cyclohexane, or piperidine.

[0070] In some forms, the solvent is trifluoroacetic acid. In other forms, trifluoroacetic acid acts as a catalyst and is the sole solvent in the reaction mixture.

[0071] In some forms, the reaction mixture is kept at a temperature ranging from 20°C to 35°C, for example, about 30°C, for up to 1 hour, up to 2 hours, up to 3 hours, or for a time ranging from 10 minutes to 1 hour, 20 minutes to 2 hours, or 30 minutes to 3 hours.

[0072] In some forms, the molar ratio of peptide to cyclic ketone reagent (peptide:cyclic ketone reagent) is 0.1 to 1, such as about 0.2.

[0073] In some forms, cyclic ketone reagents have any of the following structures:

[0074]

[0075] Wherein R1 and R3 are H, benzyl, allyl, alkyl (e.g., C1-C6 alkyl), and each of R2 is independently H, benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl. When any of R1-R3 is alkyl, the alkyl group may be substituted or unsubstituted, and the substituent(s) present may be any of the substituents disclosed herein, such as halides, azides, or alkynyl groups (e.g., -CCH or -CH2CCH).

[0076] In some forms, thioacetalized peptides have any of the following structures:

[0077]

[0078] It should be understood that, unless otherwise stated, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or specific reagents, and therefore may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0079] As used herein, “substituted” means all permitted substituents of the compounds or functional groups described herein. In the broadest sense, permitted substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally including one or more heteroatoms, such as oxygen, sulfur, or nitrogen groups, which are present in linear, branched, or cyclic forms. Representative substituents include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyhexaaryl, substituted or unsubstituted aralkyl, halogen, hydroxyl, alkoxy, phenoxy, aryloxy, silyl, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amide, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphonyl, phosphonyl, amino acid. Such substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyhexaaryl, substituted or unsubstituted aralkyl, halogen, hydroxyl, alkoxy, phenoxy, aryloxy, silyl, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amide, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphonyl, phosphoryl, phosphonyl, and amino acids can all be further substituted.

[0080] Heteroatoms, such as nitrogen, may have hydrogen substituents and / or any permitted substituents of the organic compounds described herein, wherein the substituents satisfy the valence of the heteroatom. It should be understood that “substitution” or “substituted” includes the implicit premise that such substitution must satisfy the permitted valence of the substituted atom and the substituent, and that the substitution forms a stable compound, i.e., a compound that does not spontaneously undergo transformations such as rearrangement, cyclization, or elimination.

[0081] As used herein, "alkyl" refers to a saturated aliphatic group, including straight-chain alkyl, branched-chain alkyl, and cycloalkyl (alicyclic) groups. In some forms, straight-chain or branched alkyl groups have 30 or fewer atoms in their backbone (e.g., for straight-chain C1-C1 alkyl groups). 30 For C3-C branches 30 Alkyl groups have 20 or fewer, 15 or fewer, or 10 or fewer carbon atoms. Alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Similarly, cycloalkyl groups are non-aromatic carbonyl rings consisting of at least three carbon atoms, such as having 3-30, 3-20, or 3-10 carbon atoms in their ring structure, and non-aromatic monocyclic or non-aromatic polycyclic rings having 5, 6, or 7 carbon atoms in their ring structure. Cycloalkyl groups containing polycyclic systems may have two or more non-aromatic rings, where two or more carbons are shared by two adjacent rings (i.e., "fused cycloalkyl rings"). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0082] "Substituted alkyl" refers to an alkyl moiety having a substituent for hydrogen on one or more carbons of a substituted hydrocarbon skeleton. Such substituents can be any of the substituents listed above, such as halogens (e.g., fluorine, chlorine, bromine, or iodine), hydroxyl groups, carbonyl groups (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (e.g., thioesters, thioacetic acids, or thiocarbamates), aryl, alkoxy, aralkyl, phosphonium, phosphonyl, phosphoryl, phosphate, phosphonate, hypophosphonate, amino, amide, amidine, imine, cyano, nitro, azide, oxo, sulfhydryl, thiol, alkylthio, silyl, sulfinyl, sulfate, sulfonate, aminosulfonyl, sulfonamide, sulfonyl, heterocyclic, aromatic, or heteroaromatic moiety. -NRR', where R and R' are independently hydrogen, alkyl, or aryl, and where the nitrogen atom is optionally quaternized; -SR, where R is phosphonoyl, sulfinyl, silyl, hydrogen, alkyl, or aryl; -CN; -NO2; -COOH; carboxylic acid ester; -COR, -COOR, or -CON(R)2, where R is hydrogen, alkyl, or aryl; imino, silyl, ether, haloalkyl (e.g., -CF3, -CH2-CF3, -CCl3); -CN; -NCOCOCH2CH2; -NCOCOCHCH; and -NCS; and combinations thereof.

[0083] Those skilled in the art will understand that the substituted portion of the hydrocarbon chain can itself be substituted, if appropriate. For example, substituents of the substituted alkyl group may include halogens, hydroxyl groups, nitro groups, thiols, amino groups, aralkyl groups, azide groups, imino groups, amide groups, phosphonyl groups, phosphoryl groups (including phosphonates and hypophosphonates), oxo groups, sulfonyl groups (including sulfates, sulfonamides, aminosulfonyls, and sulfonates), and silyl groups, as well as ethers, alkylthio groups, carbonyl groups (including ketones, aldehydes, carboxylic acid esters, and esters), haloalkyl groups, -CN groups, etc. Cycloalkyl groups can be substituted in the same manner.

[0084] Unless otherwise specified, “lower alkyl” as used herein refers to alkyl as defined above, but having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, in its skeletal structure. Similarly, “lower alkenyl” and “lower alkynyl” have similar chain lengths.

[0085] As used herein, “heteroalkyl” refers to a straight-chain or branched, or cyclic carbon-containing alkyl group, or a combination thereof, containing at least one heteroatom on its carbon skeleton. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized. For example, the term “heterocyclic alkyl” is a cyclic group as defined above, wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.

[0086] As used herein, the term "alkenyl" refers to a hydrocarbon group having 2 to 24 carbon atoms and a structural formula containing at least one carbon-carbon double bond. Alkenyl groups include straight-chain alkenyl groups, branched alkenyl groups, and cycloalkenyl groups. A cycloalkenyl group is a non-aromatic carbonyl ring consisting of at least three carbon atoms and at least one carbon-carbon double bond, for example, containing 3-30 carbon atoms and at least one carbon-carbon double bond, 3-20 carbon atoms and at least one carbon-carbon double bond, or 3-10 carbon atoms and at least one carbon-carbon double bond in its ring structure, as well as a non-aromatic monocyclic or non-aromatic polycyclic ring having 5, 6, or 7 carbon atoms and at least one carbon-carbon double bond in its ring structure. Cycloalkenyl groups containing polycyclic systems may have two or more non-aromatic rings, wherein two or more carbons are shared by two adjacent rings (i.e., "fused cycloalkenyl rings") and contain at least one carbon-carbon double bond. Asymmetric structures such as (AB)C=C (C'D) are intended to include both E-type and Z-type isomers. This can be inferred from the structural formula described herein, which contains an asymmetric olefin, or it can be explicitly indicated by the bond symbol C. The term "alkenyl" as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter referring to an alkenyl moiety having one or more substituents on one or more carbons of a substituted hydrocarbon skeleton. The term "alkenyl" also includes "heteroalkenyl."

[0087] The term "substituted alkenyl" refers to an alkenyl moiety having one or more substituents on one or more carbon atoms of one or more substituted hydrocarbon skeletons. Such substituents can be any of the substituents listed above, such as halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphonium, phosphonyl, phosphoryl, phosphate, phosphonate, hypophosphonate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azide, oxo, mercapto, alkylthio, sulfate, sulfonate, aminosulfonyl, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0088] As used herein, “heteroalkenyl” refers to a straight-chain or branched, or cyclic, carbon-containing alkenyl group, or a combination thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. For example, the term “heterocyclic alkenyl” is a cycloalkenyl group in which at least one carbon atom on the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.

[0089] As used herein, the term "alkynyl" refers to a hydrocarbon group having 2 to 24 carbon atoms and a structural formula containing at least one carbon-carbon triple bond. Alynyl groups include straight-chain alkynyls, branched-chain alkynyls, and cycloalkynyls. A cycloalkynyl is a non-aromatic carbonyl ring consisting of at least three carbon atoms and at least one carbon-carbon triple bond, for example, containing 3-30 carbon atoms and at least one carbon-carbon triple bond, 3-20 carbon atoms and at least one carbon-carbon triple bond, or 3-10 carbon atoms and at least one carbon-carbon triple bond in its ring structure, as well as a non-aromatic monocyclic or non-aromatic polycyclic ring having 5, 6, or 7 carbon atoms and at least one carbon-carbon triple bond in its ring structure. Cycloalkynyls containing polycyclic systems may have two or more non-aromatic rings, wherein two or more carbons are shared by two adjacent rings (i.e., "fused cycloalkynyl rings") and contain at least one carbon-carbon triple bond. Asymmetric structures such as (AB)C≡C(C''D) are intended to include both E-type and Z-type isomers. This can be inferred from the structural formulas described herein, which contain asymmetric alkynes, or it can be explicitly indicated by the bond symbol C. The term "alkynyl" as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter referring to an alkynyl moiety having a hydrogen substituent on one or more carbon atoms of one or more substituted hydrocarbon skeletons. The term "alkynyl" also includes "heterynyl."

[0090] The term "substituted alkynyl" refers to an alkynyl moiety having a substituent on one or more carbon atoms of one or more substituted hydrocarbon skeletons. Such a substituent can be any of the substituents listed above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphonium, phosphonyl, phosphoryl, phosphate, phosphonate, hypophosphonate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azide, mercapto, alkylthio, sulfate, sulfonate, aminosulfonyl, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0091] As used herein, “heteroyne” refers to a straight-chain or branched, or cyclic carbon-containing ynyl group or combination thereof containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. For example, the term “heterocyclic ynyl” is a cyclic ynyl group in which at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.

[0092] As used in this article, "aryl" refers to C5-C 26 Aromatic compounds are either polycyclic aromatic or fused aromatic ring systems. Examples of aromatic groups include benzene, naphthalene, anthracene, phenanthrene, hydroxyl, pyrene, styrene, and benzene.

[0093] The term "substituted aryl" refers to an aryl group in which one or more hydrogen atoms on one or more aromatic rings are replaced by one or more substituents, including but not limited to halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (e.g., ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphoryl, phosphate, phosphonate, hypophosphonate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azide, mercapto, imino, alkylthio, sulfate, sulfonate, aminosulfonyl, sulfoxide, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl (e.g., -CF3, -CH2-CF3, -CCl3), -CN, aryl, heteroaryl, and combinations thereof.

[0094] "Heterocycle" and "heterocyclic group" are used interchangeably, and both refer to cyclic groups consisting of non-aromatic monocyclic or polycyclic carbon or nitrogen atoms linked together by 3-30, 3-20, 3-10, or 5-6 ring atoms, wherein each ring contains carbon and 1-4 heteroatoms, each heteroatom selected from non-peroxide oxygen, sulfur, and N(Y), wherein Y is absent or is H, O, or Cl-C. 10 The group is alkyl, phenyl, or benzyl, and optionally contains 1-3 double bonds and is optionally substituted with one or more substituents. By definition, heterocyclic groups are distinct from heteroaryl groups. Heterocyclic groups can be heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkynyl, etc., such as piperazinyl, piperidinyl, piperidinoneyl, 4-piperidinoneyl, dihydrofurano[2,3-b]tetrahydrofuran, morpholinyl, piperazinyl, piperidinyl, piperidinoneyl, 4-piperidinoneyl, piperinyl, pyranyl, 2H-pyrroleyl, 4H-quinazinyl, quininecycloyl, tetrahydrofuranyl, 6H-1,2,5-thiadiazinyl. The heterocyclic group may optionally be substituted with one or more substituents as defined above for alkyl and aryl groups.

[0095] The term "heteroaryl" refers to a C5-C group in which one or more carbon atoms in one or more aromatic rings have been replaced by heteroatoms. 26A ring system of fused aromatics or polyaromatics. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetraazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzooxazolyl, benzooxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazole, benzoisooxazolyl, benzoisothiazolyl, benzimidazolinyl, carbazole, 4aH-carbazole, carbolinyl, chromanyl, chromenyl, terpineyl, and decahydroquinone. Linolyl, 2H,6H-1,5,2-dithiazinyl, furanyl, furazanyl, imidazoalkyl, imidazolinyl, imidazolyl, 1H-indazoleyl, indolenyl, indololinyl, indolazinyl, indolyl, 3H-indolyl, indigovinyl, isobenzofuranyl, isochoryl, isoindazoleyl, isoindololinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthidyl, octahydroisoquinolinyl, 1,2,3-oxadiazolyl, 1,2 ,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolylalkyl, oxazolyl, hydroxyindole, pyrimidinyl, phenanthidyl, phenanthrolinel, phenazinyl, phenothiazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purine, pyrazinyl, pyrazolylalkyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl Pyrimidinyl, pyrrolylyl, pyrrolinyl, pyrroloyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, xanthonyl. One or more rings may be substituted as defined below for "substituted heteroaryl".

[0096] The term "substituted heteroaryl" refers to a heteroaryl group in which one or more hydrogen atoms on one or more heteroaryl rings are replaced by one or more substituents, including but not limited to halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (e.g., ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphoryl, phosphate, phosphonate, hypophosphonate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azide, mercapto, imino, alkylthio, sulfate, sulfonate, aminosulfonyl, sulfoxide, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl (e.g., -CF3, -CH2-CF3, -CCl3), -CN, aryl, heteroaryl, and combinations thereof.

[0097] The term "polyaryl" refers to a chemical moiety comprising two or more fused aryl groups. When two or more fused heteroaryl groups are involved, the chemical moiety may be called "polyheteroaryl".

[0098] The term "substituted polyaryl" refers to a polyaryl group in which one or more aryl groups are replaced by one or more substituents, including but not limited to halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphoryl, phosphate, phosphonate, hypophosphonate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azide, mercapto, alkylthio, sulfate, sulfonate, aminosulfonyl, sulfoxide, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. When polyaryl is involved, the chemical part may be referred to as "substituted polyaryl".

[0099] The term "cycle" or "cyclic group" refers to a substituted or unsubstituted monocyclic or polycyclic (e.g., a ring formed from a monocyclic or fused ring system), such as a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloynyl, or substituted or unsubstituted heterocyclic group, having 3 to 30 carbon atoms where geometric constraints permit. The substituted cycloalkyl, cycloalkenyl, cycloynyl, and heterocyclic groups are substituted as defined above for alkyl, alkenyl, ynyl, and heterocyclic groups, respectively.

[0100] As used herein, the term "aralkyl" refers to an aryl or heteroaryl group having an alkyl, ynyl, or alkenyl group as defined above attached to an aromatic group, such as an aryl, heteroaryl, polyaryl, or polyheteroaryl group. An example of an aralkyl group is a benzyl group.

[0101] The term "thiol" is used interchangeably and is represented by -SR, where R can be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted carbonyl, phosphonium, phosphonyl, amide, amino, alkoxy, oxo, phosphonyl, sulfinyl, or silyl. Such substituents can be any of the substituents mentioned above, such as halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphoryl, phosphate, phosphonate, hypophosphonate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azide, mercapto, alkylthio, sulfate, sulfonate, aminosulfonyl, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0102] The disclosed compounds and substituents may independently have two or more of the groups listed above. For example, if the compound or substituent is a straight-chain alkyl group, one of the hydrogen atoms of the alkyl group may be replaced by a hydroxyl, alkoxy, etc. Depending on the chosen groups, the first group may be incorporated into the second group, or the first group may serve as a side chain of the second group (i.e., attached to the second group). For example, for the phrase "alkyl group containing an ester group," the ester group may be incorporated into the backbone of the alkyl group. Alternatively, the ester group may be attached to the backbone of the alkyl group. The nature of the chosen groups(s) will determine whether the first group is inserted into or attached to the second group.

[0103] Compounds and substituents can be independently substituted by substituents as defined in the above definition of "substituted".

[0104] The numerical range individually discloses every possible value that such a range can reasonably include, as well as any subranges and combinations thereof. For example, within a given carbon range of C3-C9, the range also discloses C3, C4, C5, C6, C7, C8, and C9, as well as any subranges between these values ​​(e.g., C4-C6), and any possible combinations of possible ranges between these values. In yet another example, a given temperature range may be from about 25°C to 30°C, wherein the range also discloses temperatures that can be independently selected from about 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C, as well as any range between these values ​​(e.g., 26°C to 28°C) and any possible combinations of ranges between these values.

[0105] The term “about” is used to describe values ​​that are higher or lower than the stated value, and modifies a value to be within the range of about + / - 10%. When the term “about” is used before a range of values ​​(i.e., about 1-5) or a series of numbers (i.e., about 1, 2, 3, 4, etc.), unless otherwise stated, it is intended to modify each value listed in the range and / or the entire series.

[0106] "Oxytochemical" refers to O.

[0107] Compounds and substituents can be independently substituted by substituents as described in the definition of "substituted" above.

[0108] The numerical range includes the range from 1 to 6. The range individually discloses every possible numerical value that such a range can reasonably include, as well as any sub-ranges and combinations thereof. For example, within a given carbon range of C3-C9, the range also discloses C3, C4, C5, C6, C7, C8, and C9, as well as any sub-ranges between these values ​​(e.g., C4-C6), and any possible combinations of possible ranges between these values. In yet another example, a given temperature range may be from about 25°C to 30°C, wherein the range also discloses temperatures that can be independently selected from about 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C, as well as any range between these values ​​(e.g., 26°C to 28°C) and any possible combinations of ranges between these values.

[0109] The term “about” is used to describe values ​​that are higher or lower than the stated value, and modifies the stated value to be within the range of about + / - 10%. When the term “about” is used before a range of values ​​(i.e., about 1-5) or a series of values ​​(i.e., about 1, 2, 3, 4, etc.), unless otherwise stated, it is intended to modify each of the two ends of the range and / or each value listed in the entire series.

[0110] When referring to a given compound, "analogue" means another compound that is structurally similar, functionally similar, or both. Structural similarity can be determined using any criterion known in the art, such as the Tanimoto coefficient, which provides a quantitative measure of similarity between the molecular descriptors of the two compounds. Preferably, the molecular descriptor is a 2D property, such as a molecular fingerprint, topological index, and maximum common substructure, or a 3D property, such as overall shape and molecular field. For different and identical molecular pairs, the Tanimoto coefficient ranges between 0 and 1, inclusive. If a compound has a Tanimoto coefficient between 0.5 and 1.0 (inclusive), preferably between 0.7 and 1.0 (inclusive), and most preferably between 0.85 and 1.0 (inclusive), it can be considered an analogue of the specified compound. A compound is functionally similar to the specified compound if it induces the same pharmacological, physiological, or both effects as the specified compound. "Analogue" can also refer to modifications of the disclosed compound, including but not limited to hydrolysis, reduction, or oxidation products. Hydrolysis, reduction, and oxidation reactions are known in the art.

[0111] The disclosed compounds and methods can be further understood through the following numbered paragraphs.

[0112] 1. A method for late-stage cyclization of a peptide, comprising:

[0113] The reaction mixture is kept at a sufficient temperature for a sufficient time to form the product.

[0114] The reaction mixture comprises a peptide containing two or more cysteine ​​residues, a cyclic ketone reagent or acetone, and a solvent.

[0115] The product comprises a thioacetalized peptide, and thioacetalization couples two cysteine ​​residues of the peptide.

[0116] 2. The method described in paragraph 1, wherein the peptide is linear or cyclic (including monocyclic, bicyclic, etc.).

[0117] 3. The method described in paragraph 1 or 2, wherein the peptide is a random peptide or a peptide drug.

[0118] 4. The method described in any of paragraphs 1-3, wherein the peptide comprises natural amino acids.

[0119] 5. The method described in any one of paragraphs 1-4, wherein the cyclic ketone reagent is a cyclic ketone reagent having the following structure:

[0120]

[0121] Where R and R' are independently alkyl groups and ----- is absent, or R and R' together form a cyclic moiety A.

[0122] Where A is a cycloalkyl, cycloalkenyl, cycloynyl, aryl, polyaryl, heteroaryl, heteropolyaryl, or heterocyclic group.

[0123] Where R'' represents a hydrogen atom or a substituent on the cyclic moiety A, and each occurrence of R'' is independently benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl.

[0124] Where n is an integer from 0 to 10, 0 to 8, 0 to 6, 0 to 4, or 0 to 2.

[0125] 6. The method described in paragraph 5, wherein R and R' are independently alkyl, such as methyl.

[0126] 7. The method described in paragraph 5 or 6, wherein A is cyclobutane, azacyclobutane, cyclopentane, fluorenone, cyclohexane, or piperidine.

[0127] 8. The method described in any of paragraphs 1-7, wherein the solvent is trifluoroacetic acid.

[0128] 9. The method described in paragraph 8, wherein trifluoroacetic acid acts as a catalyst and / or the sole solvent in the reaction mixture.

[0129] 10. The method described in any one of paragraphs 1-9, wherein the reaction mixture is kept at a temperature of 20°C to 35°C, for example, about 30°C, for up to 1 hour, up to 2 hours, up to 3 hours, or for a period ranging from 10 minutes to 1 hour, 20 minutes to 2 hours, or 30 minutes to 3 hours.

[0130] 11. The method described in any of paragraphs 1-10, wherein the molar ratio of peptide to cyclic ketone reagent (peptide:cyclic ketone reagent) is 0.1 to 1, for example about 0.2.

[0131] 12. The method described in any of paragraphs 1-11, wherein the cyclic ketone reagent has any of the following structures:

[0132]

[0133] Where R1 and R3 are H, benzyl, allyl, or alkyl (e.g., C1-C6 alkyl), and

[0134] Each R2 is independently H, benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl.

[0135] 13. The method described in any of paragraphs 1-12, wherein the thioacetalized peptide has any of the following structures:

[0136] Example

[0137] 1. AcHN-GCYIQNCPLG-CONH2+9-fluorenone (2a)

[0138]

[0139] AcHN-GCYIQNCPLG-CONH2+9-fluorenone (2a)

[0140] The purified AcHN-GCYIQNCPLG-CONH2 (1a) (11.1 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. 200 mM 9-fluorenone (36.0 mg, 20.0 equivalent) was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (15-60%, 45 min). Lyophilization yielded a white powder 2a (6.0 mg, 47%).

[0141] 2. AcHN-CAAAC-CONH2+9-fluorenone (2b)

[0142] Purified AcHN-CAAAC-CONH2(1b) (4.8 mg, 1.0 equivalent), obtained through solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. 200 mM 9-fluorenone (36.0 mg, 20.0 equivalent) was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (15-60%, 45 min). Lyophilization yielded a white powder, 2b (2.4 mg, 38%).

[0143] 3. H2N-CYFQNCPRG-CONH2+9-fluorenone (2c) (from vasopressin)

[0144]

[0145] H2N-CYFQNCPRG-CONH2+9-fluorenone (2c)

[0146] The purified H2N-CYFQNCPRG-CONH2 (1c) (10.8 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. 9-fluorenone (90.0 mg, 50.0 equivalent) was added to the solution at a concentration of 200 mM, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H2O and purified by preparative HPLC (10-50%, 45 min). Lyophilization yielded a white powder, 2c (3.4 mg, 27%).

[0147] 4. H2N-GGGCYFQNCPKG-CONH2+9-fluorenone (2d) (from terlipressin)

[0148]

[0149] H2N-GGGCYFQNCPKG-CONH2+9-fluorenone (2d)

[0150] The purified H2N-GGGCYFQNCPKG-CONH2 (1d) (12.3 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in TFA at a final concentration of 10 mM. 200 mM 9-fluorenone (36.0 mg, 20.0 equivalent) was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H2O and purified by preparative HPLC (10-50%, 45 min). Freeze-drying yielded a white powder, 2d (4.0 mg, 31%).

[0151] 5. AcHN-RC(D-)AH(D-)FRWC-CONH2+9-fluorenone (2e) (from Smeinopeptide)

[0152]

[0153] AcHN-RC(D-)AH(D-)FRWC-CONH2+9-fluorenone(2e)

[0154] Purified AcHN-RC(D-)AH(D-)FRWC-CONH2(1e) (11.2 mg, 1.0 equivalent), obtained through solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. 200 mM 9-fluorenone (36.0 mg, 20.0 equivalent) was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H2O and purified by preparative HPLC (10-50%, 45 min). Lyophilization yielded a white powder, 2e (3.8 mg, 30%).

[0155] 6. H2N-CYIQNCPLG-CONH2+9-fluorenone (2f) (from oxytocin)

[0156]

[0157] H2N-CYIQNCPLG-CONH2+9-fluorenone (2f)

[0158] The purified H2N-CYIQNCPLG-CONH2 (1f) (10.1 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in TFA at a final concentration of 10 mM. 9-fluorenone (90.0 mg, 50.0 equivalent) at 200 mM was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (15-60%, 45 min). Lyophilization yielded a white powder (2f) (4.5 mg, 38%).

[0159] 7. H2N-(D-)NaICY(D-)WKVCT-CONH2+9-fluorenone (2g) (from Lanruitide)

[0160]

[0161] H2N-(D-)NaICY(D-)WKVCT-CONH2+9-fluorenone (2g)

[0162] Purified H2N-(D-)NaICY(D-)WKVCT-CONH2 (1 g) (10.9 mg, 1.0 equivalent), obtained through solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. 9-fluorenone (90.0 mg, 50.0 equivalent) at 200 mM was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 20% ACN / H2O and purified by preparative HPLC (20-70%, 45 min). Lyophilization yielded 2 g (6.6 mg, 54%) of a white powder.

[0163] 8. AcHN-GCYIQNCPLG-CONH2+cyclobutanone (2h)

[0164]

[0165] AcHN-GCYIQNCPLG-CONH2+cyclobutanone (2h)

[0166] The purified AcHN-GCYIQNCPLG-CONH2(1a) (11.1 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. Cyclobutanone (14.0 mg, 20.0 equivalent) was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction was complete, the solvent was removed under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (15-60%, 45 min). The product was lyophilized to obtain a white powder (5.2 mg, 45%) over 2 h.

[0167] 9. AcHN-GCYIQNCPLG-CONH2+Boc-4-piperidinone (2i)

[0168]

[0169] AcHN-GCYIQNCPLG-CONH2+Boc-4-piperidinone (2i)

[0170] The purified AcHN-GCYIQNCPLG-CONH2(1a) (11.1 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. N-(tert-Butoxycarbonyl)-4-piperidinone (39.8 mg, 20.0 equivalent) was added to the solution at a concentration of 200 mM, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H2O and purified by preparative HPLC (10-50%, 45 min). Lyophilization yielded a white powder, 2i (2.3 mg, 20%).

[0171] 10. AcHN-GCYIQNCPLG-CONH2+1-(prop-2-yn-1-yl)piperidin-4-one (2j)

[0172]

[0173] AcHN-GCYIQNCPLG-CONH2+1-(prop-2-yn-1-yl)piperidin-4-one (2j)

[0174] Purified AcHN-CAAAC-CONH2 (1a) (11.1 mg, 1.0 equivalent), obtained through solid-phase peptide synthesis, was dissolved in TFA at a final concentration of 10 mM. 1-(prop-2-yn-1-yl)piperidin-4-one (42.0 mg, 20.0 equivalent) at 200 mM was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was removed under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (15-60%, 45 min). Lyophilization yielded 2 mg (5.6 mg, 46%) of a white powder.

[0175] 11. AcHN-GCYIQNCPLG-CONH2+5-Azidepentylpiperidin-4-one (2kJ)

[0176]

[0177] AcHN-GCYIQNCPLG-CONH2+5-Azidepentylpiperidin-4-one (2kJ)

[0178] The purified 11AcHN-GCYIQNCPLG-CONH2(1a) (11.1 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in trifluoroacetic acid to a final concentration of 10 mM. 200 mM of 1-(5-azidopentyl)piperidin-4-one (42.0 mg, 20.0 equivalent) was added to the solution, and the reaction mixture was stirred at room temperature for approximately 8 h. After the reaction was complete, the solvent was removed under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H2O and purified by preparative HPLC (10-50%, 45 min). Lyophilization yielded a white powder, 2k (5.6 mg, 43%).

[0179] 12. AcHN-GCYIQNCPLG-CONH2+ Acetone (3a)

[0180]

[0181] AcHN-GCYIQNCPLG-CONH2+ Acetone (3a)

[0182] The purified AcHN-GCYIQNCPLG-CONH2 (1a) (11.1 mg, 1.0 equivalent), obtained by solid-phase peptide synthesis, was dissolved in a mixed solvent (1.0 mL) of trifluoroacetic acid and acetone (1:1 v / v) to a final concentration of 10 mM. The reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was evacuated under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (10-50%, 35 min). The purified product was lyophilized to give a white powder 3a (6.6 mg, 63%).

[0183] 13. H2N-GGGCYFQNCPKG-CONH2+ Acetone (3b) (from terlipressin)

[0184]

[0185] H2N-GGGCYFQNCPKG-CONH2+ Acetone (3b)

[0186] The purified H2N-GGGCYFQNCPKG-CONH2 (1d) (12.3 mg, 1.0 equivalent) obtained by solid-phase peptide synthesis was dissolved in a mixed solvent (1.0 mL) of trifluoroacetic acid and acetone (1:1 v / v) to a final concentration of 10 mM. The reaction mixture was stirred at room temperature for about 8 h. After the reaction, the solvent was blown off under a stream of N2, and then the crude peptide product was precipitated by diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H2O and purified by preparative HPLC (10-50%, 35 min). The product was lyophilized to give a white powder 3b (8.3 mg, 65%).

[0187] 14. AcHN-RC(D-)AH(D-)FRWC-CONH2+Acetone (3c) (from Smeinopeptide)

[0188]

[0189] AcHN-RC(D-)AH(D-)FRWC-CONH2+acetone (3c)

[0190] Purified AcHN-RC(D-)AH(D-)FRWC-CONH2(1e) (11.9 mg, 1.0 equivalent), obtained through solid-phase peptide synthesis, was dissolved at a final concentration of 10 mM in 1.0 mL of a mixed solvent of trifluoroacetic acid and acetone (1:1, v / v). The reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was evaporated under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (10-60%, 35 min). The purified product was lyophilized to give a white powder 3c (8.9 mg, 77%).

[0191] 15. H2N-(D-)NaICY(D-)WKVCT-CONH2+ Acetone (3d) (from Lanrui Peptide)

[0192]

[0193] H2N-(D-)NaICY(D-)WKVCT-CONH2+ Acetone (3d)

[0194] Purified H2N-(D-)NaICY(D-)WKVCT-CONH2 (1 g) (10.9 mg, 1.0 equivalent), obtained through solid-phase peptide synthesis, was dissolved at a final concentration of 10 mM in 1.0 mL of a mixed solvent of trifluoroacetic acid and acetone (1:1, v / v). The reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was evaporated under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 20% ACN / H2O and purified by preparative HPLC (20-70%, 35 min). The purified product was lyophilized to give a white powder 3e (5.3 mg, 47%).

[0195] 16. H2N-(D-)FCF(D-)WKTCT-CH2OH+acetone(3e) (from octreotide)

[0196]

[0197] H2N-(D-)FCF(D-)WKTCT-CH2OH+acetone(3e)

[0198] Purified H2N-(D-)FCF(D-)WKTCT-CH2OH (1h) (10.6 mg, 1.0 equivalent), obtained from solid-phase peptide synthesis, was dissolved at a final concentration of 10 mM in a mixed solvent (1.0 mL) of trifluoroacetic acid and acetone (v / v 1:1). The reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was evaporated under a stream of N2, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 15% ACN / H2O and purified by preparative HPLC (15-60%, 35 min). The purified product was lyophilized to give a white powder 3e (7.5 mg, 71%).

[0199] 17. H2N-RLCRIVVIRVCR-COOH+acetone (3f) (from bovine antimicrobial peptides)

[0200]

[0201] H2N-RLCRIVVIRVCR-COOH+acetone (3f)

[0202] The purified H₂N-RLCRIVVIRVCR-COOH (1i) (7.4 mg, 1.0 equivalent) obtained from solid-phase peptide synthesis was dissolved at a final concentration of 0.5 mM in 10.0 mL of a mixed solvent of trifluoroacetic acid and acetone (1:1 v / v). The reaction mixture was stirred at room temperature for approximately 8 h. After the reaction, the solvent was evacuated under a stream of N₂, and then the crude peptide product was precipitated by passing it through diethyl ether. The residue was dissolved in 10 mL of 10% ACN / H₂O and purified by preparative HPLC (10-60%, 35 min). The product was lyophilized to give 3 f(1.1 mg, 15%) of a white powder.

[0203] 18. H2N-AGCKNFFWKTFTSC-CONH2+ Acetone (3g) (from somatostatin)

[0204]

[0205] H2N-AGCKNFFWKTFTSC-CONH2+ Acetone (3g)

[0206] The purified H2N-AGCKNFFWKTFTSC-CONH2(1j) (16.4 mg, 1.0 equivalent) obtained by solid-phase peptide synthesis was dissolved in a mixed solvent (1.0 mL) of trifluoroacetic acid and acetone (v / v 1:1) to a final concentration of 10 mM. The reaction mixture was stirred at room temperature for about 8 h. After the reaction, the solvent was blown off under a stream of N2, and then the crude peptide product was precipitated by diethyl ether. The residue was dissolved in 10 mL of 20% ACN / H2O and purified by preparative HPLC (20-70%, 35 min). The product was lyophilized to give 3 g (8.6 mg, 51%) of white powder.

[0207] It should be understood that the disclosed methods and compositions are not limited to the specific methods, schemes, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be defined solely by the appended claims.

[0208] Those skilled in the art will be able to identify or determine, using only conventional experiments, numerous equivalents of the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be covered by the appended claims.

Claims

1. A method for late-stage cyclization of a peptide, comprising: The reaction mixture is kept at a sufficient temperature for a sufficient time to form the product. The reaction mixture comprises a peptide containing two or more cysteine ​​residues, a cyclic ketone reagent or acetone, and a solvent. The product comprises a thioacetalized peptide, and thioacetalization couples two cysteine ​​residues of the peptide.

2. The method of claim 1, wherein the peptide is linear or cyclic (including monocyclic, bicyclic, etc.).

3. The method of claim 1 or 2, wherein the peptide is a random peptide or a peptide drug.

4. The method of any one of claims 1-3, wherein the peptide is formed from natural amino acids.

5. The method of any one of claims 1-4, wherein the cyclic ketone reagent is a cyclic ketone reagent having the following structure: Where R and R' are independently alkyl groups and ----- is absent, or R and R' together form a cyclic moiety A. Where A is a cycloalkyl, cycloalkenyl, cycloynyl, aryl, polyaryl, heteroaryl, heteropolyaryl, or heterocyclic group. Wherein R'' represents hydrogen or a substituent on the cyclic moiety A, and each occurrence of R'' is independently benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl, and Where n is an integer from 0 to 10, 0 to 8, 0 to 6, 0 to 4, or 0 to 2.

6. The method of claim 5, wherein R and R' are independently alkyl, such as methyl.

7. The method of claim 5 or 6, wherein A is cyclobutane, azacyclobutane, cyclopentane, fluorenone, cyclohexane, or piperidine.

8. The method of any one of claims 1-7, wherein the solvent is trifluoroacetic acid.

9. The method of claim 8, wherein trifluoroacetic acid acts as a catalyst and is the only solvent in the reaction mixture.

10. The method of any one of claims 1-9, wherein the reaction mixture is maintained at a temperature of 20°C to 35°C, for example about 30°C, for up to 1 hour, up to 2 hours, up to 3 hours, ranging from 10 minutes to 1 hour, 20 minutes to 2 hours, or 30 minutes to 3 hours.

11. The method of any one of claims 1-10, wherein the molar ratio of the peptide to the cyclic ketone reagent (peptide:cyclic ketone reagent) is 0.1 to 1, for example about 0.

2.

12. The method of any one of claims 1-11, wherein the cyclic ketone reagent has any one of the following structures: Where R1 and R3 are H, benzyl, allyl, or alkyl (e.g., C1-C6 alkyl), and Each R2 is independently H, benzyl, allyl, alkyl (e.g., C1-C6 alkyl), halogen, -CN, -CF3, -NO2, alkoxy, or aryl.

13. The method of any one of claims 1-12, wherein the thioacetalized peptide has any one of the following structures:

Images