Selenocysteine-mediated synthesis of cyclic peptides and applications thereof

Abstract

The application provides a synthesis method and application of a selenocysteine-mediated cyclic peptide. A substrate polypeptide is synthesized by a solid-phase polypeptide synthesis method, the C terminal of the substrate polypeptide exists in a hydrazine form, and the N terminal of the substrate polypeptide is selenocysteine. A cyclization method is used to add ascorbate, high-concentration tris(2-chloroethyl) phosphate (TCEP) and acetylacetone to the substrate polypeptide, and the reaction is carried out at room temperature by oscillation to obtain a corresponding target cyclic peptide. The application helps to expand the synthesis method of the cyclic peptide with a ring structure at the head and tail, and realizes efficient synthesis of the cyclic peptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide / protein synthesis, and particularly relates to a selenocysteine-mediated cyclic peptide synthesis method and application thereof. TECHNICAL BACKGROUND

[0002] With more and more naturally occurring cyclic peptides with different biological activities being discovered, cyclic peptides have attracted more and more attention, especially in the field of drug research. Chemical synthesis of cyclic peptides is of great significance for studying the structure-activity relationship. Traditional direct coupling cyclization method has certain limitations, such as sensitivity of C-terminal to isomerization, poor solubility of completely protected peptide precursor, and low yield caused by oligomerization. [1] Therefore, chemoselective ligation-mediated cyclization method has become an effective strategy for cyclic peptide synthesis.

[0003] The formation of cyclic peptides discovered in nature usually involves two reactive groups, which are reacted to obtain the cyclic structure of the cyclic peptide, and these reactions include lactamization, lactonization, thiolactonization and formation of disulfide bridge. [2-3] Based on the positions of the two reactive groups in the polypeptide, the polypeptide cyclization mode can be generally divided into four types: head-to-tail connection, head-to-side chain connection, side chain-to-tail connection and side chain-to-side chain connection. For head-to-tail connected cyclic peptides, cyclization is achieved by lactamization of the amino and carboxylate groups at both ends of the linear precursor. In addition to the two ends, the side chain amino group of Lys, the side chain carboxyl group of Asp, the side chain hydroxyl group of Glu, Ser and Thr and the side chain thiol group of Cys are also common functional groups involved in ring formation, so that the other three types of cyclic peptides can be obtained. In addition to using the functional groups present at the ends and side chains of the peptide for peptide cyclization, it is also possible to involve functional groups installed on the N-alpha atom of the main chain. This non-classical N-backbone peptide cyclization relies on the use of functionalized N-alkylated amino acids. [4-5]

[0004] At present, head-to-tail connected cyclic peptides are mainly synthesized by native chemical ligation (NCL) mediated cyclization. The specific process of the reaction is that the cysteine side chain thiol group at the N-terminus of the linear polypeptide attacks the carbonyl carbon of the C-terminal thioester to form an intermediate connected by a thioester bond through thioester transfer. Then S→N rearrangement is carried out to obtain a natural peptide bond. [6-7] However, this method has the following limitations: the reaction cannot be connected when the reaction concentration is too low, and it is reported that the reaction concentration can only be as low as millimolar, and it is difficult to observe the formation of cyclic peptides for reactions with lower concentrations; and for sites with large steric hindrance such as His, Pro, etc., the reaction speed is slow; in addition, how to efficiently obtain the C-terminal polypeptide thioester also remains to be explored. Therefore, people have carried out research on more connection methods.

[0005] Selenocysteine (Sec)-mediated NCL produces cyclic peptides containing selenocysteine by replacing N-terminal Cys with Sec and adjusting the reaction conditions. [8] Subsequent de-selenization can be performed to obtain natural Ala or Ser residues.

[0006] Sec-mediated NCL has better reaction efficiency, so it has attracted extensive research.

[0007] References:

[0008] 1. Kemp, D. S.; Rebek, J. Peptide Racemization Mechanism. Kinetic Isotope Effect as a Means of Distinguishing Enolization from Oxazolone Formation. J. Am. Chem. Soc. 1970, 92, 5792-5793.

[0009] 2. White, C. J.; Yudin, A. K. Contemporary Strategies for Peptide Macrocyclization. Nat. Chem. 2011, 3, 509-524.

[0010] 3. Davies, J. S. The Cyclization of Peptides and Depsipeptides. J. Pept. Sci. 2003, 9, 471-501.

[0011] 4. Rubin, S. J. S.; Qvit, N. Backbone-Cyclized Peptides: A Critical Review. Curr. Top. Med. Chem. 2018, 18, 526-555.

[0012] 5. Gilon, C.; Halle, D.; Chorev, M.; Selincer, Z.; Byk, G. Backbone Cyclization: A New Method for Conferring Conformational Constraint on Peptides. Biopolymers. 1991, 31, 745-750.

[0013] 6. Zhang, L.; Tam, J. P. Synthesis and Application of Unprotected Cyclic Peptides as Building Blocks for Peptide Dendrimers. J. Am. Chem. Soc. 1997, 119, 2363-2370.

[0014] 7. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of Proteins by Native Chemical Ligation. Science. 1994, 266, 776-779.

[0015] 8. Quaderer, R.; Hilvert, D. Selenocysteine-Mediated Backbone Cyclization of Unprotected Peptides Followed by Alkylation, Oxidative Elimination or Reduction of the Selenol. Chem. Commun. 2002, 0, 2620-2621. SUMMARY

[0016] The primary object of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a selenocysteine-mediated cyclic peptide synthesis method.

[0017] Another object of the present application is to provide the use of the selenocysteine-mediated cyclic peptide synthesis method described above.

[0018] The object of the present application is achieved by the following technical solutions:

[0019] A selenocysteine-mediated cyclic peptide synthesis method is obtained by adding ascorbate, a reducing agent, and acetylacetone to the polypeptide hydrazide containing selenocysteine obtained by solid-phase synthesis of polypeptides, and then shaking the reaction at room temperature, followed by deselenization, to obtain the corresponding target cyclic peptide; the specific operation preferably includes the following steps:

[0020] (1) Dissolve the polypeptide hydrazide with N-terminal selenocysteine in buffer A, add ascorbate, dissolve; then add a diselenide bond reducing agent, dissolve, react, to obtain polypeptide reaction liquid B; in this step, the diselenide bond reducing agent reduces the diselenide bond, thereby obtaining free selenol groups to participate in the reaction;

[0021] (2) adding acetylacetone to the polypeptide reaction solution B obtained in step (1) and reacting to obtain a polypeptide reaction solution C; in this step, the acetylacetone activates the C-terminal hydrazide group to obtain a pyrazole intermediate, which is easily attacked by a nucleophile and then undergoes a cyclization reaction;

[0022] (3) adding glutathione (GSH) and tris (2-chloroethyl) phosphate (TCEP) to the polypeptide reaction solution C obtained in step (2), adjusting the pH, and reacting; purifying the reaction solution to obtain the target product; in this step, under the action of the reducing agent and glutathione, the cyclic peptide is in-situ deselenized to obtain a natural Ala residue.

[0023] The N-terminal selenium-cysteine polypeptide hydrazide in step (1) is preferably obtained by using 2-chlorotrityl chloride resin to acylate the polypeptide hydrazide and then sequentially condensing Fmoc-protected amino acids from the C-terminal to the N-terminal by using the Fmoc solid-phase polypeptide synthesis method.

[0024] The buffer A in step (1) preferably has the following composition: 5-7 mol / L GdmCl, 0.1-0.3 mol / L Na2HPO4, pH = 2.9-3.1; more preferably, the following composition: 6 mol / L GdmCl, 0.2 mol / L Na2HPO4, pH = 3.0, wherein the GdmCl is guanidine hydrochloride, CAS No.: 50-01-1.

[0025] The amount of the buffer A in step (1) is preferably 0.5-2 mmol / L of the N-terminal selenium-cysteine polypeptide hydrazide in the system; more preferably, 1 mmol / L.

[0026] The amount of the ascorbate in step (1) is preferably 0.1-0.3 mmol / L in the system; more preferably, 0.2 mmol / L.

[0027] The diselenide bond reducing agent in step (1) is preferably tris (2-chloroethyl) phosphate (TCEP).

[0028] The amount of the diselenide bond reducing agent in step (1) is preferably 180-220 equivalents of the N-terminal selenium-cysteine polypeptide hydrazide; more preferably, 200 equivalents.

[0029] The reaction condition after adding the diselenide bond reducing agent in step (1) is preferably shaking at room temperature for 4-6 min; more preferably, shaking at room temperature for 5 min. The room temperature in the present application refers to 20-30°C.

[0030] The acetylacetone used in step (2) is preferably used in an amount of 2-3 times the equivalent amount of the polypeptide hydrazide with N-terminal selenocysteine; more preferably, in an amount of 2.5 times the equivalent amount.

[0031] The reaction condition after the acetylacetone is added in step (2) is preferably shaking at room temperature for 30-90 min; more preferably, shaking at room temperature for 60 min.

[0032] The amount of glutathione used in step (3) is preferably 50-70 mmol / L in the system; more preferably, 60 mmol / L.

[0033] The amount of phosphorus acid tris (2-chloroethyl) ester used in step (3) is preferably 50-70 mmol / L in the system; more preferably, 60 mmol / L.

[0034] The pH in step (3) is preferably 6.3-6.7; more preferably, 6.5.

[0035] The purification in step (3) is preferably achieved by reverse phase liquid chromatography (HPLC); the mobile phase of the reverse phase liquid chromatography is acetonitrile / water mixed solution containing 0.1% trifluoroacetic acid.

[0036] The application of the above-mentioned selenocysteine-mediated cyclic peptide synthesis method in polypeptide / protein synthesis.

[0037] The present application has the following advantages and effects relative to the prior art:

[0038] The present application activates the C-terminal hydrazide group by acetylacetone to form acylpyrazole, and realizes efficient cyclization reaction by using the extremely strong nucleophilicity of selenol group of selenocysteine, greatly shortening the reaction time, and the reaction can be carried out at a lower pH and lower concentration, providing a new idea for the synthesis of difficult-to-dissolve cyclic peptides.

[0039] The present application does not need polypeptide thioester / selenoester intermediates to participate in the reaction, and the hydrazide group is directly attacked by selenol group to form a ring after being activated to acylpyrazole, reducing the reaction steps and operation process. In addition, since a large amount of reducing agent is added in the reaction, the formation of various complex dimer structures is also avoided, the reaction product is single, and the liquid phase purification of the target cyclic peptide is greatly simplified. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 HPLC and LC-MS schematic diagram for simulating the cyclization reaction process of the peptide MPA (1 is a linear simulation peptide, and 2 is a target cyclic peptide).

[0041] Figure 2HPLC and LC-MS schematic diagram for simulating the cyclization reaction process of the peptide MPL (1 is a linear simulation peptide, 2 is a target cyclic peptide, 3 is a hydrolysis byproduct).

[0042] Figure 3 HPLC and LC-MS schematic diagram for simulating the cyclization reaction process of the peptide MPG (1 is a linear simulation peptide, 2 is a target cyclic peptide).

[0043] Figure 4 HPLC and LC-MS schematic diagram for simulating the cyclization reaction process of the peptide MPK (1 is a linear simulation peptide, 2 is a target cyclic peptide, 3 is a hydrolysis byproduct).

[0044] Figure 5 HPLC and LC-MS schematic diagram for simulating the cyclization reaction process of the peptide MPH (1 is a linear simulation peptide, 2 is a target cyclic peptide, 3 is a hydrolysis byproduct).

[0045] Figure 6 HPLC and LC-MS schematic diagram for simulating the cyclization reaction process of the peptide Cyclonellin (1 is a linear simulation peptide, 2 is a target cyclic peptide, 4 is the cyclic peptide Cyclonellin).

[0046] Figure 7 Reaction flow schematic diagram of the method of the present application. DETAILED DESCRIPTION

[0047] The present application will be further described in conjunction with the following examples and drawings, but the embodiments of the present application are not limited thereto.

[0048] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0049] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0050] The structure of selenocysteine used in the following examples is as follows:

[0051]

[0052] The HPLC used in the following examples uses an Agilent 1260 instrument, a Phenomenex C18 column, and a mobile phase of water and acetonitrile (containing 0.1% TFA).

[0053] The reagent names and abbreviations used in the following examples are as follows:

[0054] DMSO: dimethyl sulfoxide

[0055] TCEP: tris (2-chloroethyl) phosphate

[0056] GSH: glutathione

[0057] ACN: acetonitrile

[0058] TFA: trifluoroacetic acid.

[0059] In the following examples, when the substrate polypeptide is the peptidomimetic MPX, X represents the C-terminal amino acid in the amino acid sequence of MPX, which is alanine (Ala), leucine (Leu), glycine (Gly), lysine (Lys), and histidine (His), respectively. The peptidomimetics are obtained by solid-phase polypeptide synthesis, and the N-terminal amino acid is selenocysteine. The final polypeptide hydrazide fragment (named MPA, MPL, MPG, MPK, and MPH, respectively) has the amino acid sequence UGYLKSLX-NH2.

[0060] In the following examples, when the substrate polypeptide is the cyclic peptide Cyclonellin, it is obtained by solid-phase polypeptide synthesis, and the N-terminal amino acid is selenocysteine (Sec). The final polypeptide hydrazide fragment has the amino acid sequence UNPRYPYT-NHNH2. After cyclization, the selenocysteine residue can be removed to obtain an in situ alanine residue.

[0061] Example 1: Attempt at cyclization of peptidomimetics

[0062] (1) Reductive treatment of polypeptide dimer

[0063] The synthesized peptidomimetic MPX is dissolved in a buffer solution of 6 mol / L GdmCl, 0.2 mol / L Na2HPO4, and pH 3, to a concentration of 1 mmol / L of MPX in the system. Then 0.2 M ascorbate is added, and the solution is fully dissolved to a light yellow color. Then, tris(2-chloroethyl)phosphate (TCEP) is added in an amount of 200 equivalents of the polypeptide, and the reaction is allowed to proceed at room temperature for 5 min to ensure that the diselenide bond in the polypeptide is broken. The reaction can be monitored by reverse-phase HPLC. In the method 20%-50% in 25 min (the concentration of ACN is increased from 20% to 50% in 25 min), it is determined whether the reaction is complete according to whether only the target molecular peak appears. After the reaction is completed, the product is purified by preparative reverse-phase HPLC using the same method as before.

[0064] In the peptidomimetics, MPX is MPA, MPL, MPG, MPK, and MPH, respectively.

[0065] (2) Activation of the hydrazide end and cyclization reaction

[0066] To the above solution, acetylacetone was added in an amount of 2.5 times the amount of polypeptide, and the reaction was stirred at room temperature for 60 min. The reaction was monitored by reverse HPLC and MALDI-TOF.

[0067] The results are shown in Table 1. When X is different amino acids, the reaction time of the analog peptide is different, but all of them have shown that the reaction is complete at 60 min. Different analog peptides can have a small amount of hydrolysis by-products. Figures 1-5

[0068] Example 2: Synthesis of cyclic peptide Cyclonellin

[0069] Referring to the method of Example 1, the cyclic peptide Cyclonellin with selenol group was prepared, and then the following operations were performed:

[0070] (3) In situ deselenization to alanine

[0071] The above reaction solution was taken, and glutathione (GSH) and tris (2-chloroethyl) phosphate (TCEP) were added to a final concentration of 60 mmol / L and 60 mmol / L, respectively. The pH of the reaction solution was adjusted to 6.5 using 0.5 M NaOH, and the reaction was stirred at 37°C for 16 h. The reaction was monitored by reverse HPLC and LC-MS. After centrifugation and filtration, preparative reverse HPLC was used for preparative purification, freeze-drying, weighing, and storage at -20°C.

[0072] Figure 6 The HPLC and LC-MS diagrams of the reaction process are shown in Table 2, and the yield is 52.28%.

[0073] The above deselenization reaction can also be oxidative deselenization, and the corresponding site can be obtained as a serine residue after selective deselenization.

[0074] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.​

Claims

1. A method for synthesizing selenocysteine-mediated cyclic peptides, characterized in that: Includes the following steps: (1) Dissolve the N-terminal selenocysteine-containing polypeptide hydrazide in buffer A, add ascorbate, and dissolve; then add diselenyl bond reducing agent, dissolve, react, and obtain polypeptide reaction solution B; the diselenyl bond reducing agent is tris(2-chloroethyl) phosphate. (2) Add acetylacetone to the polypeptide reaction solution B obtained in step (1), react, and obtain polypeptide reaction solution C; (3) Add glutathione and tri(2-chloroethyl) phosphate to the polypeptide reaction solution C obtained in step (2), adjust the pH, and react; purify the reaction solution to obtain the target product; The composition of buffer A in step (1) is as follows: 5-7 mol / L GdmCl, 0.1-0.3 mol / L Na2HPO4, pH=2.9-3.1; wherein, the GdmCl is guanidine hydrochloride, CAS number: 50-01-1.

2. The method for selenocysteine-mediated cyclic peptide synthesis according to claim 1, characterized in that: The composition of buffer A mentioned in step (1) is as follows: 6 mol / L GdmCl, 0.2 mol / L Na2HPO4, pH=3.

0.

3. The method for selenocysteine-mediated cyclic peptide synthesis according to any one of claims 1-2, characterized in that: The amount of buffer A mentioned in step (1) is based on the concentration of the polypeptide hydrazide with selenocysteine ​​at the N-terminus in the system being 0.5 to 2 mmol / L; The amount of ascorbate used in step (1) is calculated based on its concentration in the system being 0.1–0.3 mmol / L; The amount of diselenyl bond reducing agent mentioned in step (1) is calculated as 180 to 220 times the equivalent of a polypeptide hydrazide with selenocysteine ​​at the N-terminus.

4. The method for selenocysteine-mediated cyclic peptide synthesis according to claim 1, characterized in that: The amount of buffer A mentioned in step (1) is based on the concentration of the polypeptide hydrazide with selenocysteine ​​at the N-terminus being 1 mmol / L in the system; The amount of ascorbate used in step (1) is calculated based on its concentration in the system being 0.2 mmol / L; The amount of diselenyl bond reducing agent mentioned in step (1) is calculated as 200 equivalents of a polypeptide hydrazide with selenocysteine ​​at the N-terminus.

5. The method for selenocysteine-mediated cyclic peptide synthesis according to claim 1, characterized in that: The amount of acetylacetone used in step (2) is calculated as 2 to 3 equivalents of a polypeptide hydrazide with an N-terminus of selenocysteine; The amount of glutathione used in step (3) is based on its concentration in the system being 50-70 mmol / L; The amount of tri(2-chloroethyl) phosphate used in step (3) is based on its concentration in the system being 50-70 mmol / L; The pH value mentioned in step (3) is 6.3 to 6.

7.

6. The method for selenocysteine-mediated cyclic peptide synthesis according to claim 5, characterized in that: The amount of acetylacetone used in step (2) is calculated as 2.5 times the equivalent amount of a polypeptide hydrazide with an N-terminus of selenocysteine; The amount of glutathione used in step (3) is based on a concentration of 60 mmol / L in the system; The amount of tri(2-chloroethyl) phosphate used in step (3) is based on a concentration of 60 mmol / L in the system; The pH value mentioned in step (3) is 6.

5.

7. The method for selenocysteine-mediated cyclic peptide synthesis according to claim 1, characterized in that: The reaction conditions described in step (1) after adding the diselenyl bond reducing agent are a shaking reaction at room temperature for 4 to 6 minutes; The reaction conditions described in step (2) after adding acetylacetone are a shaking reaction at room temperature for 30 to 90 minutes.

8. The method for selenocysteine-mediated cyclic peptide synthesis according to claim 1, characterized in that: The purification described in step (3) is achieved by reversed liquid chromatography; the mobile phase of the reversed liquid chromatography is an acetonitrile / water mixture containing 0.1% trifluoroacetic acid.

9. The application of the selenocysteine-mediated cyclic peptide synthesis method according to any one of claims 1 to 8 in cyclic peptide synthesis.

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